Receptor-Mediated Protein Transport into Cells. Entry Mechanisms for Toxins, Hormones, Antibodies, Viruses, Lysosomal Hydrolases, Asialoglycoproteins, and Carrier Proteins

Receptor-Mediated Protein Transport into Cells. Entry Mechanisms for Toxins. Hormones. Antibodies. Viruses. lysosomal Hydrolases. Asialoglycoproteins. and Carrier Proteins DAVID M . NEVILLE. J R. AND TA-MIN CHANG Section on Biophysical Chemistry Lobo ra tory of Neu rochemistry National Institute of Mental Health Bethesda. Maryland

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 68 I1 . Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . DiphtheriaToxin . . . . . . . . . . . . . . . . . . . . . . . . 68 B . Abrin and Ricin . . . . . . . . . . . . . . . . . . . . . . . . 76 80 C . Tetanus Toxin . . . . . . . . . . . . . . . . . . . . . . . . . 86 D . Botulinum Toxin . . . . . . . . . . . . . . . . . . . . . . . . 91 E . Cholera Toxin . . . . . . . . . . . . . . . . . . . . . . . . . F . Colicins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 101 111. Carrier Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Transcobalamin I1 . . . . . . . . . . . . . . . . . . . . . . . 101 B . Transferrin . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 C. Low-Density Lipoprotein . . . . . . . . . . . . . . . . . . . . 107 111 IV . Asialoglycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . A . Structural Requirements for Transport . . . . . . . . . . . . . . 111 B . Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 C . Pinocytotic Mechanism? . . . . . . . . . . . . . . . . . . . . 114 V . Fibroblast Lysosoinal Hydrolases . . . . . . . . . . . . . . . . . . 115 VI . Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 A . Maternal-to-Young Transfer . . . . . . . . . . . . . . . . . . . 116 B . Transport into IInmunological Cells . . . . . . . . . . . . . . . 119 C . Retrograde Axonal Transport . . . . . . . . . . . . . . . . . . 120 120 VII . Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Evidence for Receptor-Mediated Entry . . . . . . . . . . . . . 120 B . General Transport Mechanisms . . . . . . . . . . . . . . . . . 121 VIII . Growth Factors and Hormones . . . . . . . . . . . . . . . . . . . 122 A . Nerve Growth Factor . . . . . . . . . . . . . . . . . . . . . . 122 B . Glycoprotein Hormones . . . . . . . . . . . . . . . . . . . . . 126 65

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C. Lactogenic Hormones . . . . . . . . . . . . . . . . . . . . . . D. I n s u l i n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Epidermal Growth Factor . . . . . . . . . . . . . . . . . . . . IX. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Intracellular Localization Following Transport . . . . . . . . . B. Mechanisms of Transport . . . . . . . . . . . . . . . . . . . . C. Unique Functions of Receptor-Mediated Protein Transport . . . X. Pharmacological Implications of Receptor-Mediated Protein Transport References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . .

129 130 131 131 131 132 135 137 139

I. INTRODUCTION

Many cells are capable of transp0rtin.g certain proteins from the extracellular environment to an intracellular. compartment by a process which has an initial obligatory binding step to a cell surface receptor. The process is distinct from bulk fluid pinocytosis which does not require receptor binding. We refer to this process as receptor-mediated protein transport. Receptor-mediated protein transport is widespread in nature and is responsible for a variety of biological effects. Several examples of proteins transported by this process are transcobalamin I1 (TC 11)-cobalamin complex, immunoglobulins, desialylated glycoproteins, nerve growth factor (NGF),and a variety of bacterial and plant seed toxins. A common feature of each example is the high degree of cell type specificity present in the transport process. Cells lacking the specific receptor are incapable of transport. A major difference among the examples is the ultimate localization of the transported protein. Some proteins are transported through the cell to another extracellular compartment. Others localize in the lysosomes, while a few exert profound effects within the cell cytosol compartment. Consideration of these facts leads to the posing of several questions. Do all cell surface receptors participate in receptormediated transport, or is entry mediated by a class of receptors with unique properties? Are receptors divisible into certain classes which mediate entry to particular intracellular compartments? Of the receptors which mediate entry is the binding process sufficient to initiate entry, or is a more complex interaction between the receptor and the transported protein required? These questions are posed as a first step in constructing simple models of receptor-mediated protein transport. In the following sections we discuss the available data on a variety of receptor-mediated protein transport systems and attempt to answer these questions. The formal literature search for this article was completed in December 1976, although a few 1977 papers have been included. In

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order to keep the size manageable other reviews or well-referenced research papers are sometimes cited instead of the original reports. This review is limited to examples of protein transport into cells which have been demonstrated to occur b y receptor-mediated processes and which cause a change in the physiological state of the cell or organism. We have attempted to include all such examples which have been described but undoubtedly have missed some. The article is written primarily for experimentalists interested in one or more areas of receptor-mediated protein transport. Therefore pertinent experimental details and numbers derived from experiments are given. The diverse nature of this material and the presumed diversity of the readership has impelled us to include background physiology and pathology for many subject areas. These factors have produced a lengthy review. However, each section is complete within itself, and readers may choose from the table of contents any desired subject or ordering of subjects. The particular focus of this article is the result of our interest in utilizing receptor-mediated protein entry processes for pharmacological purposes, by the construction of artificial hybrid proteins. These concepts are discussed in Section X. Beyond a simple understanding of the transport process and its attendant specificity and compartmentalization is the broader question of the utility of the process to the organism. Carrying vitamin B12into a human cell interior is easily grasped as a useful event, but carrying diphtheria toxin is not. We consider the possibility that toxin transport systems are used for other purposes which have so far escaped detection. A further question for consideration is the utility humans can make of receptor-mediated protein transport. Because of the high degree of cell type specificity present in the process, and the profound effects of certain enzymes after transport to the cell cytosol, we have suggested that a whole new class of cell type-specific pharmacological reagents could be constructed which utilize this process (Chang and Neville, 1977). Attempts to construct such reagents by the formation of artificial protein hybrids are analyzed in terms of the present knowledge of receptor-mediated protein transport systems. Many receptor-mediated protein transport systems are operative at extremely low extracellular protein concentrations and transport minute quantities of protein, in the range of tens of molecules per hour. Were it not for the profound intracellular toxicity of some of these proteins, such systems would escape detection. However, since toxicity is a quantifiable end point in the process, it provides investigators with a means of studying these low-capacity transport

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systems. It is not surprising then that various bacterial and seed toxins constitute the best known examples of receptor-mediated transport. Of these diphtheria was the first to be studied. II. TOXINS A. Diphtheria Toxin

1. GENERALCHARACTERISTICS Human diphtheria is characterized by an upper respiratory infection which progresses to severe localized necrosis, forming a pseudomembrane which may cause death by suffocation. Profound muscle weakness and lethargy also occur, and death is often ascribed to heart failure. An exotoxin was identified as the causative agent in 1888. It is secreted by Corynebacterium diphtheriae lysogenic for or infected with bacteriophage p carrying the tox+ gene. The toxin is easily purified and may account for 5% of the total bacterial protein synthesized. The purified toxin is a 62,000-dalton protein. Twenty-five nanograms injected into a guinea pig causes lethargy and muscle weakness in 3-4 days and death within 5 days. Only certain animal species are sensitive to diphtheria toxin. Humans, rabbits, chickens, and guinea pigs are equally sensitive on a weight basis. Mice and rats are resistant, requiring over 1000 times the dose before toxicity is noted. Today we have an almost complete understanding of the pathogenesis of this disease in molecular terms. The one remaining gap concerns the transport process. We outline the present state of knowledge as briefly as possible and suggest that the reader consult the reviews of Collier (1975) and Pappenheimer and Gill (1973) for a fuller appreciation of how the mystery of this disease process was unraveled.

2. INTRACELLULAR SITE OF ACTION In 1959 Strauss and Hendee discovered that the toxicity of diphtheria toxin toward intact sensitive cells was due to an inhibition of protein synthesis. Following exposure to the toxin there is a lag period followed by an exponential fall in protein synthetic rate as a function of time. The higher the toxin concentration, the shorter the lag period up to a limiting lag period which depends on cell type and incubation conditions (Uchida et al., 1973). Cell-free protein synthetic systems reconstituted from ribosomes

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and various cell cytosol soluble fractions were later tested. Diphtheria toxin was inhibitory in these systems, and a cofactor requirement was found which proved to be NAD+ (Collier and Pappenheimer, 1964). Shortly thereafter Honjo and co-workers (Honjo et al., 1968) showed that diphtheria toxin was an enzyme which catalytically inactivated elongation factor 2 (EF-2), a necessary soluble protein component of the protein synthetic machinery. Inactivation was accomplished by transferring 1 mole of adenosine diphosphoribose from NAD+ to 1 mole of EF-2: N A D + + EF-2 (active)

.

diphtheria toxin

.ADPR-EF-2 (inactive)

+ nicotinamide + H+

The cause of the extreme toxicity of diphtheria toxin was apparent. There are only about 1.2 molecules of EF-2 per ribosome, and the turnover of EF-2 is slow. The equilibrium of the ADP-ribosylation reaction under intracellular conditions is such that the reaction goes virtually to completion (Collier, 1975). Calculations reveal that a steady-state concentration of only a single diphtheria toxin molecule per cell is required to inactivate the cell's supply of EF-2 within 1 day. Since the cellula,r EF-2 concentration is not normally rate limiting for protein synthesis, the dose-dependent segment of the lag period represents the time taken to reduce the EF-2 to a rate-limiting concentration (Pappenheimer and Gill, 1973). In the intact cell EF-2 cycles between the cell cytosol and its ribosome-binding site. Studies had shown that ribosome-bound EF-2 was immune to inactivation by toxin (Collier, 1975). This established the cell cytosol as the site of action of diphtheria toxin. A mechanism was then needed to transport diphtheria toxin from the extracellular fluid across the cell membrane to the cell cytosol.

3. STRUCTURAL

AND

FUNCTIONAL INTERRELATIONSHIPS

Knowledge of the mechanism of entry of diphtheria toxin into the cell cytosol was greatly increased by elucidation of the structural and functional relationships of the toxin molecule. It was discovered that diphtheria toxin was susceptible to site-specific proteolytic cleavage and reduction by thiols, generating two fragments of 39,000 daltons (fragment B) and 21,000 daltons (fragment A) (Gill and Dinius, 1971; Collier and Kandel, 1971). The B fragment was devoid of enzymic activity, while the A fragment was highly active. T h e separated fragments showed no toxicity toward intact cells. By mixing A and B frag-

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ments and removing thiol, toxicity toward cells was reestablished. The hypothesis was advanced that only the A fragment was needed within the cytosol to produce toxicity and that the B fragment was somehow involved in the entry mechanism. To test this hypothesis Uchida and co-workers (Uchida et al., 1973) studied various purified nontoxic proteins which are serologically related to diphtheria toxin (CRM proteins) and are obtained from culture filtrates of C. diphtheriae lysogenized with phages which are mutant in the toxin structural gene (tox-CRM+P).Two nontoxic CRM proteins, CRMIOand CRM,,, were found which contained normal A fragments, as judged by enzymatic activity and electrophoretic mobility, and which lacked large regions of the N-terminal portion of the B fragment. When the altered B fragment was replaced by a normal B fragment or one from CRMlWby forming the hybrid protein &5B197 or Ad5Bwildtype, 55% of the wild-type toxicity returned. Clearly the B fragment performed a necessary function in cell intoxication which was separate from the enzymatic activity of the A fragment. Further studies showed that the interaction of the B fragment with the cell occurred via receptors. Ittelson and Gill (1973) demonstrated that nontoxic CRM19,which contained an enzymatically inactive A fragment and a normal B fragment competitively blocked the toxicity of wild-type toxin toward HeLa cells with K , = lo-* M ,Saturation kinetics of toxin-promoted inhibition of protein synthesis were also noted with saturation at 5 x lo-’ M toxin (Uchida et al., 1973).The phenomenon of saturation and competitive inhibition requires the presence of a finite number of saturable binding sites, and such sites are called receptors. [Throughout this review we use the term “membrane receptor” in a phenomenonological sense, as originally defined by Langley (1905)to describe a process which can be explained by binding that exhibits saturation and competition with related substances. Thus receptor binding can be approximated by the term

(or a sum of such terms), where B1 is the amount bound, n1 is the number of sites, K1 is the equilibrium affinity constant, and F is the free ligand concentration. Nonspecific binding refers to nonsaturable binding which over the concentration range specified can be represented by B2 = K2F (Neville, 1974).1 B-Chain toxin receptors appear to be localized to the external surface of the cell membrane. When cells are exposed to toxin, toxin is

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rapidly bound to them. The binding step precedes the transport step. The dissection of these two events was made possible by the discovery of Kim and Groman (1965) that the presence of ammonium ion blocks the toxicity of toxin toward cells but does not affect the binding step. The binding is sufficiently strong to resist washing of the cells with buffer. Cells can be treated with toxin in the presence of 4 x M NH4+, washed, and reincubated with and without NH4+.Toxicity develops only in cells washed free of NH4+. However, when the washing out of NH4+is preceded by a 30-minute exposure to antitoxin antibody, the cells are protected from toxicity (Duncan and Groman, 1969; Ivins et al., 1975). Antitoxin antibody is without a protective effect when added after toxin in the absence of NH4+ and presumably cannot cross the membrane to reach the cytosol. The conclusion is that the antitoxin antibody is neutralized at the toxin-binding site on the external surface of the membrane. T h e presumption is that this binding site is identical to the receptor inferred from competition and saturation experiments.

4. THE TRANSPORT PROCESS

The mechanism of transport of diphtheria toxin has been investigated by following the uptake of 1251-labeleddiphtheria toxin by tissue-cultured cells. Bonventre et al. (1975) observed the uptake b y a sensitive cell line, HEp-2, and a resistant line, L929, using concentrations of diphtheria toxin in the range of M . The resistant cell line had a higher uptake than the sensitive line. The presence of poly-Lornithine at 10 pg/ml doubled uptake in the sensitive line. However, subsequent experiments showed that poly-L-ornithine inhibits the toxicity of diphtheria toxin. Uptake studied in the presence and absence of 4 x M ammonium chloride, which fully protected the sensitive line from toxicity, revealed no differences in the rate of uptake. Since ammonium ion does not block binding yet must block transport to the cytosol compartment of active fragment A, it can be concluded that 90% or more of the uptake observed in these experiments is localized to a compartment other than the cell cytosol, and that this transport process is not related to toxicity. This uptake decreases with time, a situation encountered with other examples of presumed adsorptive or receptor-mediated pinocytosis (Steinman et a1., 1974). [Pinocytosis is the process by which cells take up fluid from the external medium in bulk by the internalization of small portions of plasma membrane. As Fawcett (1965) points out, a variety of different kinds of surface activity lead to this result. Several examples are the

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invagination of microscopically visible tubes in the ameba, the development of apical canaliculi of submicroscopic size in the absorptive epithelium of the kidney, and the formation of minute vesicles just below the surface membrane of a variety of cells. The last-mentioned process is subdivided into two distinct morphologies, vesicles formed from smooth-surfaced membrane and vesicles formed from specialized membrane. The specialized membrane often exhibits an indentation in which both surfaces are thickened. These are called coated regions, and the vesicles so derived are called coated vesicles (Roth and Porter, 1964; Fawcett, 1965). The terms “macropinocytosis” and “micropinocytosis” were originally defined by the resolution of the light microscope. Many investigators use these terms in a relative sense without an absolute size cutoff. Uptake of solubilized macromolecules by cells undergoing pinocytosis has shown that certain proteins are taken up in direct proportion to the fluid engulfed. This uptake process is called bulk fluid pinocytosis. Other proteins are taken up in excess of the fluid engulfed, and uptake exhibits saturation and competition with analogs. The latter process is explained by receptors on the surface membrane, which concentrate the macromolecule (Steinman et al., 1974). This process is called adsorptive pinocytosis or receptor-mediated pinocytosis, as distinguished from bulk fluid pinocytosis.] Uptake of lZ5I-labeleddiphtheria toxin has been studied as a function of concentration over the range 5 x 10-9-10-7 M b y Boquet and Pappenheimer (1976). These experiments were performed using HeLa cells at 30°C in the presence and absence of excess unlabeled diphtheria toxin. At each concentration point the uptake of lz5Ilabeled toxin at 1 hour was greater in the absence of excess unlabeled toxin. In an attempt to delineate the number of receptor sites, points obtained in the presence of excess unlabeled toxin have been fitted with a straight line, whereas a curved line has been used to fit the points obtained in the absence of unlabeled toxin. Points obtained by subtracting cold plus hot points from hot points have been fitted to a hyperbolic function. This is called specific binding. The number of receptor sites per cell calculated from the point of saturation is 3500. Considering the scatter of the data and the forcing of the fit, it is unlikely that these data can be interpreted as providing a direct demonstration of saturable binding sites with a K of 10“M-’ (which agrees with the competition experiments using cytotoxicity as an end point.) In addition it is not clear in these experiments how much of the observed uptake at 1 hour represents binding and how much constitutes transport into a compartment which is not in equilibrium with the external media. However, the data do show that a portion of the

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toxin uptake occurs b y a saturable process. Saturable uptake was not found in a toxin-insensitive cell line. It is tempting to consider this finding as support for the proposition that insensitive cell lines lack the specific cell surface receptor. However, it is not known whether or not the saturable uptake measured in the sensitive cell line is the process productive of toxicity. Until this relationship can be established, the above proposition remains untested. Another type of experiment has been performed in an attempt to delineate the events occurring subsequent to the binding step. We have already mentioned experiments which have shown that the nontoxic cross-reacting protein CRMle7 can compete with toxin for entry into HeLa cells. These results were interpreted as indicating the presence of specific receptors interacting with toxin with a reversible equilibrium. Boquet and Pappenheimer (1976) were unable to obtain unequivocal evidence for a reversible equilibrium from direct uptake measurements at low temperatures or by using chase experiments with excess unlabeled toxin added after the addition of labeled toxin. These investigators postulated an initial rapid reversible interaction between toxin and receptor followed by a slow irreversible process. M was incubated with To test this hypothesis CRM,,, at 4 x HeLa cells for various intervals, followed b y the addition of M toxin. A time-dependent protection against toxicity was noted, and after 1 hour of preincubation with CRM almost complete protection was achieved. Thus CRMls7 appeared to block the toxin entry sites. This conclusion appears valid, but the irreversible nature of the process may simply be due to a very slow rate of dissociation of CRM,,, from its receptor. Without knowledge of the forward rate constant of association and the rate constant of dissociation it is impossible to interpret whether this experiment dealt with a single-step binding process or with a subsequent irreversible process which occurred after binding. Further insights into the mechanism of the binding and transport of diphtheria toxin have been provided by studying materials which block cytotoxicity. These experiments are performed by incubating cells with toxin plus another agent, followed by washing out the toxin and the agent. At this point the cells are divided into two groups, one of which is treated with a diphtheria antitoxin wash. In this way the action of a protective agent can be determined to occur at the binding site or during the transport step. Toxin bound but not transported by the action of a protective agent is neutralized by antitoxin. Agents interfering with binding do not require antitoxin treatment to elicit their protective effect during the cytotoxic assay. I n this way, Duncan and Groman (1969) showed that cations were essential for the binding

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of diphtheria toxin to HeLa cells. The effectiveness of cations in mediating binding was related to their charge. Cells incubated in isoosmolar sucrose were completely protected against toxin and 0.1 M sodium ion was required to achieve significant toxicity. Calcium at M was more effective, and M aluminum could replace calcium. In a similar manner, a pH value above 9.5 was also found to inhibit binding. Binding was not inhibited by a l-hour exposure to 1% trypsin, 1% chymotrypsin, pronase, lysozyme, neuraminidase, or hyaluronidase. Metabolic inhibitors of respiration, cyanide, and 2,4dinitrophenol failed to protect against cytotoxicity. However, M sodium fluoride gave complete protection. Under these conditions glycolysis fell 60%.Iodoacetate, another glycolytic inhibitor, gave no protection at 1.3 x M . Higher concentrations were toxic to cells, and glycolysis was unfortunately not measured at the utilized concentration. Cells not washed with antitoxin, which had been pretreated with sodium fluoride, showed complete toxicity, indicating that fluoride blocked a step subsequent to binding. Protection by fluoride has been confirmed by Ivins et al, (1975) and Middlebrook and Dorland (1977a), but the mechanism of protection is not known. Fluoride may work by inhibiting the formation of ATP through the glycolytic pathway. To be effective, ATP production through respiration could occur only at low levels. Since cellular ATP levels were not measured in these studies, the problem remains unsolved. Fluoride is also known to be a potent inhibitor of a variety of phosphatases. Using the cell line HEp-2, Ivins et al. (1975)found that cyanide or dinitrophenol gave partial protection against diphtheria toxin. Again, ATP levels were not measured. Sodium arsenite also gave partial protection, as did poly-L-ornithine and cytocholasin B. Sulfhydryl reagents such as p-chloromercuriphenylsulfonic acid and dithiothreitol were without effect. Middlebrook and Dorland (1977a) carried out an interesting comparative study on the effect of a variety of agents that protect against diphtheria toxin and the toxin from Pseudomonas aeruginosa, utilizing HeLa and HEp-2 cell lines. This study is particularly interesting, since Pseudomonas toxin has been found to have the same site of action as diphtheria toxin. Iglewski and Kabat (1975) showed that P . aeruginosa toxin catalyzed the transfer of ADP-ribose from NAD+ to EF-2. Since both toxins have the same intracellular site of action and utilize the same enzymic reaction, differences among protective agents reflect differences in transport or binding. Of a variety of agents tested, none protected against Pseudomonas toxin. In addition to the agents previously found to protect against diphtheria toxin, Middle-

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brook and Dorland (1977a) also noted almost complete protection by 10 p M ruthenium red. The anesthetic agents procaine hydrochloride and lidocaine also gave significant protection. Whether the protection achieved by ruthenium red is a consequence of inhibition of calcium-magnesium-dependent ATPase or by nonspecific blocking of multivalent cationic binding sites remains to be determined. Middlebrook and Dorland (1977a) tested all their protective agents in the cell-free EF-2 ADP-ribosylation system. Only salicyclic acid and procaine affected the reaction in a manner which could explain their protective effects. The differential protection afforded against diphtheria toxin toxicity b y ammonium ion, ruthenium red, and sodium fluoride, in contrast to the situation with Pseudomonas toxin, is best explained by different transport processes for these two toxins. It also appears from the studies of Middlebrook and Dorland (1977b) that these two toxins have different receptor specificities. These investigators determined the tissue culture median lethal dose for both toxins in 21 different cell lines. For diphtheria toxin the tissue culture median lethal dose varied from 0.02 ng/ml to greater than 103 ng/ml. For Pseudomonas toxin the range was 0.1 ng/ml to 300 ng/ml. For diphtheria toxin a general species hierarchy was seen, monkeys being the most sensitive, followed by hamsters and humans, with rats and mice being very insensitive. No species hierarchy was determined for Pseudornonas toxin, and in general there was little correlation between the sensitivities toward the two toxins, Pseudomonas having high toxicity for some mouse and rat cell lines. These data are particularly welcome, since most data of this sort used for comparative purposes have been obtained in various laboratories under various conditions. Middlebrook and Dorland (1977~) addressed themselves to the problems of performing quantitative comparative cytotoxic studies utilizing these toxins. In particular they found that most serum used in tissue culture displayed various degrees of inhibition toward the toxins and that fetal calf serum was preferred for the studies. The large difference in sensitivity between mouse cell lines and human cell lines toward diphtheria toxin has been used by Creagan et al. (1975)to localize the human chromosome responsible for diphtheria toxin sensitivity. These investigators studied the sensitivity toward diphtheria toxin for a variety of hybridized cell lines. Hybridization between mouse and human lines was achieved by treating cells with inactivated Sendai virus. Chromosomal distribution was studied in all lines, and a correlation was achieved between the presence of human chromosome 5 and hybrid cell sensitivity toward diphtheria toxin.

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Whether human chromosome 5 carries information for the receptor, the transport system, or both remains to be determined. Various models for toxin transport to the cytosol are considered in Section IX,B, B. Abrin and Ricin

1. GENERALPROPERTIESAND STRUCTURES Abrin and ricin are two extremely toxic proteins present in the seeds of the taxonomically unrelated species Abrus precatorius and Ricinus communis. These toxins are quite similar to each other in structure and mechanism of action and also share similarities in general structural features and mechanism of action with diphtheria toxin. Abrin and ricin both inhibit protein synthesis in eukaryotic cells following a dose-dependent lag period. The site of action of abrin and ricin is the 60s ribosome. These proteins are both two-chain structures of MW -60,000, the two chains being held together by a disulfide bond. Separated purified chains are nontoxic toward eukaryotic cells. For both proteins the higher-MW chain, 34,000 for ricin and 35,000 for abrin, is known as the B chain and is involvtd in the binding process. The A chain, 32,000 for ricin and 30,000 for abrin, catalytically inactivates 60s ribosomes in cell-free systems (Refsnes et al., 1974). Seeds of Abrus and Ricinus also contain two hemagglutinins known as Abrus and Ricinus agglutinin, which are related to the toxic proteins. These agglutinins are capable of agglutinating erythrocytes of human blood groups A, B, and 0. The MW of each agglutinin is approximately double the toxin MW, 120,000. Immunological studies indicate that the agglutinins contain two B chains indistinguishable from the toxin B chains. In addition, each agglutinin contains two chains similar but not identical to toxin A chains. In the case of Ricinus agglutinin, the A chains lack certain antigenic determinants present on the A chains of Ricinus toxin. In addition, the agglutinin A chains appear to be approximately 1000-3000 daltons smaller than the A chains of Ricinus toxin (Pappenheimer et al., 1974).

2. BINDING SITES The binding properties of abrin and ricin have been extensively studied. The receptor site presumably contains lactose or galactose, since serum proteins containing nonreducing terminal galactose residues interfere with the binding. Direct binding of lactose to ricin has

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been demonstrated. The binding constant is approximately 105 M-', and there is one site per ricin molecule. The Ricinus agglutinin molecule contains two lactose binding sites (Olsnes et al., 1974). The binding affinity for HeLa cells and erythrocytes is higher, with equilibrium association constants reported at 3 x 10s M-' for both ricin and abrin. Binding is rapid at both 0 and 37°C and is reversible. Reciprocal transformation of the binding isotherms over a concentration range of 10-'o-10-8 M is linear, indicating a single class of receptor sites. There are approximately 3 x lo7 sites per HeLa cell (Sandvig et al., 1976). The minimum concentrations of abrin and ricin producing inhibition of protein synthesis have been detected at 0.2 ng/ml for abrin and 2 ng/ml for ricin. For abrin this calculates to a molar concentration of 3.4 pM. At this concentration only about 0.01% of the total sites are occupied. The binding sites for ricin and Ricinus agglutinin are similar in that each substance competes to a considerable extent for the same sites (Nicolson et al., 1974). However, some differences may exist, since although ricin completely competed for Ricinus agglutinin, the agglutinin did not completely compete for all bound ricin. However, these studies were not performed as equilibrium competition experiments and contained a wash step separating the two incubations. Olsnes et a l . (1976) showed that the purified B chain of ricin can compete for HeLa cell binding sites with intact ricin and intact abrin. These investigators report a binding constant of ricin B chain essentially equal to that of intact ricin. However, the competition data do not indicate equal affinities. This is probably due to the lack of knowledge of the amount of binding of iodinated ricin to the cells which is nonspecific. Purified ricin B chain also protects against toxicity against ricin when present in a 200-fold weight excess. Protection by B-chain ricin against toxicity for abrin under the same conditions appears to be of borderline significance. Since ricin B chain appears to compete to some extent with the binding sites for both abrin and ricin but protects against toxicity only for ricin, all the binding sites revealed b y these techniques may not be involved in the transport process of the toxin to the cell interior. When ricin and abrin are bound to HeLa cells at 3TC, a fraction of the toxins becomes irreversibly bound as a function of time. This is determined by washing cells in 0.1 M lactose at various time points. The fractional amount becoming irreversibly bound for both toxins is 2 x per minute. Abrin and ricin bound to cells at 37°C enter an eclipse phase similar to that seen with diphtheria, cholera, tetanus, and botulinum toxins. Toxin antisera is effective in preventing toxicity only when added to the cells immediately or only shortly after addition of the toxin (Refsnes et al., 1974).

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3. INTRACELLULAR MECHANISM OF ACTION When abrin and ricin in concentrations ranging from 1 ng/ml to 10 pg/ml are incubated with HeLa cells, the kinetics of inactivation of protein synthesis are found to be similar to those seen with diphtheria toxin. The rate of protein synthesis falls (following a lag period) exponentially with time. For abrin and ricin the rate of fall increases with increasing concentration up to a limiting concentration between 1 and 10 pg/ml. This is about the expected saturating level for a binding constant of 108 M-'. Studies utilizing cell-free protein synthetic systems have shown that abrin and ricin inactivate the 60s ribosome. Ribosomes exposed to these toxins have reduced ability to bind aminoacyl-tRNA, reduced GTPase activity, and reduced affinity for binding EF-2 (Benson et al., 1975; Montanaro et al., 1975; Carrasco et al., 1975). The ability of abrin and ricin to inactivate 60s ribosomes increases 50- to 100-fold after exposure of the toxins to reducing agents. Evidently, as is the case with diphtheria toxin, the B chain in the intact molecule blocks the enzymic activity of the A chain (Olsnes et al., 1976). Olsnes and co-workers (Olsnes et al., 1976) noted in the cellfree protein synthetic system that a lag period following the introduction of intact ricin and abrin is due to the time taken to separate the A and B chains. Chain reduction was demonstrated directly by sodium dodecyl sulfate (SDS) gel electrophoresis of iodinated toxin. When purified abrin and ricin A chains are used, there is a linear relationship between the number of ribosomes inactivated per minute and the toxin A concentration. The inactivation rate increases with temperature, and the estimated activation energy is 10.6 kcal/mole. The turnover number for both abrin and ricin A chains is approximately 1500 ribosomes per minute. Interestingly, the A chain of Ricinus agglutinin had the same Michaelis constant, 0.1-0.2 p M , as abrin and ricin A chains and a turnover number of 100 ribosomes per minute (Olsnes et al., 1976). The Ricinus agglutinin used in this study was purified by a method yielding a very low toxicity for Ricinus agglutinin, 1/2300 compared to ricin (Olsnes and Pihl, 1973). Thus Ricinus agglutinin is 150-fold more active in a cell-free assay than in an intact cell assay. The B chains for ricin and Ricinus agglutinin have been found to be indistinguishable (Pappenheimer et al., 1974). The B chains are believed to mediate entry of the active A chains. The discrepancy raises interesting questions. It is possible that having a B chain which binds is not sufficient for entry. The relationship between the B and A chain may not be in a necessary specific configuration. It is also possible that bivalent B chains do not gain entry. Another possibility is that the configuration of the B chain with respect to the A chain is not sufficient to

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inhibit inactivation of the A chain within the cytosol. Thus, although we know that a configurational change must take place after reduction between the A and B chains to achieve activation of enzymatic activity, the B chain may stay associated with the A chain and protect against degradation. It is also possible that the very low i n vivo toxicity of Ricinus agglutinin reflects a very high degradation rate of the A chain within the cytosol, which is due to a structural difference within the A chain. These differences were noted above. This hypothesis places the A chain of Ricinus agglutinin in a category similar to that of the CRM17eA-chain mutant reported by Uchida et d. (1973).Th'is mutant of diphtheria toxin exhibits 9% of the wild-type fragment-A enzymatic activity and only 0.3%of the whole-animal toxicity. Fragment B of CRM,,, is normal. When HeLa cells are exposed to CRM,,, in saturating quantities ( M ) the rate of inactivation of protein synthesis M wild-type toxin. Cells treated with is equivalent to that of 5 x CRM,,, for 3 hours can be rescued b y washing, treating with antitoxin, and resuspending in toxin-free media. The protein synthetic rate continues to fall for another 9 hours and then climbs to the initial toxinfree value. However, it is impossible to rescue cells treated with 5 x M wild-type toxin. Pappenheimer and Gill (1973) interpret this experiment as indicating that CRM176has a more rapid half-time of degradation within the cytosol than wild-type toxin A. This explains the ability to rescue CRMl16 and the discrepancy between the much higher enzymatic activity of CRM,,, A chain and its in vivo toxicity. Recently, Etinger and Goldberg (1977) described an ATP-dependent proteolytic system operating within reticulocytes, which selectively degrades abnormal proteins. Systems such as this one may play a role in degrading exogenous proteins transported to the cell cytosol.

4. THE TRANSPORT PROCESS The mechanism of the transport of abrin and ricin into the cell interior which leads to toxicity is unknown. Nicolson (1974) demonstrated that ricin is taken u p and encased within pinocytotic vacuoles using ferritin-labeled ricin. However, the relationship between this process and the process which produces toxicity is unclear. Studies with metabolic inhibitors and inhibitors of various transport systems have not been reported for abrin and ricin. However, Olsnes and co-workers (Olsnes et al., 1973) have reported experiments which are highly relevant to the pertinent questions of receptor-mediated transport. These investigators purified A and B chains of ricin and then proceeded to reform the various hybrid species. They found that ricin A-abrin B

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and ricin B-abrin A were both cytotoxic, as judged by the LD,, dose for mice, In this study reconstituted abrin LD,, was 0.08 pg, and the reconstituted ricin LD5o was 0.2 pg. The hybrid abrin A-ricin B had an LD5o of 0.15 pg, and the hybrid ricin A-abrin B had an LDSoof 0.3 pg. It is interesting to note that the increased toxicity of abrin over that of ricin appears to be determined b y the A chain. This is in keeping with our previous discussion that the half-time of degradation of the A chain within the cell may be a major factor in governing cytotoxicity. The significance of this experiment is in part dependent upon whether or not the B chains of abrin and ricin utilize the same transport system. Ifdifferent transport systems are utilized, then Olsnes and co-workers (Olsnes et al., 1973) have shown that competent B chains can be recombined with A chains from other molecules and that these A chains gain entry, directed solely by the B-chain transport system. This generalization from their results is somewhat weakened because of the similarity between the abrin and ricin B and A chains. However, these chains are distinct immunologically, even if they have similar receptor binding sites and similar enzymatic activities. Antisera directed against ricin or abrin could not protect for the hybrid toxins. An indication that the two transport systems are different was obtained when it was shown that ricin B chain could block ricin toxicity without having a significant effect on abrin toxicity in HeLa cells (Olsnes et al., 1976). Olsnes and co-workers (Olsnes et al., 1973) were unable to form hybrids of ricin and diphtheria toxin. Their methodology involved dialysis of the reduced chains and presumably involved autoxidation. Hybrid formation between toxins and other putative B chains is discussed in Section X. C. Tetanus Toxin

EXPERIMENTALTOXICITY Tetanus toxin is a potent protein neurotoxin made by Clostridia tetani. In most cases bacterial entry to the body occurs through minor trauma, although in 20%of the cases no wound can be detected. The disease is characterized by muscle rigidity and reflex spasms severe enough to cause compression fractures of the spine and loss of pulmonary ventilation. Death is often the result of respiratory failure and may be induced by laryngeal spasm. The presenting symptoms are usually trismus (lockjaw) and stiffness of the neck. Rigidity of facial, pharyngeal, thoracic, abdominal, arm, and leg muscles follows. Reflex spasms increase in frequency until they follow one another in rapid 1.

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succession. This pattern (Adams, 1971) constitutes generalized tetanus and can be produced in animals by the intravenous or subcutaneous inoculation of the purified toxin. For mice the LD50 is 2.5 ng/kg (Habermann and Dimpfel, 1973).A more limited disease called localized tetanus can be produced by injecting a smaller dose intramuscularly. After a lag period (dose-dependent), rigidity and spasm result which are confined to the groups of muscles innervated by the local motor neuron. Localized tetanus is a rare clinical entity (Habermann and Dimpfel, 1973). The site of action of tetanus toxin appears to be the pre- and postsynaptic junctions of the motor neurons located in the spinal cord and brain stem. Inhibitory reflexes in these areas are impaired or abolished (Curtis, 1971).The evidence indicates that tetanus toxin reaches these sites by first being fixed at the motor neuron end terminal and then being transported into the axon and up the axosplasm (Curtis, 1971). Since axonal transport has a relatively constant rate from nerve to nerve, toxin fixed at one time reaches the motor synaptic junction at different times, depending on the length of the motor neuron. The fifth cranial nerve, being the shortest, is the first to malfunction. This explanation of the clinical syndrome has been given credence for some time but was put on a firm basis by the experiments of Habermann and co-workers who prepared biologically active tetanus toxin labeled with lZ5I (Habermann and Dimpfel; 1973, Wellhoner et al.,

1973).

When tetanus toxin is injected intravenously into mice at low dosage (3.5 ng/kg), there is a long symptomless period, and death does not ensue until 72-96 hours. With increasing dosage these time periods decrease until a limit is reached at 10 pg. Increasing the dosage beyond this point fails to kill animals sooner than 2 hours, or to produce signs of intoxication sooner than 45 minutes. Zacks and Sheff (1971), who investigated this phenomenon, reasoned that the minimum lag period represented the time needed for the toxin-promoted exhaustion of a crucial biochemical and that this process was rate limiting. By performing similar experiments with goldfish they showed that increasing the body temperature shortened the survival time. The relationship was log/linear and yielded a Qlo value of 4.2. These investigators calculate a corresponding activation energy for this process of 27 kcal/mole. The situation seen with tetanus toxin is likely to be analogous to that for diphtheria toxin. Both show a dosedependent lag period with saturation kinetics. I n the case of diphtheria toxin the saturation phenomenon is due to the receptor-mediated transport process. The lag period is the time required to exhaust the

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cell of EF-2 catalytically, a process having a large activation energy. No information is available on the biochemical process affected by tetanus toxin. However, we believe it reasonable to propose that the effect is enzymatic and that the activity resides in a fragment of the tetanus toxin molecule. Habermann and Dimpfel (1973) estimated that the spinal column concentration of tetanus toxin in an intoxicated anM . It is difficult to conceive of a nonenimal is in the range of zymatic inactivating process operative at this concentration. 2. RECEPTORS There are other similarities between the processes of intoxication by tetanus and by diphtheria toxin. In both cases antitoxin has minimal effectiveness when administered after the toxin, indicating rapid fixation of the toxin by the tissue (Zacks and Sheff, 1971). This fixation of tetanus toxin can be demonstrated in vitro by suspending nervous tissue in a solution of tetanus toxin, removing the tissue, and noting a decline in the toxicity of the solution-a phenomenon described by Wassermann and Takaki (1898). More recently Habermann (1973) developed a radioreceptor assay for tetanus toxin utilizing 1251-labeled tetanus toxin and brain homogenate. When the homogenate is iricubated with tracer in the presence of increasing amounts of cold toxin, a typical competition-displacement curve is seen. This indicates that binding of the toxin involves a finite number of saturable sites (receptors) on the brain tissue. When a tracer concentration of 8 ng/ml was used, the one-half displacement point occurred at about 40 ng/ml of cold toxin. Using a toxin MW of 160,000, we calculated an apparent equilibrium affinity constant at a half-displacement of 4 x lo9 A4-l. Van Heyningen (1973) showed that the receptor materials in brain and spinal cord are certain gangliosides containing sialidase-sensitive bonds. Gangliosides with the structure GGnSSLC or SGGnSSLC can bind tetanus toxin at low concentrations of both tetanus (50 ng/ml) and ganglioside (100 ng/ml) (van Heyningen and Mellanby, 1973). [The convention used for abbreviating gangliosides is that of McCluer (1970): G, galactose; Gn, N-acetylgalactosamine; L, lactose; S, sialic acid; C, ceramide.] Hydrolysis of the sialyl residue of GGnSSLC to GGnSLC renders the product inert toward tetanus toxin but capable of deactivating cholera toxin. Fixation of tetanus toxin by GGnSSLC does not deactivate the toxin, and complexed mixtures injected into mice are toxic. Although gangliosides can complex tetanus and cholera toxin, it is possible that the naturally occurring receptor which mediates toxicity is more complex.

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3. STRUCTURE

The structure of tetanus toxin has been investigated by Matsuda and Yoneda (1974). Toxin isolated from extracts of bacteria (intracellular toxin) has a MW of 160,000, which is unchanged by treatment with thiol. Intracellular toxin contains a trypsin-sensitive region spanned by a disulfide bond. Mild trypsinization creates a two-chained structure linked b y a disulfide bond. This material appears identical to material isolated from bacterial filtrates (extracellular toxin). Reduction of extracellular toxin or trypsin-nicked intracellular toxin with thiol in the presence of urea generates two subunits known as the a fragment (53,000 daltons) and the /3 fragment (107,000 daltons). The separated chains show no toxicity. Remixing in equimolar ratios followed by the removal of urea and dithiothreitol (DTT) by dialysis restores 100%of the toxicity (Matsuda and Yoneda, 1976). On SDS gel this material has a MW of 160,000, and only traces of the subunits are seen. Van Heyningen (1976) recently showed that only the /3 subunit binds to the ganglioside SGGnSSLC. The similarities to diphtheria toxin are striking. These similarities led us to inquire whether or not the tetanus toxin gene resides in a bacteriophage as is the case with diphtheria toxin and botulinum toxin. Prescott and Altenbern (1967) reported that all seven strains of C . tetani tested by induction with mitomycin C displayed lysis, presumptive evidence for lysogeny. However, phage plaques were not found. I n two strains phage particles were detected by electron microscopy. After induction with mitomycin C, the ratio of toxin production to total protein production was constant, indicating that the toxin gene was not translated at an increased rate. This might constitute evidence against the toxin gene being a phage gene. However, the toxin gene is under regulatory control involving the iron concentration (Mueller and Miller, 1954; Largier, 1956) (another similarity to diphtheria toxin), and the site of the regulatory gene, phage or host, is unknown. 4. RETROGRADE AXONAL TRANSPORT

Tetanus toxin reaches its site of action within the central nervous system by retrograde axoplasm transport from the peripheral motor terminals. This phenomenon can be observed by assaying nerve segments, ventral root segments, and cord ventral gray areas at various times either for toxicity in a mouse assay (Kryzhanovsky, 1973) or for radioactivity utilizing 1251-labeled tetanus toxin (Habermann and Dimpfel, 1973). Twenty-four hours after the injection of toxin into the

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DAVID M. NEVILLE, JR. AND TA-MIN CHANG

gastrocnemius muscle a steep gradient, 50-fold higher at the periphery, is found along the ipsilateral sciatic nerve. The ventral gray segments at L7 and S1 contain high concentrations of label. No gradient is observed in the contralateral nerve. Contralateral L7 and S1 ventral segments contain low concentrations of label, as do ipsilateral segments L6 and S2. Most of the transported label resides in the ventral roots of the spinal cord segments supplying the injected muscle (Wellhoner et al., 1973). This type of specificity was not seen when tetanus antitoxin was given intravenously and simultaneously with labeled. toxin or when Nalz5I was substituted for 1251-labeledtetanus toxin. A similar time-dependent appearance of labeled toxin within the motor gray areas of the brain stem and spinal cord was observed after intravenous injection of labeled toxin which induced generalized tetanus. These areas concentrated toxin before the onset of symptoms. Forebrain and cerebellum gray areas (devoid of lower motor neurons) did not concentrate toxin, although homogenates from these areas can bind toxin. It was concluded that the blood-brain barrier was impermeable to toxin, and that retrograde transport through the motor neuron explains generalized as well as localized tetanus (Habermann and Dimpfel, 1973).

5. INHIBITION

OF

NEUROTRANSMITTER RELEASE

The muscle rigidity and spasm produced b y tetanus toxin results from the loss of inhibitory reflexes impinging on the lower motor neuron. By the use of microrecordings and microinjection techniques with single cells, this loss of inhibition has been found to be the result of decreased secretion of the inhibitory neurotransmitters glycine and y-aminobutyric acid (GABA). Renshaw cells are the inhibitory interneurons of the recurrent motoneuron axon collateral pathway. By recording directly from these cells, the inhibition produced by hind paw stimulation or the local administration of glycine or GABA was observed before and after the local injection of tetanus toxin (Curtis and DeGroot, 1968). Twenty minutes after tetanus toxin administration the inhibition following hind paw stimulation decreased. Fifty minutes after tetanus toxin injection afferent inhibition was abolished. However, the neurons were still sensitive to the inhibitory neurotransmitters. Local administration of glycine and GABA produced inhibition. Since the levels of neurotransmitters in the spinal cords of toxin-treated animals were found unchanged (Osborne and Bradford, 1973), a failure in transmitter release rather than synthesis was postulated. Other workers have reported similar findings (Kano and Ishikawa, 1972; Curtis et al., 1973).

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Osborne and Bradford (1973) observed the stimulated release of neurotransmitters from synaptosomes prepared from spinal cords. These synaptosome preparations maintained respiration and concentration gradients of potassium and amino acids during the course of the experiments. Stimulation by raising the external potassium or electrical pulses in the presence of calcium provoked increases in respiration and neurotransmitter release. Synaptosomes prepared from animals intoxicated with tetanus toxin showed reduced release of neurotransmitters following electrical stimulation (glycine, 70% inhibition; GABA, 44% inhibition). Toxin added directly to synaptosomes ( lo4 mouse MLD per 5 ml) was without effect. The total duration of these experiments consisted of a 30-minute preincubation and a 10minute stimulation. Tetanus toxin in high dosage and acting over long periods of time appears to block release of the excitatory neurotransmitter acetylcholine. These effects are often masked clinically by rigidity and spasms; however, flaccid paralysis has been reported in tetanus, as well as alterations in the autonomic nervous system (Curtis, 1971). The best documented study is on the effect of intraocularly injected toxin on the sphincter pupillary muscle. The resulting dilatation was unresponsive to oculomotor nerve stimulation but was responsive to adrenergic stimulation or to local administration of acetylcholine (Curtis, 1971). In this respect tetanus toxin is similar to botulinum toxin. Mellanby (Mellanby et d., 1973) has commented on this and other similarities. In Section II,D we consider possible molecular mechanisms of action for these two toxins which inhibit neurotransmitter release. 6. TRANSSYNAPTIC MIGRATION

The inhibitory neurotransmitter blockade produced by tetanus toxin places the main site of action presynaptic to the motor neuron. Yet convincing evidence demonstrates that the toxin is taken up at the motor neuron end terminals and transported to the cell bodies and synaptic regions of the motor neurons prior to the onset of toxin action. Taken in conjunction, these two facts indicate that the toxin, once reaching the synaptic region in the motor neuron, is transported across the synapse into the presynaptic region where it interferes with inhibitory neurotransmitter release. Schwab and Thoenen (1976), using '251-labeled tetanus toxin, performed an electron microscopic, autoradiographic, morphometric study on the distribution of labeled toxin in the spinal gray areas after injection (tetanic rigidity became visible 12-13 hours after injection). Most of the labeled synaptic terminals were afferent to the motor neurons. These workers conclude that

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these observations strongly favor the assumption of a transsynaptic migration of tetanus toxin. It is known that synaptic membranes show the highest binding affinity of any tissue fragments of the spinal cord. It is not possible with present techniques to determine whether these receptors are present on both post- and presynaptic membranes. The evidence for transsynaptic migration indicates that tetanus toxin probably remains intact as a two-chain disulfide-linked structure during axonal transport and transsynaptic migration. The argument is as follows. The receptor binding function has been linked to the B chain of the toxin. The same binding function is presumably used at the presynaptic membrane, hence dissociation cannot take place before the toxin gains entry through the presynaptic membrane. Habermann et al. (1973) performed gel filtration in SDS (Sephadex G-200) and SDS gel electrophoresis on spinal cords obtained from rats intoxicated by 1251-labeledtetanus toxin. Most of the label moved coincident with marker tetanus. If the dissociation of A and B chains had occurred on entry, as has been postulated for diphtheria toxin (Boquet and Pappenheimer, 1976) and cholera toxin (Gill and King, 1975),this result would not have been obtained. D. Botulinum Toxin

1. STRUCTURE-ACTIVITYRELATIONSHIPS Botulinum toxin is a potent neurotoxin causing flaccid paralysis. Unlike most protein toxins which are active systemically, this toxin can be absorbed from the gastrointestinal tract. The estimated human oral lethal dose lies between 0.5 and 5 pg (Koenig, 1971). The basic structure of the toxin is similar to that of diphtheria and tetanus toxin. The toxin is secreted as a single polypeptide chain of about 150,000 daltons. A tryptin-sensitive region spanned by a disulfide bond is present, and following proteolytic nicking and reduction two chains of 100,000 and 50,000 daltons are obtained. These are referred to in the literature as heavy and light chains. However, in keeping with the scheme and nomenclature for other toxins we refer to the 100,000dalton chain as B and the 50,000-dalton chain as A (DasGupta and Sugiyama, 1976). Neither chain alone is active (Kozaki and Sakaguchi, 1975).Attempts to reoxidize the structure and regenerate activity have not been reported. Botulinum toxin is made by Clostridium botulinum, and six distinct strains (A, B, C, D, E, and F) are known, as defined by antigenic differences in the toxin. Strains A, B, E, and F are associated with human

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disease, while C and D produce botulism exclusively in animals (Koenig, 1971). These strains can be interconverted by curing the lysogenized phage and reinfecting from phage produced by an alternate strain (Inoue and Iida, 1968). The toxin is thus a phage gene product, like diphtheria toxin. Toxins A, B, E, and F are believed to contain a common antigen (Kozaki et al., 1975).However, a detailed immunological analysis of the B and A chains of the six toxins has not been reported. Botulinum toxin is rapidly fixed to tissues like tetanus toxin through a neuraminidase-sensitive material. Studies on the binding of separated A and B chains have not yet been reported but, presumably, as with diphtheria and tetanus toxins, binding activity resides in the heavier B chain. Since antitoxins generated from toxins A, B, C, D, E, and F show no cross-protection, w e presume that the binding chains are antigenically distinct. I n keeping with our remarks on tetanus toxin we imagine that the A chain is an enzyme active in the intracellular milieu. Botulinum toxins B and A differ from tetanus and diphtheria toxin in that they are found in culture filtrates in association with high-MW proteins which are nontoxic. The type-A toxin complex has a MW of 1 million and can be crystallized in this form. The association is weak and can be broken by chromatography with DEAE-Sephadex (Kozaki et al., 1975). The nontoxic fragment is a hemagglutinin but apparently plays no role in animal toxicity. Unlike diphtheria and tetanus toxin, botulinum toxins require proteolytic nicking to achieve full animal toxicity. DasGupta and Sugiyama (1976) showed that unnicked type-E toxin undergoes a 29-fold increase in activity following nicking. Since the sensitivity of detecting nicked species in the unnicked starting material was not given, it is possible that the activation by nicking is actually much greater. This feature is reminiscent of the nicking required to activate diphtheria toxin in the cell-free assay system, where unnicked, unreduced toxin is devoid of activity (Collier, 1975).To account for the full animal toxicity of unnicked diphtheria toxin it has been postulated that nicking occurs at the cell membrane or after entry. Evidently this process in mice is limited for botulinum toxins B, E, and F (DasGupta and Sugiyama, 1976).

2. EXPERIMENTAL TOXICITY Intramuscular injection of limited amounts of botulinum toxin produces a localized flaccid paralysis in the injected muscle. A larger intramuscular dosage causes generalized flaccid paralysis, as does sub-

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cutaneous, intravenous, or oral administration. A portion of the injected material is rapidly fixed to tissue in a compartment not accessible to specific antitoxin. However, some toxin continues to circulate, and the definitive diagnosis is made by injecting 1 ml of a suspected patient’s serum into mice, a subgroup of which has already received univalent antitoxins A, B, E, and F (Koenig, 1971).

3. RECEPTORSAND TRANSPORT Receptors for botulinum type-A toxin have been demonstrated in synaptosomes where 1251-labeledtoxin binds and competition with cold toxin is observed (Habermann, 1974). Treatment with neuraminidase virtually abolishes binding. Toxin binding to a variety of gangliosides could not be demonstrated. With the use of 1251-labeledtoxin, specific binding to the neuromuscular junction of mouse diaphragm has been reported (Hirakawa and Kitamura, 1975).The presence of receptors for botulinum toxin appears to show species specificity, as is the case with diphtheria toxin. Rats are quite resistant to botulinum toxin B, requiring 500 times the A-toxin dose for similar effects. Guinea pigs, however, show equal sensitivity to both toxins (Burgen et al., 1949). It is possible that not all humans have a receptor for botulinum toxin. There are two reports of finding toxic levels of botulinum toxin in the serum of individuals known to have consumed contaminated foods, yet these individuals remained free of toxic symptoms (Koenig, 1971). Direct evidence that botulinum toxin once bound enters the motor neuron in an active form comes from the studies of Habermann (1974), Wiegand et a l . (1976), and Wiegand and Wellhoner (1974). These investigators, using 1251-labeled toxin, demonstrated retrograde axon transport from the motor neuron of the injected muscle to the ipsilatera1 spinal cord half-segments of the motor neuron. In addition, they obtained evidence that the toxin reduced synaptic transmission from recurrent motor axon collaterals to Renshaw cells in this animal preparation. Although central effects of botulinum intoxication are not the predominant symptomatology, they are present in clinical descriptions of the disease (Koenig, 1971).

4.

INHIBITION OF

NEUROTRANSMITTER RELEASE

I n a study on isolated rat diaphragms, Burgen et al. (1949) reported the basic facts of botulinum intoxication. The toxin produces a blockade at the neuromuscular junction after a dose-dependent lag

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period. A minimum lag period of 15-30 minutes was always present, no matter how high the dose. The lag period could be lengthened for a given dose by lowering the temperature. A restudy in frogs indicated a high Qlo consistent with the activation energy required to break a covalent chemical bond. Nerve conduction was not altered. The neuromuscular junction maintained its normal sensitivity to acetylcholine, and the output of acetylcholine following nerve stimulation was reduced in intoxicated preparations. The conclusion, supported in rich detail since then, was that botulinum toxin interfered with release of the excitatory neurotransmitter acetylcholine. Burgen et al. (1949) also noted the similarity between the dose-dependent lag period in diphtheria, tetanus, and botulinum toxins.

5. BIOCHEMISTRY O F NEUROTRANSMITTER RELEASE Since both botulinum toxin and tetanus toxin appear to act by blocking neurotransmitter release, it should be useful to explore what is known about the biochemical details of neurotransmitter release. Acetylcholine is believed to be synthesized in the region of the synaptic terminals and then packaged into synaptic vesicles. Neurotransmitter release occurs by exocytosis of the synaptic vesicle or the contents of the vesicle. This process occurs at a low frequency in a random manner independent of neural conduction. It gives rise to miniature end plate potentials of a constant amplitude. The consistency of the amplitude has led to the postulate that neurotransmitter release is a quantum phenomenon and that each vesicle contains a fixed amount of neurotransmitter (Drachman, 1971). Calcium ion is known to be involved in neurotransmitter release. It has been postulated that botulinum toxin in some way interferes with calcium metabolism, and this could explain the toxin’s effect. However, both Drachman (1971) and Simpson (1971) concluded that all the available data were not consistent with this hypothesis. Recently, however, Lundh et a l . (1976) demonstrated the restoration of acetylcholine release in botulinum-poisoned skeletal muscle. Using the calcium ionophore A23187 they restored miniature end plate potentials to their normal frequency when the motor end plate was perfused with 15 mM calcium. Similarly, when calcium ions in excess of normal were allowed to enter the nerve terminal by use of the calcium ionophore, neuromuscular transmission was restored. The same effect was achieved by using tetraethylammonium, which prolongs the duration of the nerve terminal action potential and thereby increases the amount of calcium entering the terminal. These results obtained on

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the extensor digitorum longus muscle of male rats were interpreted as indicating that the botulinum toxin-poisoned muscle contained an intact neurotransmitter release mechanism which required higher than usual levels of intracellular calcium to produce activation. The relationship between neurotransmitter release, calcium ion, and contractile proteins was explored by Berl et al. (1973) and Puszkin and Kochwa (1974). These investigators reported that brain actin was found in synaptosomal membrane-enriched fractions, whereas myosin molecules were found on synaptic vesicles. They proposed that the release of neurotransmitters resulted from an interaction of vesicle, myosin, and synaptosomal membrane actin. This proposed mechanism involves contact between vesicles and presynaptic membranes, with the formation of actomyosin that contracts in the presence of calcium ion and ATP. The contractile force would induce a change in the synaptic vesicles, with release of neurotransmitters into the synaptic clefs. Puszkin and Kochwa (1974) isolated from brain synaptic membranes a protein fraction with the properties of an actin-troponintropomyosin complex. This protein fraction, when incubated with synaptic vesicles, induces the release of preloaded 14C-glutamatefrom the vesicles in the presence of calcium. In the absence of calcium, glutamate release is reduced by a factor of 5. Glutamate release evoked by the protein complex plus calcium is associated with an increase in magnesium-ATPase activity. When purified muscle actin is substituted for the protein complex isolated from brain synaptic membranes, magnesium-ATPase activity increases, and glutamate release occurs both in the presence and absence of calcium. Addition of the protein complex from the brain synaptic membranes restores the inhibition of glutamate release and magnesium-ATPase activity in the absence of calcium. These investigators conclude that, as in the case of muscle, the relaxing proteins from brain seem to be part of a calcium receptor. Because the blockage of neurotransmitter release induced by either tetanus or botulinum toxin involves a dose-dependent lag period, we believe that the site of action of these toxins occurs at a step preceding neurotransmitter release from the synaptic vesicle, and that this step is initially not rate limiting but becomes rate limiting due to the exhaustion of a component through the enzymatic activity of the toxin. It appears to us that the calcium-binding proteins which confer calcium specificity on the neurotransmitter release in the systems of Puszkin and Kochwa are a likely candidate for enzymatic inactivation b y the A chains of tetanus and botulinum toxin. It further appears that these cell-free systems may serve as an effective assay

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system for the activity of the isolated A chains of these toxins. If such activity could be demonstrated, the transfer of a labeled cofactor to the proteins involved could pinpoint the actual substrate of the proposed toxin enzyme. E. Cholera Toxin

1. EXPERIMENTALTOXICITY AND BIOCHEMICALMECHANISM Cholera is a disease characterized by watery diarrhea in which the stool production may reach 20 liters per day. This leads to severe dehydration, circulatory collapse, and death. The entity causing this disease is a toxin elaborated b y the bacterium Vibrio cholerae. The toxin has been purified, and the experimental disease can be produced in animals by injecting the toxin into the small bowel. Quantitative studies can be performed b y forming loops of small bowel sealed with ligatures and cannulated at each end. In this manner toxin and other substances can be introduced for varying periods of time, washed out, and the loop refilled with water and electrolytes. At various times water and electrolyte can be removed for sampling purposes. T h e results of such studies show that after a 10-minute contact with cholera toxin the normal transport of water and electrolyte from the bowel lumen to the bloodstream is reversed following a lag period of approximately 40 minutes. The effect is half-maximal at 1b hours, maximal at 3 hours, falls to half-maximal at 24 hours, and requires 48 hours to reach baseline values. Intestinal adenylate cyclase activity also rises after a 40-minute lag period, and these values parallel the water and electrolyte changes throughout the time course. Other agents which stimulate intestinal adenylate cyclase, such as prostaglandins and theophylline, also cause this type of alteration in intestinal water and electrolyte transport. The disease is therefore caused b y the ability of the toxin to produce an abnormally high and prolonged stimulation of intestinal adenylate cyclase activity (Guerrant et al., 1972; Finkelstein, 1973, 1975). When naturally occurring stimulators of adenylate cyclase interact with cells, the stimulation does not last much longer than the duration of the stimuli. In the case of cholera toxin stimulation of intestinal adenylate cyclase activity, the stimulation lasts for a time period equal to the turnover of the intestinal epithelium (Finkelstein, 1973). Naturally occurring substances which stimulate adenylate cyclase activity do so without a lag period.

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2. TOXINSTRUCTURE AND RECEPTOR BINDING The subunit structure of cholera toxin has been described (Finkelstein et al., 1974; Mendez et al., 1975). The toxin is an 84,000-dalton protein and consists of an A and a B subunit held together with noncovalent forces. The A subunit has a MW of 28,000 daltons and consists of two polypeptide chains of 22,000 daltons and 7500 daltons linked by a single disulfide bond. These chains are known, respectively, as the Al and A2 chains. The B-subunit MW is 56,000 daltons, and dissociating reagents reduce this MW to a single 9000-dalton species. The subunit structure of cholera toxin appears to be ABs. Each B monomer subunit contains a single disulfide bond, but no interchain disulfide bonds exist within the B subunit. Studies of the binding of cholera toxin to cells have been performed by several investigators. These studies were prompted by the finding that a sialidase-resistant monosialyl ganglioside bound to cholera toxin and inactivated its biological activity (van Heyningen, 1973). Using 1251-labeledcholera toxin and fat cells, saturable binding was observed with an equilibrium dissociation constant in the range of 2.5 x M (Cuatrecasas, 1973). Holmgren and co-workers (Holmgren et al., 1975) obtained evidence that the ganglioside GMl or GGnSLC is the biological receptor in intestinal tissue. These workers demonstrated a relationship among the endogenous GMl concentration, the number of binding sites for cholera toxin, and the sensitivity of the intestinal mucosa to the biological activity of the toxin. These investigators also demonstrated the incorporation of exogenously added GMlinto intestinal mucosa cells and found this to be associated with both an increased number of binding sites and an increased sensitivity to cholera toxin. Similar studies have been reported for fat cells (Cuatrecasas, 1973). However, it is not clear whether added GMlincreases the sensitivity of the fat cell to cholera toxin or whether it simply alters the time course of the stimulatory effect (Kanfer et al., 1976). Recently, Moss et al. (1976a) demonstrated that a mouse fibroblast line which had lost its native capacity to synthesize GMl could be restored to responsiveness to cholera toxin by the addition of exogenous GMl. These studies argue for the case that GMl is the native receptor for cholera toxin in these cell types. This generalization has been questioned b y Kanfer et al. (1976), Donta (1976), and King et al. (1976). The last-mentioned investigators added GM1to pigeon erythrocytes and studied the enhancement of adenylate cyclase activity. They observed that at least 90% of the exogenously added toxin-binding sites were nonproductive of cyclase stimulation. It is possible that productive toxin binding sites require the association of GMl with another membrane component.

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3. EVIDENCEFOR TRANSPORT The binding activity of cholera toxin appears to be associated with the B chain. Cholera toxin B subunit, also known as choleragenoid,

can block the action of cholera toxin when instilled with toxin into experimental animals (Finkelstein, 1973). The competitive nature of this effect was shown by Gill and King (1975), who incubated pigeon erythrocytes in the presence of 1pg/ml cholera toxin and various concentrations of B subunit. Adenylate cyclase activity fell with increasing B-subunit concentration, and 50% inhibition occurred at approximately 3 pg/ml of B subunit. A considerable body of evidence argues for the case that cholera toxin must be first transported through the plasma membrane before it can activate adenylate cyclase. Several facts led to the formulation of this hypothesis. First, cholera toxin is highly effective in stimulating adenylate cyclase when applied to the mucosal surface of the intestine. It is only minimally effective when injected into the bloodstream. However, adenylate cyclase is not concentrated in the brush border of the intestinal epithelium; rather it is believed to be concentrated on the blood front of the cell. I n order for cholera toxin bound to the brush border to gain proximity to adenylate cyclase it either has to be transported into the cell interior or transported around the cell membrane through mobile receptors. Once toxin is transported within the membrane to the vicinity of the adenylate cyclase it still has to interact with the cyclase molecule, whose active site is localized to the internal surface of the plasma membrane. Activation of cyclase by toxin does not occur via cyclase hormonal receptors, since toxin-treated cells do not lose their hormonal cyclase stimulation. More direct evidence for toxin transport has been obtained by observing that toxin-treated cells or tissues are accessible to the neutralizing effects of antitoxin antibodies for only short periods of time. This was observed in vivo utilizing the system of isolated intestinal loops and in vitro utilizing pigeon erythrocytes. Antitoxin added within 30 seconds after cholera toxin prevented adenylate cyclase stimulation. Antitoxin added 10 minutes after the toxin had no effect on the cyclase stimulation by cholera toxin, and 10- to 15-fold levels of stimulation were seen. Fifty percent inhibition of the toxin-stimulated cyclase activity was observed with addition at 24 minutes. An identical phenomenon is seen in the case of tetanus, botulinum, and diphtheria toxin interaction with cells. I n the case of diphtheria toxin the inference is quite strong that this eclipse period represents transport of the toxin into the interior of the cell. This is because the substrate for the toxin’s enzymatic activity is known to reside within the interior of the cell. The inference that the eclipse phase represents transport for chol-

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era toxin is less strong, since the primary site of action of the toxin is unknown. However, it is difficult to construct a model for the eclipse period which does not involve changing the relative position of the toxin molecule with respect to the surrounding membrane. This process appears to involve crossing a high-energy barrier, since the molecule is no longer accessible to antibodies present in the media bathing the cell. Therefore, on both energetic and structural grounds it appears that during the eclipse period the toxin has moved from one phase to another, and in our view this constitutes transport. Whether this transport takes place to the interior of the cell cytoplasm or simply to the interior of the plasma membrane remains to be determined.

4.

SIGNIFICANCE OF THE

LAG PERIOD

Following the application of cholera toxin to tissues or cells, there is a lag period lasting approximately 30 minutes before the rise in adenylate cyclase activity is seen. .4 similar lag is observed in diphtheria toxin, tetanus toxin, and botulinum toxin effects. However, in the case of these toxins, the lag period is dose-dependent. In addition, the temperature dependence of the lag period is high, and the Qlo.is in the range of that required for breakage of a covalent bond. The usual explanation for a dose-dependent lag period involves the postulation of a preceding step in the reaction process, which is not initially rate limiting but becomes rate limiting as a result of application of the reagent in question. In the case of diphtheria toxin this postulate was shown to be accurate. The observable in this case was the rate of protein synthesis, while the substrate for the enzymatic activity ofthe toxin was EF-2. Under normal conditions the concentration of EF-2 is not rate limiting. Thus the lag period, particularly the long lag period seen at low concentrations of toxin, is a function of the time it takes to reduce the EF-2 concentration to a rate-limiting value. It should be pointed out that long lag periods due to this process are seen only when the rate of turnover of the substrate in question is long compared to the observation period. The lag periods associated with cholera toxin do not show the marked dose dependency exhibited by diphtheria, tetanus, and botulinum toxin, and no dependency may in fact exist. Studies on pigeon erythrocytes reported by Gill and King (1975) failed to show a dose-dependent lag period. However, these studies were not designed to answer this question, and more data points at early times would be desirable. Studies on fat cells reported by Kanfer and co-workers (Kanfer et al., 1976) measure the rate of toxin-stimulated glycerol release. The half-maximal glycerol release

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occurs earlier at high toxin dosage; however, the curves are sigmoidal in shape and nonparallel, and no obvious difference in the initial lag can be detected. An alternative explanation of a lag period which does not display dose dependency is that it represents the time required to transport the toxin from its binding site to the compartment where it displays its activity. This explanation does not rule out the possibility of a preexisting step (before adenylate cyclase) where the toxin exerts its effect. This step may simply have a rapid turnover which is short compared to the lag period. The hypothesis that the lag period represents the time required to transport toxin from the binding site on the plasma membrane to its intracellular site of action has been proposed (Parkinson et al., 1972). Recently, Gill and King (1975) showed that lysates of pigeon erythrocytes are sensitive to the stimulation of adenylate cyclase b y cholera toxin. The kinetics of this system differ from those of the intact cell system in that no lag period is present and that the maximal rates of cyclase activation are much higher than in the intact cell preparation. These investigators believe that the absence of a lag period in the broken cell system is due to the lack of a requirement for transport through the membrane in this system.

5. IS

THE

A,

CHAIN AN

ENZYME?

Recently, Gill and King (1975) showe'd, utilizing the pigeon erythrocyte broken cell system, that the A, chain of cholera toxin is active in stimulating adenylate cyclase. Cholera toxin was dissociated into B monomeric subunits and an A subunit by S D S and purified on gels. Toxin subjected to prior treatment with DTT was also run, and the A2 chain was dissociated from the A subunit. Both the A subunit and the A, chain were effective in stimulating adenylate cyclase activity more than 10-fold in the broken cell pigeon erythrocyte system. The intact B8 subunit was inactive. It had been previously shown with intact cell systems and tissue systems that purified B and A fragments were inactive. These results are then similar to the general findings observed with the A and B chains of diphtheria toxin, abrin, and ricin. The question then arose whether or not the A, fragment of cholera toxin was an enzyme, as is the case with diphtheria toxin. A requirement for N A D as a cofactor was demonstrated by Gill and King, which is consistent with the enzymatic hypothesis. When the increasing amounts of A, chain are added to lysed pigeon erythrocytes, the rate of cyclase activation increases, which is also consistent with the presence of an enzyme. Gill and King (1975) also estimated that 10 to 50 copies of a subunit

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per erythrocyte volume give the same rate of cyclase activation in lysates as saturating amounts of toxin in the intact cell system. This number is less than the estimated number of cyclase molecules per cell (several hundred to 1000)and implies some type of amplification. Most biological amplification systems involve an enzyme at some step. However, if adenylate cyclase is the substrate for the A, chain, the ratio of A, chains per erythrocyte to total cyclase molecules per erythrocyte gives a very low presumptive turnover number (Gill and King, 1975). Recently, Moss and co-workers (Moss et al., 197613) demonstrated NADase activity of cholera toxin A subunit. I n addition, they showed that in the presence of NAD the cholera A subunit catalyzes the ADPribosylation of arginine.

F.

Colicins

1. GENERALCHARACTERISTICS Colicins are bacteriocidal proteins synthesized by certain strains of enteric bacteria, including Escherichia coli, and are active against other strains of the same or related bacterial species (Nomura, 1967). Sensitivity or resistance to a colicin is determined by the presence or absence of a receptor for the colicin on the outer membrane of the bacterium. Various colicins have the same receptor specificity, and these are grouped under a common letter. Thus colicins E2 and E3, although different, utilize the same receptor and are bacteriocidal toward all strains of bacteria carrying this receptor, with the exception of strains producing the homologous colicin. Escherichia coli carrying the E receptor contain about 250 receptor sites (Bradbeer et al., 1976).Strains of E . coli producing a colicin are said to be colicinogenic. Colicinogenic cells carry a plasmid, a small circular DNA called colicinogenic factor, which confers the ability to produce the colicin and also the immunity for the corresponding colicin in that particular strain. Immunity differs from resistance, since immune cells retain receptors and adsorb homologous colicins. However, colicinogenic cells are sensitive to very high concentrations of homologous colicin.

2. INTRACELLULAR SITE

OF

ACTION

Colicins E2 and E3 have their site of action within the bacterial cell cytoplasm. Colicin E3 produces a specific cleavage of 16s rRNA

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within the 30s subunit at a point approximately 50 nucleotides away from the 3’ end. This cleavage inactivates protein synthesis. This process has been shown to be catalytic (Sidikaro and Nomura, 1974). Ribosomes from eukaryotic cells are equally sensitive to colicin E 3 (Turnowsky et al., 1973).The cell-free assay of colicin E3 activity involves incubation of colicin with 70s ribosomes at 37°C for 45 minutes, following which the components necessary for assay of poly-U-directed polyphenylalanine synthesis are added. Colicin E 2 is an endonuclease with broad specificity toward a variety of DNAs. When covalently closed circular DNA is used as a substrate, open circles appear as the initial product. With higher concentrations of colicin E 2 small linear fragments are formed. Agarose gel electrophoresis serves as a cell-free assay system for colicin E 2 activity (Schaller and Nomura, 1976). Colicin E, is a single-chain 62,000-dalton protein. No detectable carbohydrate is present. The protein contains a single sulfhydryl group (Glick et al., 1972). There are no reports in the literature of attempts to separate the enzymatic activity and the binding activity of the colicin molecule by limited proteolysis. Both colicins E 2 and E 3 are secreted from bacteria in tight association with a 9500-dalton peptide (Jakes and Zinder, 1974; Sidikaro and Nomura, 1974; Schaller and Nomura, 1976). This peptide confers immunity to the strain secreting the colicin and is known as the immunity protein. Immunity is conferred by blocking the enzymatic activity of the colicin. This has been observed in the cell-free assay systems for colicins E2 and E3. The blockage is reversible, since in the presence of dissociating agents such as 6 A4 guanidine hydrochloride the colicin can be separated from the immunity protein. The increase in activity is on the order of 10-fold. Sensitive strains of bacteria (noncolicinogenic) are equally as sensitive to colicin complexed with immunity protein as purified colicin. The conclusion from this result is that the immunity protein does not cross the plasma membrane in an active form. It is inferred that immunity is conferred on a colicinogenic strain by a high concentration of immunity protein within the bacterial cytoplasm. In diphtheria toxin and in the seed toxins abrin and ricin the B chain performs two functions. It binds the toxin to the cell surface receptor, and it inhibits the enzymatic activity of the toxin until chain separation occurs. In colicins E 2 and E3 the immunity protein serves only the latter function of inhibiting enzymatic activity. It would be interesting to determine if these separate activities in the B chain of diphtheria toxin can be separated. Two early termination mutants in the B chain have been reported which lack binding ability, and in CRM,, enzy-

9%

DAVID M. NEVILLE, JR. AND TA-MIN CHANG

matic activity is present prior to reduction (Pappenheimer and Gill, 1973). Investigations of point mutations within the B chain might be of interest.

3. SHARED TRANSPORT SYSTEMS

a. Colicins B , D , 1, and V, Complexed Zron, and T Bacteriophages. Direct studies of colicin transport into bacteria have been hampered by the minute quantities of intracellular colicin required to elicit bacteriocidal effects. The same problem occurs in direct studies of bacterial toxin transport into eukaryotic cells. However, considerable evidence concerning colicin transport is available from genetic manipulations. The striking feature to emerge from these studies is that the colicins utilize physiological transport mechanisms of low-molecular weight metabolites. Even more striking is the fact that these same transport mechanisms or parts of them are utilized by certain bacteriophages. In E . coli K-12, iron transport systems have been found to be associated with colicin transport systems. One of the iron transport systems transports iron chelated by enterochelin. Enterochelin is a cyclic trimer of 2,3-dihydroxybenzoylserine.Enterochelin is made by the bacterium and secreted into the external environment where it chelates iron tightly. This chelate is then transported via a transport system. The first step in transport involves the binding of the ferrienterochelin to a cell surface receptor. Ferrienterochelin is then transported actively into the cell interior. This process is inhibited by 2,4-dinitrophenol or azide. Several workers noted that various mutants ofE. coli K-12 that are resistant to colicins B and D also hyperseCrete enterochelin. This led to investigation of the relationship between ferrienterochelin transport and the colicins (Pugsley and Reeves, 1976a). By performing binding studies in the presence of dinitrophenol, binding could be separated from the uptake step. Ferrienterochelin receptors were found to be increased by a factor of 7 when the bacteria were grown in iron-poor media. This culture condition was also associated with a 10- to 20-fold increase in two outer membrane polypeptide components in the 100,000-MWrange. These polypeptides appear to be related to the receptor function and are cvidently derepressed by iron starvation. These observations suggest that colicins B and D and ferrienterochelin share a common receptor. The fact that ferrienterochelin can block the bacteriocidal action of colicin B and D supports this hypothesis (Davies and Reeves, 1975). In addition, colicins B and D can block ferrienterochelin binding to the

RECEPTOR-MEDIATED PROTEIN TRANSPORT INTO CELLS

99

bacterial outer membrane. One-half displacement occurs at about 75 pg/ml of colicin D. With a MW of 50,000, this represents a colicin D concentration of 1.5p M . Thus the colicin appears to have a somewhat higher affinity for the receptor than ferrienterochelin. Pugsley and Reeves (1976a) investigated four classes of E . coli K-12 colicin Bresistant mutants. All four mutant classes, cbt, exbC, exbB, and tonB, were defective in the uptake of ferrienterochelin. Ferrienterochelin binding was demonstrated in outer membranes in the exbB, erbC, and tonB mutants but was missing in the cbt mutant. The cbt mutant has been reported to show colicin binding, and the tonB mutant is defective in all other forms of chelated iron transport which in E . coli involves complexes with ferrichrome, citrate, and rhodotorulic acid. All these mutants show increases in the two membrane polypeptides when grown under iron starvation conditions. The complete relationship between the receptor functions, transport functions, and these mutations remains to be determined. Pugsley and Reeves (1976b) recently reported the purification of colicin B and D receptor activities following solubilization of the outer bacterial membrane with Triton X-100. The fractions containing the highest purification, which amounted to a 40-fold increase in binding activity toward colicins B and D, contained two protein peaks corresponding to the protein species which are increased in the outer membrane under conditions of iron sta.rvation. Hancock and Braun (1976) have described mutants in E . coli K-12 which are defective in ferrienterochelin uptake, which they call feu mutants. ThefeuA mutant is resistant to colicins I and V. In strain VR-42, three proteins of MW 83,000, 81,000, and 74,000, residing in the outer membranes, markedly increase under iron starvation conditions. These proteins may be analagous to the proteins previously described by Pugsley and Reeves in their strain P1552. In contrast to the mutants report by Pugsley and Reeves thefeuA mutant in strain VR-42 lacks one of the iron starvation-derepressed proteins. This protein is presumably the colicin I receptor and functions in enterochelin-mediated iron transport. These investigators also partially purified the iron-derepressed proteins after solubilization in Triton X-100. Another class of E . coli K-12 mutants defective in ferrichromemediated iron transport has been described (Hancock and Braun, 1976). These mutants, known as tonA, lack an 85,000-daIton polypeptide in the outer membrane. Ferrichrome protects sensitive cells against the bactericidal activity of colicin M. I n addition, it protects sensitive cells against phages T5, T1, and $30. The tonA polypeptide

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DAVID M. NEVILLE, JR. AND TA-MIN CHANG

is probably the receptor for ferrichrome, T5, and colicin M (McIntosh and Earhart, 1976). Manning and Reeves (1976) described a group of mutants known as t s x mutants which are resistant to T6-like bacteriophages and to colicin K. They demonstrated that these mutants lack an outer membrane protein of MW 32,000. These mutants are unable to adsorb either the bacteriophages or the colicin, and it is suggested that t s x protein is the receptor for both the T6 phages and colicin K. b. E Colicins, Vitamin Biz, and Phage BF23. Bradbeer et al. (1976) studied the vitamin B12transport system in E. coli. They demonstrated that the E. coli outer envelope normally contains about 250 B12 receptors and that these receptors function both in Blz transport and as receptors for E colicins (DiMasi et al., 1,973).Bradbeer et a l . (1976) later presented convincing evidence that this same receptor is involved in the adsorption and transport of phage BF23. Blz can decrease the rate of phage adsorption 50-fold. The 50% inhibition occurs at 0.8 nM, close to the dissociation constant for B12 binding. Phage BF23 can block the transport of B12 into cells. This occurs at the binding step and, by using cell envelopes and observing the binding of B12 at various concentrations in the presence of various phage concentrations, classical competitive inhibition was observed. The K, for the phage was calculated to be 0.2 nM. These workers described a mutant btuB69 which contains only about half a receptor, on the average, per cell for both BF23 and Blz. In E. coli, bacteriophages share receptors with metabolites other than heavy metals. Hazelbauer (1975) noted that chemotaxis toward maltose is specifically defective in many strains ofE. coli carrying mutations affecting lamB, the gene coding for the outer membrane receptor for bacteriophage lambda. The receptor involved functions as a high-affinity system over the range 0.1-10 pM for maltose transport. Blockage of high-affinity maltose transport is responsible for the defect in chemotaxis. 4. SIGNIFICANCEFOR EUKARYOTIC CELLS

The finding that bacteriophages and colicins, both of which can be destructive toward bacteria, utilize physiological receptors strengthens our belief that the receptors for toxins on eukaryotic cells may also have physiological functions. To date no one has succeeded in demonstrating an adverse effect upon a eukaryotic cell, either in tissue culture or in viva, from the application of a toxin-binding chain

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protein. However, it is possible that these toxins utilize receptors involved in trace-metal transport and that adverse effects would not be demonstrated until the trace metal or other material became limiting. Such limiting conditions are not usually encountered in tissue culture or in whole-animal preparations unless they are specifically provoked. Alternatively, toxin receptors may function as receptors for protein or peptide growth factors or factors responsible for maintaining certain states of differentiation. These functions may be overlooked in tissue culture, because of the undifferentiated nature of the tissue-cultured cells under the usual logarithmic growth conditions. In whole animals it may be necessary to administer B chains of toxins over a prolonged period of time to elicit a biological effect. 111.

CARRIER PROTEINS

A. Tranrcobalamin It

1. STRUCTURE AND FUNCTION Vitamin B n or cobalamin is a water4oluble vitamin of MW 1355, containing tightly complexed cobalt. After absorption from the gut Blz is transported to all tissues, where it serves as a cofactor either as methylcobalamin or 5'-deoxyadenosylcobalamin in methyl or other group transfers involving a carbon 1,2-hydrogen shift. Uptake from the gut is a receptor-mediated process. The uptake is facilitated by intrinsic factor, a glycoprotein which forms a stable complex with BIZ. The binding of the B,,-intrinsic factor complex to receptor sites on the membranes of intestinal microvilli is the first step in the uptake process. Receptor sites for the B12-intrinsic factor complex were first identified on intestinal microvilli by Donaldson et al. (1967). In human blood B12is carried by two proteins, transcobalamin I (TC I) and T C TI. T C I carries 75%of the serum Biz., and TC I1 carries the remainder. The significance of T C I is unknown, since a congenital deficiency of T C I in humans is asymptomatic and does not result in reduced levels of Blz in tissues (Carmel and Herbert, 1969). T C I1 is required for the transport of B,2 to peripheral tissues, since a congenital lack of TC I1 leads to severe manifestations of B12deficiency (Hakami et al., 1971). TC I1 is a 40,000-dalton protein. The affinity of T C I1 for cobalamin is very high, with a reportedK, of 10" M-' (Hippe and Olesen, 1971).

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DAVID M. NEVILLE, JR. AND TA-MIN CHANG

TC I1 has been found in a wide variety of mammals with similar functions. Considerable evidence indicates that B12 is transported into cells in the form of the TC II-BlZ complex. 2. TRANSPORT INTO LEUKEMIA CELLS The uptake of vitamin Blz has been extensively studied in mouse leukemia cells L1210 using 57Co-labeledBlz (DiGirolamo and Huennekens, 1975). Labeled Blz in the presence and absence of TC I1 has been incubated with washed mouse leukemia cells. Concentrations of Blz ranged between 30 and 200 pM. The cells were spun down, washed, and then counted. In the presence of TC 11, uptake increased by over 50-fold. A plot of uptake in the presence of TC I1 versus time was biphasic, with a rapid initial rate for the first 1 minute and a slower rate which leveled off to a steady state at 45 minutes. The initial rate represents the binding of TC I1 to the surface membrane receptors and can be inhibited by EDTA or reversed by EDTA. Labeled material eluted from the cells after the primary incubation period is in the form of the TC II-cobalamin complex as judged by chromatography on Sephadex G-100. The secondary process represents transport into the cell interior. This process is temperature-dependent and is inhibited by azide and 2,4-dinitrophenol. No efflux is noted from the intracellular compartment. When cells are incubated with TC II-B1z for 2 hours, washed in EDTA to remove receptor-bound label, and then homogenized in saline and subjected to sucrose density gradient fractionation, seven label peaks were found. The largest, 31-47%, was in the soluble fraction, while the next highest, 17-20%, coincided with a mitochondria1 fraction. The soluble fraction label cochromatographed with TC II-Bl2 (Rye1 et al., 1974). 3. TRANSPORT INTO LIVERAND KIDNEY

The results reported with mouse leukemia cells differ from those reported in liver and kidney (Pletsch and Coffey, 1971,1972; Newmark, 1971). In these tissues labeled cobalamin is first associated with a plasma membrane vesicular fraction and then moves to the lysosomal fraction. Smaller amounts of label in these tissues appear in the soluble fraction, and larger quantities in the soluble fraction do not appear until after 72 hours, at which time the label is associated with a macromolecule with a MW greater than lo5. In liver vitamin B,S appears to also be transported as a TC I1 complex. Lysis of the plasma membrane vesicular fraction by Triton X-100, followed by chromatography on

RECEPTOR-MEDIATED PROTEIN TRANSPORT INTO CELLS

103

RECEPTOR-MEDIATED PINOCYTOTIC TRANSPORT MODEL

3

FIG. 1. In this model high-affinity surface membrane receptors (notched squares) concentrate the specific protein at the membrane (1). Membrane invagination and pinching off result in a pinocytotic vesicle containing receptor-bound protein (2 an d 3). Vesicle fusion with a lysosome (4a) directs the protein to this compartment. When the protein is known to enter the cytosol, a second transport mechanism out of the vesicle (arrow, 4b) is required. Uptake proceeds by the continuous addition of receptor to the surface membrane. (See Sections II,A,4 and IX,B.)

Sephadex G-100 in the presence of Triton shows that the BI2 label cochromatographs with TC II-Bl2. The same is true of the lysosomal label at early time points, but lower-MW B12label appears at the later time points. These data have given rise to the following model of Biz transport (see Fig. 1). Binding occurs via TC II-BI2 complex. The complex is internalized by receptor-mediated pinocytosis. Fusion of pinocytotic vesicles with lysosomes leads to the degradation of TC 11, freeing Blz. B12 is then bound to a second intracellular binding protein.

4. THE PINOCYTOTICMODEL

The preceding model does not appear to be applicable to L1210 leukemia cells, because these cells exhibit high initial concentrations of soluble B,, as compared to the particulate fractions. In addition, the kinetic data derived b y DiGirolamo and Huennekens (1975) for mouse leukemia cells is inconsistent with receptor-mediated pinocytosis unless receptor clustering at pinocytotic sites is invoked. From

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DAVID M. NEVILLE, JR. A N D TA-MIN CHANG

double reciprocal plots these investigators calculated V,,, for the secondary transport process as 0.4 pmoles per minute per los cells. The number of receptor sites for the primary process was found to be 400 sites per cell. If these sites are evenly distributed, then pinocytosis of one receptor requires pinocytosis of 1/400 of the membrane. The maximum velocity of transport calculated in units of molecules per cell per hour is 14,400.To pinocytose this number of sites requires turning over the entire membrane 36 times in 1 hour. This figure is much higher than the reported rates of membrane turnover. Fibroblast surface membrane turnover has been reported as 30% per hour (Steinman et al., 1974). Receptor clustering identified by morphological techniques has also been described (see Section 1117C,3). It appears then that B12 is transported into leukemia cells, liver, and kidney as TC II-B12 complex. This is further supported by disappearance curves from serum of TC II-B12 labeled in both the B12and TC 11. The half-times of disappearance from the serum are identical, being 1.5 hours for both molecules of the complex (Schneider et al., 1976). The mechanism of transport in mouse leukemia cells does not appear to occur via the pinocytotic model a in Fig. 1, since at early time points TC II-B12 is not lysosome-associated. If the pinocytotic model is correct, a mechanism for escape from the lysosome before degradation ensues is required, as diagramed in Fig. 1, step 4b. Transport in liver and kidney appears to occur via pinocytosis in the studies cited. Whether or not a nonpinocytotic transport mechanism is present in liver or kidney remains to be determined. Fiedler-Nagy et al. (1975) studied the binding of TC II-B12 complex to isolated rat liver plasma membranes. Their affinity constant for the binding is in the same range as that reported for mouse leukemia cells (5.5 x lo9 A4-l). Binding isotherms gave a linear Scatchard plot with 7.2 x 1O'O sites per milligram of membrane protein. This appears to be even less than the number of sites observed on mouse leukemia cells. We calculate, assuming the cell membrane contributes 2% of the total cellular pro tein (Neville, 1976), 1300 x 1O'O sites per milligram of protein for mouse leukemia cells. With such a low number of specific receptor sites it is difficult to see how receptor-mediated pinocytosis could incorporate B12 into liver unless receptor clustering at pinocytotic sites is postulated (see Sections II1,C and IV,C). However, the experiments in liver and kidney were done at saturating concentrations of TC II-BI2, and nonspecific binding processes may bind considerably more material than specific receptor sites. It would be interesting to repeat the experiments of Pletsch and Coffey under nonsaturating receptor conditions. This may require a replacement transfusion uti-

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lizing serum depleted of TC II-BIz. Uptake of TC II-Bl2 has also been studied in human fibroblasts (Rosenberg et al., 1975). These studies do not further reveal the mechanism of transport but do show the role of a second intracellular binding protein for BIZ.After incubation for 76 hours most of the labeled cobalamin present is bound to a protein of MW 120,000. A mutant fibroblast line unable to synthesize cobalamin coenzymes showed no defect in transport but was unable to retain high intracellular concentrations of cobalamin at late times. The mutant line was found to be deficient in the intracellular binding protein. 6. tranrferrin

1. STRUCTURE

AND

FUNCTION

Transferrin is the major iron-binding protein of the serum and serves to distribute iron to the peripheral tissues. Transferrin is a glycoprotein of MW 78,000. Iron uptake from transferrin has been extensively studied in reticulocytes and nucleated erythroid cells from bone marrow. The uptake of Iz5I-transferrin and 59Feby these cells proceeds by a biphasic process displaying an initial rapid uptake which is independent of temperature and a slower uptake rate which is highly temperature-dependent. The phenomenon appears similar to that seen with Blz uptake, and the rapid initial uptake is believed to represent binding to receptors, while the slower rate represents transport into the cell. When uptake of transferrin labeled with iodine in the protein component and radioactive iron is studied in nucleated erythroid cells as a function of time, iron uptake increases linearly over a 45-minute period. lZ5I-Transferrinuptake rises rapidly and then levels off, net uptake ceasing at 7 minutes (Kailis and Morgan, 1974). These studies and many others of a similar nature have led to the following scheme. Transferrin containing iron is initially bound to a cell surface receptor. The transferrin-iron complex is transferred into the cell interior where the iron is removed. Transferrin is returned to the extracellular medium in an intact and functional state. Data derived from intact animal preparations support this hypothesis. Labeled iron bound to transferrin is rapidly cleared from the serum with a half-life on the order of 60-90 minutes. The half-life of transferrin, however, is between 8 and 10 days (Awai and Brown, 1963). Nonenzymatic dissociation of iron from transferrin cannot explain the differences in half-life, since the affinity of iron for transferrin is reported at 1V1M-I (Aasa et al., 1963).

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2. RECEPTORS Evidence that receptors are involved in transferrin uptake comes from two types of data, Transferrin uptake has been shown to be a saturable process in bone marrow erythroid cells. The uptake rate falls one-half when the transferrin concentration is increased from 40 pM to 100 pM (Kailis and Morgan, 1974). The initial uptake of doubly labeled transferrin can be reduced 10-fold b y short incubations with pronase or trypsin. Reticulocytes exposed to doubly labeled transferrin at 0°C and then incubated with trypsin release transferrin into the medium containing the same specific activity of iron and 1251as in the incubating media. If, however, a 37°C incubation follows the initial 0°C incubation, treatment with trypsin fails to release significant amounts of transferrin. At 37°C both labels in transferrin enter a compartment not accessible to trypsin (Hemmaplardh and Morgan, 1976).

3. TRANSPORT The details of the entry process have been investigated by Fielding and Speyer (1974),utilizing timed and chaser experiments. After incubation with labeled transferrin, reticulocytes are homogenized and fractionated into a cytosol and membrane fraction. The membrane fraction is dissolved in Triton X-100 and chromatographs on Sepharose with a MW of 230,000. This complex presumably represents the transferrin cell membrane-receptor complex. At later time points transferrin is found in the cytosol compartment. Transferrin within the cytosol compartment chromatographs with a MW of 95,000 (Sly et al., 1975). This presumably represents a complex with an intracellular binding protein of MW 20,000. By increasing the external transferrin concentration, cytosol transferrin appears at MW 78,000. This process presumably represents saturation of the intracellular carrier protein. The mechanism of transferrin transport is unknown. Although pinocytosis has been proposed, no direct or strong inferential evidence to support this proposal exists. The uptake of transferrin is highly temperature-dependent, and the activation energy of the forward rate constant has been calculated at approximately 20 kcal/mole (Kailis and Morgan, 1974). Inhibitors of oxidative metabolism depress the uptake of the temperature-dependent transport process. Inhibitors of microtubules also depress the uptake process (Hemmaplardh et d., 1974). A major difficulty for the pinocytotic mechanism is to explain how transferrin escapes degradation in lysosomes. Studies on fibroblasts have shown that virtually all pinocytotic vesicles fuse with lyso-

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somes (Hubbard and Cohn, 1975; Steinman et al., 1974). T h e situation may b e different with reticulocytes and nucleated erythroid cells. C. low-Density lipoprotein

1. STRUCTURE AND FUNCTION In the absence of dietary cholesterol the liver synthesizes more than 90% of the total daily cholesterol requirement (Dietschy and Wilson, 1970), yet the peripheral tissues, such as fibroblasts, skin cells, and aortic media cells are capable of high rates of cholesterol synthesis. Therefore a regulatory mechanism must exist linking these disparate sites of synthesis (Brown and Goldstein, 1976a). Cholesterol is transported in the serum tightly bound to proteins. The major cholesterol-carrying protein in human serum is known as low-density lipoprotein (LDL). LDL is a high-MW lipoprotein (2-3.5 x lo6) carrying a core of neutral lipid, mainly esterified cholesterol. The core is surrounded by a polar coat that contains phospholipid, free cholesterol, and a 500,000-dalton protein known as apoprotein B (Goldstein and Brown, 1977). LDL carries, generally on a weight basis, a ratio of cholesterol to protein of 1.6: 1, 70% of which is esterified. Goldstein and Brown (1976) and co-workers showed in a series of articles that LDL in addition to being a carrier protein functions as a regulator of peripheral cholesterol synthesis, and that this process is receptormediated. Fibroblasts grown in tissue culture in the absence of LDL exhibit profound changes in cholesierol metabolism when LDL is added to the medium: (1)The cellular cholesterol pool is expanded, (2) cholesterol synthesis is suppressed because of a reduction in the activity of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG CoA reductase), ( 3 ) cholesterol esterification is activated, and (4) cell surface LDL receptor number is reduced. All these effects were achieved in the absence of LDL by adding cholesterol to the medium in the presence of ethanol. Evidently, cholesterol itself initiated these feedback mechanisms when it gained entry to the cell, and LDL was the physiological vehicle for cholesterol entry. Fibroblasts obtained from patients suffering from the disease familial hypercholesterolemia exhibited high rates of cholesterol synthesis which were unchanged by the addition of LDL. However, these cells returned to normal levels of synthesis after cholesterolethanol was placed in the medium. The mutation apparently involved a failure in LDL-mediated cholesterol entry.

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2. FIBROBLAST RECEPTORS AND TRANSPORT Binding studies were performed on normal cultured human fibroblasts utilizing lZ5I-labeledLDL. Saturable binding was detected, half-saturation occurring in the region of 1pg/ml and complete saturation occurring in the range of 10-20 pg/ml of lz5I-labeledLDL at 4°C. When fibroblasts were incubated with 1251-labeledLDL at 37"C, uptake was noted, and degradation products in the form of trichloroacetic acid (TCA)-soluble iodine accumulated in the cell culture medium. Degradation products increased at a linear rate with time and, when near saturating conditions of LDL were used, the rate of degradation was found to be 180 ng of LDL per hour per milligram of fibroblast protein (Basu et al., 1976). Hydrolysis of cholesterol esters paralleled the rate of protein degradation. Both processes were inhibited 90%by chloroquine. Since chloroquine is believed to inhibit lysosoma1 hydrolases, the site of degradation was presumed to be the lysosome. When the same type of study was repeated on fibroblasts from cell lines carrying the homozygous familial hypercholesterolemia mutation, binding at 4°C was decreased 50-fold and was linear with concentration, indicating the absence of detectable high-affinity binding sites. At 37°C uptake was decreased by more than a factor of 10, and no detectable degradation products in the medium were noted. Thus the overproduction of cholesterol seen in familial hypercholesterolemia appears to result from an absence of feedback inhibition, which is the consequence of a defect in the receptor for LDL required for cholesterol transport into the cell. 3. CLUSTERED RECEPTORS AT COATED REGIONS

The localization of LIjL receptors on the plasma membrane of fibroblasts has been studied utilizing a ferritin-conjugated LDL (Anderson et al., 1976). The striking feature to emerge from this study is that the receptors are not uniformly distributed but rather are clustered at thickened indentations in the membrane which appear to be sites in the early stages of pinocytosis. Such sites are known as coated regions (see Fig. 2). Seventy percent of all receptors visualized were at these sites which accounted for only 1.4% of the total membrane surface area. Controlled studies on cells carrying the homozygous mutation for familial hypercholesterolemia failed to show binding sites. The ferritin technique revealed between one-half and one-quarter the number of sites detected by the 1251-labeledLDL-binding technique. This clustering of sites does not occur by a process similar to that seen

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RECEPTOR-MEDIATED PROTEIN TRANSPORT INTO CELLS

CLUSTERED RECEPTOR-MEDIATED PINOCYTOTIC TRANSPORT MODEL

,Bound Protein

J 1

Lysosome

2m ez3 A

3

Coated Vesicle

Secondarv Lysosome

FIG.2 . In this model receptors are clustered at specialized small areas of the surface membrane known as coated regions which are predestined to become sites for pinocytosis. Uptake proceeds as in Fig. 1. Clustering allows for a high ratio of receptormediated uptake to membrane internalized. As shown here, clustering is promoted by a second insertion protein or a second specialized region on a single polypeptide chain receptor (solid circles) which has a high affinity for the coated regions. (See Sections II1,C and IX,B. (Redrawn from Anderson et ul., 1977b.)

in lymphocytes following binding of antiimmunoglobulin, since the LDL-binding studies are performed at 4"C, a temperature which does not permit transmembrane receptor migration. By performing LDL-ferritin binding to human fibroblasts at 4"C, followed b y rapid warming to 37"C, sequential quantitative analysis of the internalization process was performed (Anderson et d.,1977a). Within 10 minutes, 98% of the LDL-ferritin bound to coated regions was internalized, predominantly into coated pinocytotic vesicles formed b y the invagination and pinching off of the coated membrane regions. With increasing time the coated vesicles were observed to migrate through the cytoplasm, to lose their cytoplasmic coat, and to fuse with either primary or secondary lysosomes (see Fig. 2). LDLferritin cores initially bound to noncoated regions of the membrane also became reduced in number with time. Pinocytosis from these regions also occurred, but these vesicles did not contain LDL-ferritin. These findings raise the interesting possibility that LDL receptors can migrate from noncoated regions of the membrane to become inserted and concentrated in coated regions. This proposition receives further support from studies of a second type of mutation producing familial

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hypercholesterolemia. This mutant (J. D.) described by Brown and Goldstein (1976b)is unable to internalize LDL like the other familial hypercholesterolemic mutation, yet binding of LDL displays normal kinetics and number of sites. Evidently two alleles are required for internalization, one specifying binding and a second related to internalization (see Fig. 2). Two findings indicate that the second allele may serve to insert LDL receptors at coated regions: (1)J. D. cells form coated pinocytotic vesicles like normal cells, and (2) although the kinetics of binding and receptor number are normal in J. D. cells, LDL-ferritin cores are not found in coated regions; rather they are distributed diffusely over the surface membrane (Anderson et al., 1977b). Clustering of sites seems to be necessary to explain the relatively high rate of LDL uptake if receptor-mediated pinocytosis is the only process operating. The rate of LDL internalized and degraded is 180 ng per hour per milligram of fibroblast protein, whereas the amount bound is half this quantity. This indicates that the plasma membrane must turn over two times each hour. Reported rates for fibroblast plasma membrane turnover are one-quarter of the membrane per hour (Steinman et al., 1974; Hubbard and Cohn, 1975).Hence the uptake of LDL appears to be eight times more rapid than the maximum possible rate calculated for receptor-mediated pinocytosis assuming a uniform receptor distribution. The maximum rate of protein uptake into fibroblasts by nonreceptor-mediated pinocytosis (bulk fluid pinocytosis) has been ascertained by Steinman and co-workers (Steinman et al., 1974) utilizing studies with horseradish peroxidase. This type of uptake is nonsaturable and is linearly related to the protein concentration in the external medium. The rate is 100 ng/mg of cell protein per hour for external protein of 1 mg/ml. In the uptake studies of LDL cited above, the LDL concentration was in the range of 5 pg/ml. This would lead to a non-receptor-mediated uptake of 0.5 ng/mg of cell protein per hour. This figure is 400-fold less than the uptake found in normal fibroblasts but is in the range of uptake reported for the mutant fibroblasts. Therefore the data are consistent with two modes of uptake, clustered receptor-mediated pinocytosis occurring in normal cells and bulk fluid pinocytosis occurring at a much lower level in both normal and mutant cells. Since bulk fluid pinocytosis uptake of a soluble macromolecule is linearly related to the macromolecular concentration, normal levels of LDL protein uptake can be achieved in mutant cells b y raising the LDL concentration to the appropriate level. The surprising result obtained is that this maneuver fails to increase the cholesterol pool, and consequently abnormal levels of synthesis and es-

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terification are unchanged (Goldstein and Brown, 1976).The failure of the cell to accumulate cholesterol acquired by this uptake route points up the specificity of the uptake process in defining the final physiological result. Recently, Basu et a l . (1976) found it possible to achieve uptake of LDL by fibroblasts lacking LDL receptors by incorporating positive charges into the LDL. This was done b y mixing LDL with N , N dimethyl-1,3-propanediamine.Cationized LDL, when incubated with mutant fibroblasts, induced a fall in HMG CoA reductase. In addition, labeled degradation products of the cationized LDL were found to accumulate in the medium at a rate approaching that for normal fibroblasts and native LDL. These workers believe that the cationized LDL was bound to receptors present on the surface membrane for cationic groups, pinocytosed, and delivered to the lysosome and hydrolyzed, similar to the proposed scheme for native LDL. It is not known whether the cation receptors are diffusely distributed or localized to coated regions; however, cationized ferritin is bound diffusely. The end result of introducing cholesterol via cationized LDL into cells lacking LDL receptors is a marked accumulation of cellular cholesterol in the form of large lipid droplets. Presumably this is because the receptors involved (now cationic receptors) are not reduced in concentration (down-regulated) as the cholesterol pool increases, as would occur with LDL receptors (Goldstein and Brown, 1977). The concept that down-regulation of receptors, subsequent to specific ligand binding, serves to stabilize an intracellular pool of the ligand or a fragment of the ligand may have general applicability. Down-regulation of receptors was first reported for the insulininsulin receptor system (Neville et al., 1973; Gavin et al., 1974); however, the physiological consequences have not been fully explored. IV.

ASIALOGLYCOPROTEINS

A. Structural Requirements for Transport

Desialylated glycoproteins are rapidly cleared from the circulation

by a receptor-mediated transport process. This process has been studied in detail by Ashwell and Morel1 (1974) and collaborators. The initial studies were performed with ceruloplasmin. The half-life of native ceruloplasmin in the rabbit was known to be approximately 56 hours. However, when the terminal sialic acid residues of this glycoprotein were removed with neuraminidase, the injected protein was

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found to clear from the rabbit serum with a half-life of approximately 3 minutes. The removal of terminal sialic acid left galactose residues as the terminal sugar on this glycoprotein. Rapid clearance of glycoprotein was found to be dependent upon terminal galactose. Treatment of asialoceruloplasmin with galactose oxidase or 0-galactosidase resulted in prolongation of the half-time of survival by a factor of 10 over that of the desialylated glycoprotein. The rapid disappearance of asialoceruloplasmin from the serum was found to be accompanied by an equally rapid accumulation of labeled protein within the liver. Less than 1% of the labeled protein was found in kidneys, spleen, lungs, and heart combined. By oxidizing asialoceruloplasmin, followed by a reduction of the resultant aldehyde derivative with tritiated borohydride, tritiated asialoceruloplasmin was prepared. A second label was introduced by using 64Cu.Cellular distribution within the liver was monitored by serial histoautoradiography. Tritium was located exclusively in the parenchymal cells. The initial uptake of doubly labeled asialoceruloplasmin by the liver occurred via the intact molecule. Six minutes after doubly labeled asialoceruloplasmin was injected into a rat, 75% of the dose was recovered from the liver with no change in the ratio of tritium to copper. The intracellular site of transport was found to be the lysosome. Sucrose density gradient fractionation of liver homogenates revealed that labeled immunoprecipitable asialoceruloplasmin migrated together with the lysosomal marker enzymes P-galactosidase and acid phosphatase. At late time points copper was shown to be cleaved from the protein, and the protein underwent catabolism within the lysosome. Further studies showed that the exposure of any two galactosyl residues was sufficient to achieve prompt removal of the glycoprotein from the plasma. The phenomenon of rapid clearance of desialylated ceruloplasmin from the plasma was found to be general for a wide variety of asialoglycoproteins. Clearance curves followed a first-order process, and half-times varied from 3 minutes for asialoceruloplasmin to 40 minutes for asialothyroglobulin. B. Receptors

The receptor nature of this process was demonstrated in vivo by showing that the rapid first-order clearance of a given desialylated glycoprotein could be blocked by higher doses of another glycoprotein or higher doses of the same unlabeled glycoprotein. The receptors were found to be localized to the cell surface membrane by performing binding studies on isolated purified plasma membrane preparations. The binding studies indicated a close parallel between the in vitro

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binding system and the in vivo clearance system. Rank orders for binding affinity and clearance for various asialoglycoproteins were identical. In addition, the binding system also showed requirements for the asialo derivative and for an intact galactose residue. Competitive binding was demonstrated, and the 50% inhibition level spanned several factors of 10 for a variety of asialoglycoproteins. The highest-affinity asialoglycoprotein was orosomucoid. Saturation of specific binding sites occurred at 4 ng/ml. This corresponds to an equilibrium dissociation constant of 10-l0 M . The maximal binding capacity was estimated to be 7.5 pmoles of asialoorosomucoid per milligram of membrane protein (Pricer and Ashwell, 1971). The receptor site on the plasma membrane was also shown to be a glycoprotein. Terminal sialic acid on this glycoprotein was required for the binding process. Removal of the terminal sialic acid by neuraminidase treatment of the plasma membranes resulted in a diminution of binding. Resialylation was performed by incubating membranes with CMP14C-sialicacid, and binding was restored proportional to the incorporation of sialic acid residues. Binding was also shown to be dependent upon the presence of calcium ion. Recently, Kawasaki and Ashwell (1976a) isolated the receptor binding protein and studied its properties. The glycoprotein exhibits concentration-dependent self-association. The smallest oligomer with asialoglycoprotein-bindingproperties appears to be 2.6 x lo5 daltons. Following extensive treatment with SDS, two subunits are found in a ratio of 1:2 with MWs of 48,000 and 40,000. Two glycopeptidex have been isolated from the binding glycoprotein and have been sequenced. The terminal residues for glycopeptide 1 are sialic acid, galactose, and N-acetylglucosamine (Kawasaki and Ashwell, 1976b). Recently, Pricer and Ashwell (1976) examined the subcellular distribution of the hepatic binding protein. Initially it was thought that the binding protein was restricted to a plasma membrane localization. However, with the experience gained in isolation of the binding protein using Triton X-100, the application of this technique to subcellular fractions revealed considerable amounts of binding protein present in the Golgi apparatus, smooth endoplasmic reticulum, and lysosomes. These investigators raise the question whether or not this localization reflects a biosynthetic cycle originating in the endoplasmic reticulum, progressing to the Golgi apparatus and on to the plasma membrane and then back to the lysosomes. In the last step from plasma membrane to lysosome the asialoglycoprotein-binding protein may function as a stable shuttle. However, no real evidence exists concerning the mechanism of transport from the cell surface re-

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ceptor into the lysosome. Regardless of the mechanism the critical information in this cell-ligand system is in specific carbohydrate structures. Ashwell and Morell (1977) believe that these same structures may provide the critical determinants for certain cell-cell and subcellular organelle interactions. C. Pinocytotic Mechanism?

It may appear at first glance that the transport process occurs by pinocytosis of receptor-bound asialoglycoprotein. This seems likely, since pinocytotic vesicles are known to fuse with lysosomes. However, it appears to us that the rates of receptor-mediated pinocytosis judged to exist are insufficient to transport asialoglycoproteins at the rates determined by Morell et al. (1971)unless a marked degree of receptor clustering occurs. The following calculation is instructive. The maximum rate of pinocytosis in fibroblasts has been determined by Steinman et a l , (1974) as 25% of the plasma membrane per hour. Pinocytotic rates do not appear to be higher than this in liver, as judged b y plasma membrane turnover data (Schimke, 1969). We therefore can calculate the amount of plasma membrane internalized by the liver per hour as follows. We assume a 7-gm liver for a 200-gm rate, which is 20% protein. A generous estimate for the amount of plasma membrane protein per total liver protein is 2% (Neville, 1976).This gives 28 mg of plasma membrane protein and, assuming one-quarter internalization per hour, gives 7 mg of plasma membrane internalized per hour. Ashwell and Morell (1974) have determined that there are 7.4 pmoles of asialoglycoprotein receptor per milligram of plasma membrane protein. With the assumption of an even distribution of receptors, this leads to a maximum rate of receptor internalization of 52.5 pmoles per hour. If each receptor internalizes one molecule of ceruloplasmin, 7.8 mg of ceruloplasmin can be internalized per hour by this process. Morell et al. (1971) have reported plasma clearance data for ceruloplasmin after the injection of 9.3 mg of ceruloplasmin into a 200-gm rat. The half-time of clearance is about 3 minutes, and the process appears to be first-order. Dividing the logarithm of 2 b y the half-time of clearance gives a first-order rate constant of 0.23 per minute. The maximum transport rate is the first-order rate constant times the initial load, giving a figure of 4.1 mg per hour, almost 500 times the rate calculated on the basis of a receptor-mediated pinocytotic process. The same calculation performed on orosomucoid data yields an even greater discrepancy (2000 times). Receptor clustering at the site of pinocytosis could raise the rate for the pinocytotic process. Such clus-

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tering may b e induced by binding of the asialoglycoprotein to the receptor in a manner similar to that reported for IgG binding to lymphocytes. However, such clustering does not achieve the degree of segregation of receptor relative to other membrane components required for this process (Gonatas et al., 1976). Clustered receptor-mediated pinocytotic models (see Sections III,C,3 and IX,B) imply a much higher rate of turnover for the receptor than for components of the membrane at large, unless mechanisms exist to extract receptors from pinocytotic vesicles and reinsert them in the plasma membrane. Widely discrepant turnovers of membrane proteins analyzed b y SDS gel electrophoresis have not yet been reported (Tweto and Doyle, 1976). In any case, the clustering hypothesis is open to test. If clustering does not occur, then a nonpinocytotic mechanism of transport exists, operating from the membrane receptor site to the lysosome. V.

FIBROBLAST LYSOSOMAL HYDROLASES

Considerable evidence indicates that fibroblast lysosomal glycosidases and sulfatases gain entry to the lysosome after first being excreted extracellularly. Uptake from the extracellular compartment proceeds through a receptor-mediated process. The secretionrecapture hypothesis and its experimental foundation have recently been reviewed b y Neufeld and co-workers (Neufeld e t al., 1977). Much of the evidence comes from studies of inherited mucopolysaccharide storage diseases which result in the lysosomal accumulation of dermatan sulfate or heparin sulfate. Two general types of diseases exist. One type, exemplified by Hurler’s syndrome, is characterized b y a deficiency in one of a group of enzymes necessary to cleave the variety of linkages found in these polymers of uronic acid and sulfated hexosamine. In Hurler’s syndrome the deficient enzyme is a - ~ iduronidase. In the second type several enzymes are deficient within the lysosome but are present and active in the extracellular fluid. I-Cell disease is an example of this type of disorder (Neufeld et al., 1975). When Hurler fibroblasts are grown with normal cells or with media conditioned by normal cells, the abnormal accumulation of polysaccharide is reversed. The corrective factor found in the media proved to be a form of a-L-iduronidase. As much as 25% of the added a - ~ iduronidase was taken up in 2 days. Uptake of active enzyme was demonstrated to be a saturable process, with one-half saturation occurring at lov9 M (Neufeld et al., 1977). Not all samples of a - ~ iduronidase were capable of rapid, saturable uptake. High-uptake

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forms of enzyme were converted to low-uptake forms by treatment with dilute periodate. This conversion appeared to destroy the uptake recognition factor. Six other lysosomal glycosidases and sulfatases were found to exist in both high- and low-uptake forms. Kaplan and co-workers (Kaplan et al., 1977) showed that uptake of the high-uptake form of P-glucuronidase was competitively inhibited by mannose 6phosphate and that the high-uptake form could be converted into the low-uptake form by treatment with alkaline phosphatase. Similar findings have been reported for a-L-iduronidase (Neufeld et al., 1977). The recognition marker appears to be a phosphorylated carbohydrate residue (Kaplan et al., 1977). The hydrolytic enzymes not present in fibroblast lysosomes but secreted by I-cell fibroblasts are all low-uptake forms. I-Cell fibroblasts can internalize high-uptake enzymes with the same kinetic constants as normal cells and retain these enzymes with the same half-life. The secretion-recapture hypothesis of Neufeld and co-workers postulates for I-cell disease a single mutation in the synthesis of the common recognition marker, explaining the resulting failure of uptake and accumulation of a group of active extracellular enzymes. The mechanism of entry of lysosomal hydrolases from the extracellular environment into the lysosome is unknown, although receptormediated pinocytosis has been postulated (see Fig. 1). The pinocytotic vesicle containing receptor-bound hydrolases could then be considered either a primary lysosome (if substrate was also engulfed) or a secondary lysosome (Neufeld et al., 1977). VI.

ANTIBODIES

A. Maternal-to-Young Transfer

Active production of antibodies is lacking in newly born mammals, and these mammals receive their immunity from the transfer of yglobulins from the maternal to the fetal circulation. In the rabbit, guinea pig, rhesus monkey, human, and gray squirrel transmission of y-globulin occurs prior to birth. Goat, sheep, pig, horse, and cat receive y-globulins after birth via colostrum and milk. In the dog, mouse, and rat both processes occur (Wild, 1973). The transfer process is highly specific for certain types of globulins and also exhibits species specificity. The route for this process was shown by Brambell (1970) to be across the yolk sac splanchnopleur to the vitelline circulation (Wild,

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1973). [Toward the end of gestation the rabbit embryo is attached to the uterus via the chorioalantoic placenta. On the .opposite side, the

embryo projects into the uterine lumen and is covered by a series of membranes, the outermost being the yolk sac splanchnopleure. Beneath this membrane lie the vitteline vessels which enter the embryo via the yolk sac stalk. Thus the embryo is in contact with maternal fluids through two distinct membranes and circulatory paths (Wild, 1973)l Thus labeled antibodies injected into the uterine lumen were found to reach the fetal circulation, a process that could be i’nterrupted b y ligaturing the yolk sac stalk. In order for proteins to reach the vitelline circulation they must cross the yolk sac endoderm and basement membrane, the vascular mesenchyme, and the endothelial cells of the vitelline capillaries. I n order to explain selective uptake and competition between various species of globulins, Brambell and co-workers proposed that uptake occurred via pinocytotic vesicles and was mediated by specific receptors. These receptors, originally located on the microvilli of the endodermal cells, become internalized in the pinocytotic vesicle and protect all bound proteins from degradation within the vesicle. Fusion of pinocytotic vesicles with lysosomes would result in the degradation of unbound protein within the vesicle. On reaching the basal portion of the endodermal cell, the vesicle would fuse with the basement membrane and release the bound protein (Wild, 1975). Wild (1975), using a combination of fluorescent microscopy, electron microscopy, and autoradiography observed that a wide variety of proteins becomes localized in pinocytotic vesicles within the yolk sac endoderm. No selectivity is shown in this process. Moreover, these pinocytotic vesicles, called macropinocytotic vesicles by Wild, have never been observed to fuse with the basal plasma membrane of the endodermal cell. Wild noted a smaller vesicle (0.07-0.15 pm), called a micropinocytotic vesicle, often apparently fusing with the basal plasma membrane of the endodermal cell. These vesicles are also observed to b e generated at the base of microvilli on the lumenal side of the endodermal cell. Such vesicles are so small that they seem to consist entirely of membrane, and they may contain no aqueous phase. These vesicles do not fuse with lysosomes, as do macropinocytotic vesicles. Wild believes that they may b e involved in the selective transport of globulins from the maternal to the fetal circulation. Since only approximately 12% of the protein injected into the uterine cavity is transported intact into the fetal circulation, the rest being degraded, another process must b e invoked to explain degradation. According to Wild, this would occur in the macropinocytotic vesicles which fuse

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with lysosomes. Thus macropinocytotic vesicles would contain receptor-bound protein and soluble protein originally present in the extracellular environment, and both would eventually be catabolized. Mi'cropinocytotic vesicles containing no aqueous phase would contain only receptor-bound protein, and this would be transported across the endodennal cell. No direct evidence for this process has been obtained. However, receptors for human IgG and human Bence-Jones A proteins have been demonstrated using 1251-labeledproteins in a 60,000 g membrane fraction derived from human placenta (Gitlin and Gitlin, 1974). Jones and Waldmann (1972) studied the transport of proteins from the small intestine into the circulation of neonatal rats. Using tracer amounts of 1251-labeledproteins instilled into the small intestine, they demonstrated that 21 -35% of the instilled protein was transported to the circulation in a TCA-precipitable form for rabbit, human, rat, and mouse IgG. In contrast, less than 5% of the instilled amounts of human albumin, human transferrin, human IgA, human IgM, and polyvinylpyrolidone was transported to the circulation. The specificity for this transport appeared to reside in the Fc piece of IgG, since 12.6%of this material was transported to the circulation in contrast to 1.7%of the Fab piece. These investigators also demonstrated the saturability of this transport process. When 0.5 mg of unlabeled protein was instilled with the tracer, IgG transport to the circulation was reduced 50%.When unlabeled protein was instilled in amounts ranging from 1 to 8 mg, transport of the tracer was reduced 96%.The amount of total protein transported from the intestine to the circulation became constant when more than 1 mg was instilled into the intestine. The limiting protein transport during the 4-hour study period was 0.12 mg. These investigators also observed that the labeled proteins studied were bound to small intestine microvillous preparations in direct proportion to their transport rate. Tracer binding was competed for by cold protein in cases where high degrees of transport were observed. The ability of the neonatal rat jejunum to absorb functional antibodies selectively into the bloodstream is lost by the twenty-second postpartum day (Halliday, 1955). Immunoglobulin transport from the maternal circulation into milk was studied in lactating mice by Gitlin and co-workers (Gitlin et al., 1976). Lactating mothers were injected intravenously with 1311iodinated proteins, and the disappearance of counts in the mothers and the appearance of counts in nursing litters were followed by whole body counting. Over 80% of the counts recovered from the stomachs of nursing young were in a TCA-precipitable form. Trans-

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port of labeled proteins from a lactating mother to young is given as a daily turnover percent of the maternal body pool. These transmammary transfer percentages were low for mouse IgM (5.9%)and human albumin (4.6%),and below detection for human IgA. Higher values were observed for mouse IgG (30%) and human IgG (22%). The highest values were obtained with the K and A chains of Bence-Jones proteins, being 580% and 866%, respectively. Tracer transfer for IgG was inhibited 50% when excess unlabeled human IgG was injected into the maternal circulation. Maternal pools of cold IgG were increased to 90, 180, and 295 mg. Tracer transfer remained constant but, by assuming the validity of the tracer assumption, total transfer increased proportionately to the pool size ratios. This indicates that a portion of the transfer process is nonsaturable. B. Transport into immunological Cells

Taylor and co-workers (Taylor et aZ., 1971) reported that plasma membrane surface immunoglobulins of lymphocytes incubated with fluorescein-labeled antiimmunoglobulin antibodies aggregate into patches, form a polar cap, and undergo pinocytosis. This process is temperature-dependent and is inhibited by dinitrophenol, cyanide, and sodium fluoride. Cytochalasin B, which disrupts microfilaments associated with the plasma membrane, also inhibits this process (Nicolson and Poste, 1976). Taylor and co-workers (Taylor et al., 1971) proposed that the clustering and capping phenomena were prerequisites for the activation of lymphocytes. It is known that each B-type lymphocyte contains about lo5 immunoglobulin molecules as an integral component of the plasma membrane. These molecules function as receptors for antigens. Each individual lymphocyte contains a different species of immunoglobulin specific for a different antigen. When bivalent antigen interacts, the lymphocyte is triggered to proliferate and differentiate (Singer, 1974). Singer (1974) concludes that clustering and capping are not essential for lymphocyte activation and actually may inhibit the process. If this is the case, capping followed by pinocytosis may be a mechanism for ridding the cell surface of the stimulating antigen or antiimmunoglobulin antibody. The internalization of bound antiimmunoglobulin on lymphocytes has been confirmed by a quantitative autoradiographic study (Gonatas et al., 1976). The site of internalization is presumed to b e the lysosome, since most pinocytotic vesicles are observed to fuse with lysosomes in fibroblasts. The generality of this observation is, however, unknown. Lewis and co-workers (Lewis et al., 1974) have presented fractiona-

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tion data indicating that lymphocytes exposed to labeled antibody directed against transplantation antigen (anti-HL-A antibody) internalize the antibody predominantly at the nucleus. Isolated nuclei were shown to bind anti-HL-A antibody in amounts 10-fold greater than those bound by intact lymphocytes on an equal number basis. When cells were incubated with labeled anti-HL-A in tracer amounts, nuclear localization was stable over a 96-hour period. However, when the cells were exposed to large amounts of cold anti-HL-A antibody 30 minutes after exposure to labeled antibody, label disappeared from the nuclei at 72 hours. This time was correlated with the onset of blastogenesis. The localization data supplied by Lewis and co-workers (Lewis et al., 1974) can be criticized for its lack of data relating to the purity of the fractions. In addition, the nature of the nuclei-associated labeled antibody in terms of its integrity was not considered. However, these studies raise the question whether or not antibodies can be transported into cell compartments where immunological processes are operative. C. Retrograde Axonal Transport

The selective uptake and retrograde axonal transport of antibodies to dopamine P-hydroxylase was recently demonstrated by Fillenz et al. (1976). The experimental design and data obtained are similar to the reported studies with NGF (see Section VII1,A).

VII.

VIRUSES

A. Evidence for Receptor-Mediated Entry

For many viruses the first step in the infection of a eukaryotic cell involves binding of the virus to a specific cell surface membrane receptor (Fenner et al., 1974). For influenza and polyoma viruses the receptor has been found to be a glycoprotein with a terminal sialic acid residue. Treatment of cells with neuraminidase renders them resistant to the viral infection. Competition for the cell surface membrane has been observed between adenoviruses and the type I1 fiber antigen isolated from adenoviruses. The membrane’s receptors for avian tumor viruses are genetically determined, and in chickens the receptor is controlled by a single dominant autosomal gene. Infection of cells b y picorna viruses, polio virus being an example, is limited to

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human and simian cells because only these cells carry the specific receptor. However, the adsorption of pox viruses to cells does not require a specific receptor. 8. General Transport Mechanisms

There appear to b e at least three different mechanisms for the entry of animal viruses into eukaryotic cells. In all three mechanisms the viral protein coat remains cell-associated, although the compartmentalization of the protein varies among the mechanisms. Pox viruses, which do not require specific receptors, enter the cell by pinocytosis. The pinocytotic vesicle rapidly fuses with the lysosome, forming a phagocytotic vacuole. Within this vacuole the outer membrane of the virus is rapidly degraded, and the virus is converted to a subviral particle called the core. The membrane of the vacuole then undergoes dissolution, and the core particle is released into the cytoplasm (Fenner et aZ., 1974). A second mechanism of entry is seen with adenoviruses, in which the viral particle crosses the cell membrane and appears in the cytoplasm devoid of any association with a membranous particle. Although the viral particle appears intact, density gradient centrifugation has shown that the virus has lost approximately 5% of its protein. Viral particles move rapidly to the nucleus where further loss of protein occurs and DNA is released. This process requires only 1hour (Morgan et al., 1969). Coincident with this process of entry, some adenovirus particles are also found to enter the cell in phagocytic vesicles. The ratio of the number of viral particles entering b y pinocytotic vesicles to the number entering directly varies with the cell type and the genetic strain of the virus (Chardonnet and Dales, 1970). A third mechanism of entry has been observed in Newcastle disease virus, a member of the paramyxovirus group. Entry for this group is presumed to be receptor-mediated since, like influenza viruses, paramyxoviruses have a hemagglutinin and a neuraminidase in the outer viral envelope. Evidence exists that Newcastle disease virus nucleoprotein is released into the cell cytosol following fusion of the viral envelope with the plasma membrane. The fusion process is believed to be aided b y a hemolysin carried by the paramyxovirus. Herpes virus may also enter cells by a fusion process (Fenner et al., 1974). Most of the above observations have been obtained by morphological techniques, and knowledge of the biochemistry of the transport process is meager. Evidence exists that certain viruses, notably rabies and herpes, can gain entrance to the central nervous system from the

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subcutaneous compartment by retrograde axonal transport. The reverse process, centrifugal transmission from the dorsal root ganglion cells to the skin and mucous membrane, most likely explains the mode of spread of recurrent herpes simplex and herpes zoster (Fenner et al., 1974). VIII.

GROWTH FACTORS AND HORMONES

A. Nerve Growth Factor

1. STRUCTURE AND FUNCTION

P-NGF is a protein of MW 26,500 present in the serum of all vertebrates. NGF is necessary for development of the sympathetic nervous system in young animals. The injection of antiserum directed against NGF into newborn mice results in selective and permanent destruction of 90-95% of the sympathetic nerve cells (Levi-Montalcini and Angeletti, 1968). NGF is also necessary for the maintenance of tissue-cultured sympathetic neurons in serum-free media. When dorsal root ganglions from 8-day chick embryos are incubated with NGF, neurite outgrowth is observed which is dependent upon the presence of NGF. This constitutes the usual bioassay, and the maximum response for purified preparations occurs at 0.26 nM. In the mouse the serum concentration of NGF is reported to be 0.4 nM (Hendry and Iversen, 1973). NGF is synthesized and highly concentrated in the salivary gland of the mouse and in the venom gland of vipers. The material is produced elsewhere, since extirpation of these glands does not produce a deficiency disease. Considerable evidence exists that NGF is fixed by receptors located primarily at the sympathetic nerve cell terminals, transported across the plasma membrane, and transported by retrograde axonal transport up the axon to the region of the cell body where it exerts its stimulatory effects. P-NGF is isolated from a higher-MW complex, 'IS-NGF, in the mouse submaxillary gland. It is associated with two other polypeptide chains, one of which has proteolytic activity (Baker, 1975). NGF is prepared by dissociation of 7s-NGF. It contains two identical polypeptide chains held together by noncovalent forces. Each chain contains three intrachain disulfide bonds (Stach and Shooter, 1974). The sequence of P-NGF has been compared to the sequence of human proinsulin by Frazier and co-workers (Frazier et al., 1972). These investigators conclude that there are sequence similarities and that the two proteins are evolutionarily related,.

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2. MEMBRANERECEPTORS

NGF has been successfully labeled with 1251,and binding studies with dorsal root nerve cells and membranes derived from sympathetic ganglion have revealed the receptor nature of the binding process. Eight-day chick embryo dorsal root ganglion cells contain approximately 2 x lo4 receptors per cell. The specific binding reaches halfsaturation at 0.26 nM (Herrup and Shooter, 1973). A crude vesicular membrane fraction isolated from the sympathetic ganglia of young rabbits exhibited a 10-fold increase in binding activity over that of the homogenate. An apparent equilibrium dissociation constant was calculated to be 0.2 nM. No cross-reactions with insulin or epidermal growth factor were found. Specific binding was limited to membrane preparations from sympathetic ganglia (Banerjee et al., 1973). The binding of NGF to these membranes has been found to be dependent upon calcium. Low concentrations of trypsin and phospholipase A from Vipera russelli decrease NGF binding (Banerjee et al., 1975). 3. BIOCHEMICALEFFECTS

The treatment of newborn rats with NGF enhances growth and induces differentiation in the sympathetic neurons. Thoenen and coworkers (Thoenen et al., 1971) showed that NGF stimulates the activity and concentration of two enzymes, tyrosine hydroxylase and dopamine P-hydoxylase, which are localized exclusively in adrenergic neurons and which are concerned with the synthesis of adrenergic neurotransmitters. The activity of these enzymes increases 13- to 18-fold after 10 days of treatment with NGF. The specific activity rises 4-fold. Monoamine oxidase and dopa decarboxylase activities rose 1.2and 1.5-fold, respectively. Combined extracts from controls and treated animals gave additive activities, indicating that formation of an activator or loss of an inhibitor was not responsible for the phenomenon. These investigators conclude that NGF evokes a selective induction of tyrosine hydroxylase and dopamine P-hydroxylase. These enzymes are known to be synthesized in the cell body of the sympathetic neurons and transported in an orthograde manner to the axon e n d terminals where the neurotransmitters are synthesized and packaged. 4. RETROGRADE AXONALTRANSPORT Studies on the binding of NGF to homogenates of sympathetic ganglia and dorsal root cells do not provide information on the anatomical localization of the receptor sites. The following evidence indicates that NGF specifically interacts at the end terminals. When

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1251-labeledNGF is injected intraocularly, counts are observed to rise in the ipsilateral superior cervical ganglion as a function of time, peaking at 8 hours. The number of counts is two to three times higher on the ipsilateral side compared to the contralateral noninjected side. When the same experiment is repeated with a variety of proteins, such as cytochrome c, insulin, horseradish peroxidase, or bovine albumin, counts on the contralateral and ipsilateral sides are identical and the time course follows a decay curve rather than a peak preceded by a lag period. The time required for the peak to be achieved with NGF is consistent with the known rates of retrograde axonal transport (Stockel et al., 1974). When 1251-labeledis given intravenously, there is a rise in the number of counts in the superior cervical ganglion, which is half complete at 3 hour and complete at 1hour. Counts are constant until 4 hours, when a further, larger rise takes place which again peaks at 8 hours, giving a specific activity seven times that of the blood. Other organs observed, such as heart muscle, show a simple decay curve paralleling the decay curve for blood (Stoeckel et al., 1976).It is believed that the initial, low-level rise represents uptake from the blood by the cell bodies within the superior cervical ganglion. The later, more profound rise has the same time course as retrograde axonal transport and represents this phenomenon. Most of the NGF reaching the superior cervical ganglion arrives by the retrograde route from the end terminals of the axon. This is understandable, since the ratio of the cross-sectional area of the sympathetic end terminals to the cell body has been estimated to be 100: 1(Stoeckel et al., 1976).The specificity of this process was further demonstrated by showing that NGF was not taken up and transported retrograde in the lower motor neuron, as is the case with tetanus toxin (Stockel et al., 1974).

5. INTRACELLULAR VERSUS MEMBRANESITE

OF

ACTION

Evidence that the stimulatory effect of NGF on tyrosine hydroxylase is mediated through retrograde axonal transport was obtained by Paravicini and co-workers (Paravicini et al., 1975). These investigators administered large doses of NGF and lz5I-1abeled NGF systemically to 8-day-old rats. Experimental animals had the axon to one superior cervical ganglion sectioned. At 48 hours tyrosine hydroxylase activity was assayed in the superior cervical ganglions. The stimulation of tyrosine hydroxylase activity seen on the intact side was twice that on the axotomized side. With the use of lZ5I-labeledNGF the counts were also reduced by one-half on the axotomized side. NGF appears to arrive at the superior cervical ganglia after retro-

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grade transport in a largely intact form. SDS gel electrophoresis 16 hours after NGF injection shows that labeled NGF migrates coincidentally with marker NGF. It is not known whether NGF is transported in a free form within the axoplasm or whether it is transported within a vesicle. However, fractionation of the ganglia at 16 hours reveals 18% of the NGF associated with the microsomal fraction and 36%associated with the nuclear fraction. In contrast, at 2 hours, a time at which NGF reaching the ganglion must do so independently of retrograde transport, 72% of the NGF is in the supernatant fraction and only 3% is in the microsomal fraction (Stoeckel et al., 1976). The evidence that NGF enters the axoplasm and is transported in a retrograde fashion is strong. Supporting evidence exists that part of the stimulation of tyrosine hydroxylase activity evoked by NGF occurs via axonal retrograde transport. Together these facts make a compelling argument that NGF exerts its biological activity at an intracellular site. However, it has been proposed that NGF acts on the surface membrane (Frazier et al., 1973). The evidence supporting this proposal comes from studies which show that NGF coupled to Sepharose beads is capable of displaying activity in the neurite outgrowth bioassay. The control for this experiment, which attempts to show that stimulation is not due to NGF dissociated from the Sepharose beads, has ganglia and beads in separate adjacent clots connected by media. The bead environment is thus different in the control and experimental cases. An adequate control for this experiment requires coupling of high-specific-activity labeled NGF to the Sepharose on the order of 1 mole of iodine per mole of NGF. A demonstration that dorsal root ganglion cells did not accumulate iodine would constitute adequate evidence that NGF did not leave the Sepharose phase and enter the cell. Such an experiment appears difficult to perform, necessitating coupling at the tracer level or the use of large amounts of radioactivity. This type of experiment in general has other pitfalls, most notably the fact that it is not amenable to testing by a quantitative model in which the stimulating variable is changed while the response is noted. The difficulty arises from the fact that the active growth factor or hormone is localized only at the periphery of the bead and the interaction with the cell membrane is not a simple function of either bead or cell number. Early experiments of this type with insulin have been criticized on quantitative grounds (Katzen and Vlahakes, 1973; Butcher et al., 1973). Subsequent experiments showed that solubilization of insulin from Sepharose-insulin occurred at levels sufficient to explain the stimulatory effects (Garwin and Gelehrter, 1974; Kolb et al., 1975; Davidson et d.,1973). These criti-

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cisms have never been adequately dealt with in the literature by investigators utilizing this technique. B. Glycoprotein Hormones

1. FEATURES SHARED

WITH

CHOLERA TOXIN

The glycoprotein hormones of mammals have many functional and structural characteristics in common. These hormones evoke differentiation and differentiated responses in their target tissues b y stimulating adenylate cyclase activity. They are thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and (human) chorionic gonadotropin (hCG). The response of the target tissues is quite specific; for example, the relative activity of LH in the thyroid hormone release bioassay is less than 0.1%(Wolff et

al., 1974).

These hormones consist of two dissimilar polypeptide chains held together by strong noncovalent forces. The chains have been separated and purified, and it has been possible to form artificial hybrids of the type TSH,-hCG, and hCG,-TSH,. When the hybrids are bioassayed, nearly complete activity is recovered for bioassays of psubunit-type activity. Conversely, the hybrids show little activity when assayed for a-subunit-type activity. Thus TSH,-hCG, is as effective as TSH in a T S H bioassay (Pierce et al., 1971). Separated p and a subunits lack in vivo bioactivity. Structural studies revealed that the a chains of LH and TSH had nearly identical amino acid sequences, whereas the /3 chains showed marked differences in their amino acid sequences (Liao and Pierce, 1970). Since the common feature of these hormones is cyclase stimulation and the variable feature is target specificity, it seemed reasonable that the p chains were involved in binding to the specific target organ receptors, while the a chains were involved in stimulating adenylate cyclase. On these grounds, glycoprotein hormones are similar to cholera toxin. Subsequently, it was shown that additional similarities existed. It is for this reason that glycoprotein hormones are considered in this article. At the present time it is not known whether the glycoprotein hormone or its a subunit is transported into the cell cytosol or even into the interior of the membrane as apparently happens with cholera toxin. However, we briefly consider some of the similarities between cholera toxin and glycoprotein hormones. The knowledge in this area is accumulating rapidly and has been recently reviewed (Kohn et al., 1977).

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Evidence for the simple scheme of separate functions for glycoprotein hormone subunits could not be obtained from studies of binding and adenylate cyclase stimulation using isolated bovine thyroid membranes. The p subunits of TSH and LH both bind to the TSH receptor, having 2%of the activity of TSH. Both the p subunits of TSH and LH, and LH alone, stimulated adenylate cyclase activity ranging from 2 to 8% of the stimulations achieved by TSH (Wolff et al., 1974). The investigators state that neither this binding activity nor the biological activity of the p subunits could be accounted for by TSH contamination. Either the system is more complicated than the proposed model or experimental difficulties obscure its simplicity.

2. INTERRELATIONSHIPS BETWEEN TSH, CHOLERA TOXIN, AND TETANUSTOXINRECEPTORS

The relationship between the TSH receptor and the cholera toxin receptor has been investigated by performing studies involving the binding of lZ5I-labeledTSH to thyroid plasma membranes in the presence of varying amounts of cold cholera toxin. Additions of cholera toxin in the range of 5 x loF0M to 5 x lo-' M produce a gradual increase in the amount of tracer TSH binding. Tracer binding is competed with additions of cholera toxin greater than M , and a maximal inhibition of 40% is achieved at 2 x M (Mullin et al., 1976). Thus, although competition for receptor sites is seen at high cholera toxin concentrations, cooperativity must be invoked to explain the enhancement of tracer binding at low concentrations. Further insights into this process were achieved by studying the inhibition of TSH binding by various gangliosides. The most potent inhibitor of TSH binding was GGnSSLC. It should be remembered from the previous section that this ganglioside is the most potent inhibitor of tetanus toxin binding to its receptor. The ganglioside GGnSLC was 10-fold less effective in inhibiting TSH binding. A similar situation exists with tetanus toxin. The latter ganglioside, however, is a potent inhibitor of cholera toxin binding. These gangliosides inhibit binding by complexing with the toxin and invoking conformational changes. A similar conformational change in TSH has been noted upon binding to GGnSSLC (Kohn et al., 1977). It is believed that the interaction between gangliosides and these glycoproteins reflects to some degree the interaction of the glycoproteins on the cell membrane with gangliosides or with glycoproteins having a similar structure. Thus TSH appears to have two types of ganglioside receptors differing in affinity by approximately a factor of 10. The lower-affinity receptor for TSH

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and the receptor for cholera toxin share the same site. The failure of cholera toxin to produce more than 40% inhibition of TSH binding to thyroid membranes could be explained by two separate TSH receptors, only one of which reacts with cholera toxin. Direct evidence that GGnSLC is the receptor for cholera toxin in thyroid plasma membranes has been obtained by Mullin and coworkers (1976). These investigators oxidized the terminal galactose residues in thyroid plasma membranes with galactose oxidase. Labeling of the exposed galactose residues was achieved by incubating the membranes with borotritide. Thyroid plasma membranes reacted in this fashion showed heavy labeling of GGnSLC. Other gangliosides were labeled to a minor extent. Thyroid plasma membranes containing bound cholera toxin were similarly treated with borotritide. GGnSLC labeling was markedly reduced under these conditions. However, an unexpected finding was the increased labeling of four other ganglioside components, among them SGnSSLC. The presence of bound cholera toxin enhanced the reactivity of these gangliosides. Mullin and co-workers consider this result a direct demonstration of the ability of cholera toxin when bound to its receptor to induce a conformational change within the plasma membrane. This conformational change exposes other gangliosides, notably SGnSSLC, which has the highest affinity for TSH. It appears that this experiment is a direct demonstration of the cooperative phenomena seen in binding studies with tracer TSH, cold cholera toxin, and the thyroid plasma membrane. Tetanus toxin shows the same relative binding affinities toward GGnSSLC and SGnSSLC as TSH. Ledley and co-workers (Ledley et aZ., 1977) showed that cold tetanus toxin competes with 1251-labeled TSH for receptors on thyroid plasma membranes. And conversely, cold TSH competes for 1251-labeledtetanus toxin on thyroid membranes. Thus it appears that TSH and tetanus toxin share the same receptor. The same receptor appears to be utilized by interferon (Kohn ,-' d., 1976). The relationship between the receptors defined by the ling studies and the functional activity of these proteins remains de established. It is possible that only a small proportion of the receptors produces biological activity, and that these have a more complex structure. A glycoprotein has been isolated from thyroid membranes which binds TSH (Meldolesi et al., 1977). The interrelationship between this glycoprotein, GGnSSLC, and cyclase stimulation has not yet been determined (Kohn et al., 1977). It will be of considerable interest to see if TSH can block the neurotoxicity of tetanus toxin. Kohn and co-workers (1977) have speculated that the tachycardia, la~

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bile blood pressure, and pyrexia seen in tetanus intoxication treated with curarization and positive pressure ventilation may represent thyroid storm. The previously accepted interpretation is that these symptoms are the result of inhibition of acetylcholine release within the autonomic nervous system.

3. DIFFERENCESIN TSH,

CHOLERA, AND

TETANUS TOXIN

Although TSH and tetanus toxin share the same receptors and one of these is shared by cholera toxin, the functional similarities appear to cease at this level. Cholera toxin stimulates adenylate cyclase, a stimulation requiring NAD. This was recently demonstrated in thyroid plasma membranes (Kohn et al., 1977). However, TSH does not require NAD for tlie stimulation of adenylate cyclase. In fact, NAD inhibits this stimulation. There are no reports in the literature of tetanus toxin stimulating adenylate cyclase activity. The available evidence indicates that tetanus toxin functions by inhibiting neurotransmitter release, particularly the inhibitory neurotransmitters, and that this function is accomplished within the cell cytosol (see Section 11,C). A characteristic of these toxins, which are known to have an intracellular site of action and which are transported across the membrane by a receptor-mediated process, is a dose-dependent lag period which reaches a minimum value at a saturating dose of toxin. Cholera toxin, which may penetrate only to the interior of the membrane, also has a lag period which does not appear to b e dose-dependent. The effect of TSH on nonconfluent thyroid cells in tissue culture does not exhibit an observable lag period. The addition of TSH to this system causes an increase in the mitotic index and an increase in the rate of thymidine incorporation into DNA. Thymidine incorporation is linear over a 6-hour period in untreated cells. Treatment of cells increases the slope of incorporation with time, and the extrapolated line passes through the zero-time origin (Winand and Kohn, 1975). C. Lactogenic Hormones Prolactin has been found in the milk of a variety of mammals by radioimmunoassay (McMurty and Malven, 1974). The concentrations vary between 50 and 500 ng/ml. These concentrations are equal to or somewhat higher than the concentrations found in serum. Since prolactin is synthesized only in the pituitary gland, it must cross both the vascular endothelium and the mammary gland epithelium to gain

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entry to the milk. Grosvenor and Whitworth (1976) have reported that, during an intravenous infusion of prolactin into lactating rats, the prolactin concentration in the milk rose and reached a steady-state value of 250 ng/ml. After termination of the infusion, plasma prolactin levels fell rapidly to the preinfusion level, but the milk prolactin level fell more slowly. Nolin and Witorsch (1976) have reported the presence of prolactin within the alveolar cells in milk-containing ducts of lactating rats, using histological techniques dependent on a reaction involving antiprolactin antibodies and a second chromogenic antibody. These two studies indicate that prolactin can enter mammary cells from the circulation and be transported across these cells into the milk ducts. Although prolactin receptors in mammary tissues have been documented, it is not known whether or not prolactin transport across the gland is a receptor-mediated process (McMurty and Malven, 1974). Bioassays of radioimmunoassayable milk prolactin have not been reported. D. Insulin

Insulin has many diverse actions on cells. Some actions, such as stimulation of glucose transport, can be explained by a direct effect on the membrane subsequent to receptor binding. Other actions such as inhibition of protein degradation and stimulation of DNA synthesis are intracellular events and require either a second messenger or the entry into the cell of active insulin or an active insulin fragment. Since a second messenger has not been convincingly demonstrated, the latter alternative has been proposed (Goldfine, 1977; Steiner, 1977). Cellular uptake of insulin has been demonstrated in cultured lymphocytes, and 10% of the uptake is localized to receptors found in the nuclear fraction. Uptake by this fraction is a saturable process and displays a slower time course than surface membrane uptake (Goldfine et al., 1977a). In an experiment of this type it is usually difficult to exclude an alternative interpretation-surface membrane contamination of the nuclear fraction. However, a major difference between nuclear receptors and surface membrane receptors has been reported. Surface membrane receptors avidly bind an antiinsulin antibody (isolated from an insulin-resistant patient), while nuclear receptors exhibit little affinity for the antibody (Goldfine et d., 1977b). At present the physiological significance of intracellular insulin is unknown, and a large fraction of the internalized insulin may constitute a degradative pathway (Terris and Steiner, 1976).

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E. Epidermal Growth Factor

Epidermal growth factor (EGF), a MOO-dalton mitogenic peptide is also bound to high-affinity receptors, internalized, and then degraded. However, as is the case with insulin, it is not known whether internalization, with or without degradation, is required to elicit a mitogenic response (Carpenter and Cohen, 1976). Most of the internalized EGF appears to be degraded within the lysosome, since degradation is blocked by chloroquine. Degradation is also blocked by inhibitors of metabolic energy production, various local anesthetics and, interestingly, ammonium ion (see Section 11,A74).When EGF is bound to fibroblasts at 4"C, followed by rapid warming to 37"C, the kinetics of entry can be followed by sequentially incubating cells with '251-labeledanti-EGF. The t1,2of entry is about 2 minutes, and no surface EGF accessible to antibody is found at 8 minutes (Carpenter and Cohen, 1976). Anderson et d.(1977a) point out that the kinetics of entry of human EGF into human fibroblasts are strikingly similar to those observed for LDL. These workers speculate that EGF may also enter via clustered receptors in coated regions (see Section 111,C73). IX.

SUMMARY

A. intracellular localization Following Transport

We have reviewed the literature on several proteins which are transported from the extracellular environment to the cell interior by receptor-mediated processes. These proteins have different functional activities and different intracellular sites following transport. The proteins we have considered can be classed into various groups. Thus there are toxins, exemplified by diphtheria toxin, abrin and ricin, tetanus toxin, botulinum toxin, and cholera toxin. In addition there is the bactericidal toxin colicin. Growth factors and hormones represent a second group, and among these are NGF, glycoprotein hormones, and lactogenic hormones. TC 11, transferrin, and LDL are grouped as carrier proteins. In addition, we have considered antibodies, desialylated glycoproteins, lysosomal hydrolases, and viruses. The data on receptor-mediated protein transport can also be organized with respect to the intracellular localization of physiologically active proteins following transport. Thus diphtheria toxin, abrin and

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ricin, colicins, and some viruses are known to be physiologically active within the cytosol following transport. Other proteins are known to be active at intracellular sites. However, the specific intracellular site of action has not been determined. The site of action is believed to be extralysosomal for some of these proteins, since they are transported by retrograde axonal transport long distances away from their site of uptake and survive destruction during this process. NGF, tetanus toxin, and botulinum toxin fit into this group. Transferrin is transported into the cell and then out of the cell, without being structurally or functionally altered (except for the loss of iron), and the presumption is that the intracellular site must b e extralysosomal. The interior of the plasma membrane may be the site of action of cholera toxin, or this protein may be transported to the cytosol. Glycoprotein hormones appear to be similar to cholera toxin in some respects. Proteins transported to the lysosomes are LDL, TC 11, fibroblast lysosomal hydrolases, and desialylated glycoproteins. Some viruses are also transported into lysosomal vesicles but subsequently lyse these vesicles and escape into the cytoplasm. Finally, there is a group of proteins which are transported across the cell to another compartment. Prolactin and antibodies are both transported across mammary gland cells into the milk. Tetanus toxin appears to be transported across the presynaptic junction. Transferrin crosses the cell membrane twice and is the only reported example of bidirectionality among the proteins discussed here. The state of the proteins following transport into the cell is largely unknown, but in two cases proteins are complexed to intracellular binding proteins (TC I1 and transferrin). 8. Mechanisms of Transport

The biochemical mechanisms of transport for these proteins are largely unknown. Intuition tells us that a very large energy barrier must be exceeded to pull a large hydrophilic protein across a hydrophobic lipid bilayer. Therefore specialized processes to lower this energy barrier are expected. Receptor-mediated pinocytosis is such a process and has been postulated for many of the proteins covered in this article. This model is particularly attractive for cases in which the protein is known to be localized and then degraded in the lysosomes, since considerable evidence exists that various types of pinocytotic vesicles fuse with lysosomes (Steinman et al., 1974). The model is pictorially represented in Fig. 1, steps 1 through 4a. The receptors serve to concentrate the protein on the surface membrane so that the uptake is higher than that achievable by bulk fluid pinocytosis. Uptake pro-

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ceeds as new receptors are supplied to the surface membrane. Proteins which gain entry to the cytosol to elicit their effects, such as the toxins described in Section 11, or proteins which escape degradation, such as transferrin, require a second transport process to escape from the lysosomal compartment. This is pictorially represented in Fig. 1, step 4b. No supporting evidence for this latter model exists. Sufficient data to evaluate the receptor-mediated pinocytotic model are available for five systems: asialoglycoprotein uptake by liver, TC I1 uptake by liver and leukemic cells, LDL uptake by human fibroblasts, and E G F uptake by human fibroblasts. In each case uptake is too rapid for a model having an even distribution of membrane receptors. For these cases the model must be modified to include clustering of receptors at pinocytotic sites, as shown in Fig. 2. Very convincing evidence for this model has been obtained b y Anderson et al. (1977a) (Section III,C,3) for LDL. The model as drawn requires a second receptor protein site which serves to cluster the receptors in the coated regions, a condition required by genetic data. Two types of protein transport models not involving pinocytosis are shown in Fig. 3. Support for nonpinocytotic models comes from colicin data (Section 11,F,3), which indicate that proteins can share transport systems designed for low-MW substances such as chelated iron. The upper part of Fig. 3 shows a model in which any substance bound to the receptor enters the cell as the receptor is pulled in b y some mechanism. This is a single-interaction model, and binding is a sufficient condition for entry. In the lower part of Fig. 3 the act of binding opens a gate through which the bound protein, or a fragment of the bound protein, passes, but not the receptor. This may be a single-interaction model, the receptor undergoing a flip-flop motion through the gate, leaving the protein in the intracellular compartment. Alternatively, two interactions may be required, one with the receptor and the other with the gate. The second interaction may be negative; i.e., only proteins of a limited size or configuration can pass through the gate. Or the interaction may be positive and the gate may actually be a shuttle mechanism, accepting the protein from the receptor. No evidence for these models exists. They are presented merely as the most general types of testable models we could devise. At present, however, sufficient data to test these models are not available. As we have indicated, tests of nonpinocytotic models which provide entry to the cytosol compartment are complicated by high levels of pinocytosis going to the lysosomal compartment. Therefore, as is the case with the toxins, the more interesting transport process producing toxicity is masked by a degradative pathway.

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1

1

transport mechanism

1

2

mechanism

mechanism

FIG.3. Top: A nonpinocytotic entry model in which any substance bound to the receptor enters the cell as the receptor is pulled in by some mechanism. This is a single-interaction model; binding is a sufficient condition for entry. Bottom: The act of binding opens a gate through which the bound protein, or a fragment of the bound protein, passes, but not the receptor. This could be a single-interaction model, the receptor undergoing a flip-flop motion through the gate, leaving the protein in the intracellular compartment. Alternatively, two interactions may be required, one with the receptor and the second with the gate. Transport models involving gates have been proposed to explain the active transport of small ions (Shamoo and Goldstein, 1977).

Finally, it should be realized that there are non-receptor-mediated transport processes for high-MW polymers across epithelial surfaces. These processes are nonsaturable. The transport of polymers higher than 50,000 daltons has been observed. The amount of transport is inversely related to size and is bidirectional, and a linear relationship between log MW and log clearance has been observed (Loehry et al., 1970). A distribution of pore sizes, possibly in epithelial tight junctions, has been proposed to explain these findings (Smulders and Wright, 1971). Studies with inhibitors have provided some data for and against various transport models. Inhibitors of oxidative phosphorylation and glycolysis inhibit the transport of many of these proteins into cells. For diphtheria toxin several specific inhibitors have been found. Ammonium ion and poly-L-ornithine both inhibit transport. This raises the question whether or not this toxin shares an amine or polyamine transport system. Ruthenium red is also an inhibitor of diphtheria

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toxin transport, which suggests that calcium-magnesium-ATPase may b e involved. Cytochalasin B depresses the transport of many of these proteins, but the significance of this is not understood. It appears, however, that contractile proteins very similar to muscle actin and myosin are localized beneath the cell membrane in unique arrays, and that these arrays effect membrane receptor distribution (Ash and Singer, 1976).Whether or not these contractile proteins play a role in receptor-mediated entry processes remains to be determined. The most detailed data on protein transport across cell membranes exist for colicin transport systems which are shared by iron transport, vitamin Bl2 transport, and certain bacteriophage transport. Most of these data have been derived from genetic manipulations. It is known that at least two gene products are involved, one of which is the receptor for the specific colicin. The second gene product affects not only the specific colicin transport but a group of transport processes also. It is likely that an understanding of receptor-mediated transport in eukaryotic cells will require a genetic approach. C. Unique Functions of Receptor-Mediated Protein Transport

The requirement for an obligatory binding step to a cell surface receptor preceding transport achieves a high degree of cell type specificity for the transport process. Cells lacking the specific receptor are incapable of transporting the protein to the intracellular site. Thus asialoglycoproteins are removed from the bloodstream almost exclusively b y the liver. The uptake of NGF is largely limited to sympathetic neurons. It has been postulated in the latter case that trophic phenomena may also occur. Thus local release of NGF may stimulate neurite outgrowth along particular paths, and this process may function to establish unique neuronal connections. The degree of cell type specificity achieved by some receptor-mediated transport processes is enormous. Certain eukaryotic cells which are insensitive to diphtheria toxin are over 10,000times more insensitive than sensitive cells. This sensitivity involves the receptor-mediated transport process, since the active fragment of the toxin is totally active in broken cell preparations. The high degree of selectivity may involve other mechanisms besides transport, such as protection of the active chain from degradation. A second unique characteristic of receptor-mediated protein transport is the specificity of intracellular compartmentalization achieved. Thus asialoglycoproteins are degraded within the lysosomes, whereas transferrin escapes this fate and is transported out of the cell to func-

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tion again. NGF, after crossing the plasma membrane, is transported in a retrograde fashion and finally localized in the nucleus of the sympathetic neuron. Other proteins, notably toxins, become localized in the cell cytosol. The biochemical mechanism of intracellular compartmentalization is unknown. Few data are available to indicate whether this process is directed by the specific receptors involved or whether it is a function of a portion of the transported molecule. The specificity for compartmentalization appears to be localized to only a portion of either the transported molecule or the receptor. Rogers and Kornfield (1971) synthesized hybrid molecules of fetuin and albumin and fetuin and lysozyme. Lysozyme and albumin are not normally taken up by the liver and degraded in hepatic lysosomes. However, following coupling with fetuin and treatment with neuraminidase to remove the sialic acid residues, the hybrid molecule is processed similarly to desialylated fetuin, and the lysozyme and albumin components are degraded within the hepatic lysosomes. The remarkable similarities in the structure of diphtheria toxin, abrin, ricin, tetanus toxin, and botulinum toxin suggest that the common structural features are in some way necessary for the function of these toxins. The functions appear quite diverse, however, cytosol localization may be a common feature. A puzzling feature of toxins is how these organisms evolved and then maintained biochemical processes so highly integrated.with those of eukaryotic cells (Pappenheimer and Gill, 1973: Collier, 1975). Since a number of the toxin genes are phage-specific, evolvement could have come about by way of genetic transfer in either direction. Maintenance implies a selective advantage for both organisms. For the plant seed toxins abrin and ricin an obvious selective advantage exists, since oral ingestion of the seeds leads to death. For bacterial toxins, the death of a host under certain conditions may be a selective advantage. It is interesting to note that diphtheria toxin, botulinum toxin, and tetanus toxin genes are all regulated by the media iron content. Toxins are made in high quantity only under conditions of limiting iron. Iron is probably quite limiting for nondestructive bacteria within a vertebrate host, because of the high binding constant with which iron binds transferrin. The liberation of toxin leading to the destruction of a host places the bacterial population in a high-iron evironment as the decomposition of host iron proteins proceeds. The utility for a vertebrate organism transporting these toxins to their intracellular site of action is difficult to perceive. However, just as colicins utilize transport systems for metabolites, the same may be

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true for these toxin transport systems. Since these are low-capacity transport systems, they may have escaped detection, especially if they are involved in trace-metal transport. Another possibility is that toxin transport systems were originally derived for peptide and protein transport similar to that for NGF. There are probably many such systems in existence which have escaped detection because of the low levels of these circulating materials. X. PHARMACOLOGICAL IMPLICATIONS OF RECEPTOR-MEDIATED PROTEIN TRANSPORT

The unique feature of receptor-mediated protein transport is that certain proteins are fixed by specific cell types and then transported to a specific intracellular site where the protein (or a fragment of the protein) exerts its biological effects. These effects can be quite profound-inhibition of protein synthesis for diphtheria toxin, abrin, and ricin, and stimulation of adenylate cyclase activity for cholera toxin. The evidence previously summarized indicates that NGF, botulinum toxin, and tetanus toxin also act intracellularly. NGF induces two key enzymes; botulinum toxin and tetanus toxin inhibit the release of stimulatory and inhibitory neurotransmitters, respectively. We have suggested (Chang and Neville, 1977), that the phenomena associated with receptor-mediated protein transport can be exploited to achieve the synthesis of an entirely new class of pharmacological reagents which would exhibit cell type-specific activities. This could be achieved by constructing hybrid- proteins, utilizing the binding chain of one protein (or the more specific receptor recognition factor when known) and the active chain of a different protein. An obvious application of such hybrids is cell type-specific cancer chemotherapy. For example, if the binding portion of NGF were hybridized with the active chain of diphtheria toxin, the resulting reagent would inhibit protein synthesis specifically in sympathetic neurons or related cell types. Such a reagent might be effective in destroying tumors of sympathetic neuronal origin, such as neuroblastomas. Metastatic lesions would be equally susceptible, as long as they carried NGF. Hybrids containing binding chains directed at tumor-specific antigens may also be effective as tumor-specific reagents. Hybrid protein reagents may have considerable application in the mental health field. It is quite likely that the high degree of differen-

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tiation exhibited by the nervous system is mirrored in receptor differences in various groupings of neural nets and nuclei within the central nervous system. By combining the active chain of tetanus toxin with the right binding chain it might be possible to depress inhibitory neurotransmitter release in particular portions of the brain. Similarly, by using the active fragment of cholera toxin, localized stimulation of adenylate cyclase might be achieved in a distribution independent of the receptors normally linked to this enzyme. Many other possibilities also exist. In order to achieve these ends, considerably more must be known about the interrelationships between receptor binding and protein transport. Do all receptors at some low level mediate entry to the cytosol compartment? Or does only a certain class of receptors with unique properties have this function? Is entry of the protein or its active chain determined uniquely by the binding chain, or does the active chain require certain properties essential for entry? The available data summarized in this article shed little light on these questions. As an initial attempt to answer some of these questions, we have devised a general method for the synthesis of disulfide-linked artificial protein hybrids in high yield (Chang and Neville, 1977; Chang et al., 1977). The method involves the reaction of an exogenously introduced alkyl thiosulfate group on one protein with a sulfhydryl group on another. This reaction is more rapid than any intraspecies competing reactions and avoids gross contamination with homopolymers, which usually occurs with conventional bifunctional cross-linking reagents (Chang and Neville, 1977). By utilizing this methodology, the hybrid human placental lactogen-SS-diphtheria toxin fragment A has been synthesized. The hybrid maintains 26% of its binding activity toward lactogenic receptors and one-third of its toxin A enzymic activity as assayed in a cell-free system. However, the hybrid is without detectable effect on the protein synthetic rate of organ-cultured lactating mammary gland explants carrying the lactogenic receptor (Chang et aZ., 1977). Although the artificial protein hybrid we have synthesized is a structural analog of diphtheria toxin with an altered binding chain specificity, it does not behave as a functional analog. The reasons for this are not yet apparent, We do not know whether or not the lactogenic receptor mediates entry to the cell cytosol where the A chain must localize to be functional. Prolactin transport into milk may occur via another intracellular compartment. Our disulfide-linked hybrid may split at the membrane binding site because of lack of protection of

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the disulfide bond, which is provided in diphtheria toxin by the diphtheria toxin-binding chain. The toxin A fragment may be inactivated in the artificial hybrid, whereas protection of the active site is probably provided by the B chain in the native toxin. Olsnes et al. (1974) constructed functional hybrids of abrin and ricin A and B chains. Although these molecules are closely related in binding and enzymic properties, they are nevertheless immunologically distinct. Further work will be required to understand the range of possibilities and limitations existing for the synthesis of functional protein hybrids which utilize receptor-mediated transport processes. REFERENCES Aasa, R., Malmstrom, B. G., Saltman, P., and VanngBrd, T. (1963).The specific binding of iron(II1) and copper(I1) to transferrin and conalbumin. Biochim. Biophys. Acta 75,203-222. Adams, E. B. (1971). The clinical effects of tetanus. In “Neuropoisons; Their Pathophysiological Actions” (L. L. Simpson, ed.), Vol. 1, pp. 213-224. Plenum, New York. Anderson, R. G. W., Goldstein, J. L., and Brown, M. S. (1976). Localization of lowdensity lipoprotein receptors on plasma membrane of normal human fibroblasts and their absence in cells from a familial hypercholesterolemia homozygote. Proc. Natl. Acad. Sci. U.S.A.73, 2434-2438. Anderson, R. G. W., Brown, M., and Goldstein, J. L. (1977a).Role of the coated endocytotic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts. Cell 10, 351-364. Anderson, R. G. W., Goldstein, J. L., and Brown, M. S. (1977b). A mutation that impairs the ability of lipoprotein receptors to localize in coated pits on the cell surface of human fibroblasts. Nature (London) 270, 695-699. Ash, J. F., and Singer, S. J. (1976). Concanavalin-A-induced transmembrane linkage of concanavalin A surface receptors to intracellular myosin containing filaments. Proc. N n t l . Acad. Sci. U.S.A.73,4575-4579. Ashwell, G., and Morell, A. G. (1974). The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adu. Enzymol. Relat Areas M o l . Biol. 41,99-128. Ashwell, G., and Morell, A. G. (1977). Membrane glycoproteins and recognition phenomena. Trends Biochem. Sci. 2, 76-78. Awai, M., and Brown, E. B. (1963). Studies of the metabolism of 1131-labeledhuman transferrin. /. Lab. Clin. Med. 61, 363-396. Baker, M. E. (1975).Molecular weight and structure of 7 S nerve growth factor protein. /. B i d . Chem. 250, 1714-1717. Banejee, S. P., Snyder, S. H., Cuatrecasas, P., and Greene, L. A. (1973). Binding of nerve growth factor receptor in sympathetic ganglia. Proc. Natl. Acad. Sci. U.S.A. 70,2519-2523. Banejee, S. P., Cuatrecasas, P., and Snyder, S. H. (1975).Nerve growth factor receptor binding. Influence of enzymes, ions and protein reagents. 1. Biol. Chem. 250, 1427-1433.

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Fiedler-Nagy, C., Rowley, G. R., Coffey, J. W., and Miller, 0. N. (1975). Binding of vitamin B,,-rat transcobalamin I1 and free vitamin BIZto plasma membranes isolated from rat liver. Br. J. Hematol. 31,311-321. Fielding, J,, and Speyer, B. E. (1974). Iron transport intermediates in human reticulocytes and the membrane binding site of iron-transferrin. Biochim. Biophys. Acta 363,387-396. Fillenz, M., Gagnon, C., Stoeckel, K., and Thoenen, H. (1976). Selective uptake and retrograde axonal transport of dopamine-P-hydroxylase antibodies in peripheral adrenergic neurons. Brain Res. 114,293-303. Finkelstein, R. A. (1973). Cholera. Crit. Rev. Microbiol. 2,553-623. Finkelstein, R. A. (1975). Observations on the cholera enterotoxin (choleragen).Jpn. J. Med. Sci. Biol. 28, 76-78. Finkelstein, R. A., Boesman, M., Neoh, S. H., LaRue, M. K., and Delaney, R. (1974). Dissociation and recombination of the subunits of the cholera enterotoxin (choleragen).J. Zmmunol. 113, 145-150. Frazier, W. A., Anyeletti, R. H., and Bradshaw, R. A. (1972). Nerve growth factor and insulin. Structural similarities indicate an evolutionary relationship reflected by physiological action. Science 176,482-488. Frazier, W. A,, Boyd, L. F., and Bradshaw, R. A. (1973). Interaction of nerve growth factor with surface membranes: Biological competence of insolubilized nerve growth factor. Proc Natl. Acad. Sci. U.S.A. 70,2931-2935. Gavin, J. R., 111, Roth, J., Neville, D. M., Jr., De Meyts, P., and Buell, D. N. (1974). Insulin-dependent regulation of insulin receptor concentrations: A direct demonstration in cell culture. Proc. Natl. Acad Sci. U.S.A. 71, 84-88. Gamin, J. L., and Gelehrter, T. D. (1974). Induction of tyrosine amino-transferase by Sepharose-insulin.Arch. Biochem. Biophys. 164,52-59. Gill, D. M., and Dinius, L. L. (1971). Observations on the structure of diphtheria toxin. J. Biol. Chem. 246,1485-1491. Gill, D. M., and King, C. (1975). The mechanism of action of cholera toxin in pigeon erythrocyte 1ysates.J. Biol. Chem. 250,6424-6432. Gitlin, J. D., and Gitlin, D. (1974). Protein binding by specific receptors on human placenta, murine placenta, and suckling murine intestine in relation to protein transport across these tissues.J. Clin. Invest. 54, 1155-1166. Gitlin, J. D., Gitlin, J. I., and Gitlin, D. (1976). Selective transfer of plasma proteins across mammary gland in lactating mouse. Am. J. Physiol. 230, 1594-1602. Click, J. M., Kerr, S. J., Gold, A. M., and Shemin, D. (1972). Multiple forms of colicin E 3 from Escherichia coli. Biochemistry 11, 1183-1188. Goldfine, I. D. (1977). Does insulin need a second messenger? Diabetes 26, 148-155. Goldfine, I. D., Smith, G. J., Wong, K. Y.,and Jones, A. L. (1977a).Cellular uptake and nuclear binding of insulin in human cultured lymphocytes: Evidence for potential intracellular sites of insulin action. Proc. Natl. Acad. Sci. U.S.A. 74, 1368-1372. Goldfine, I. D., Vigneri, R.,Cohen, D., Pliam, N. B., and Kahn, C. R. (1977b). Evidence that intracellular binding sites for insulin are immunologically distinct from those on the plasma membrane. Nature (London) 269,698-700. Goldstein, J. L., and Brown, M. S. (1976). The LDL pathway in human fibroblasts: A receptor-mediated mechanism for the regulation of cholesterol metabolism. Curr. Top. Cell. Regul. 11, 147-181. Goldstein, J. L., and Brown, M. S. (1977). The low density lipoprotein pathway and its relation to atherosclerosis. Annu. Rev. Biochem. 46,897-930. Goldstein, J. L., Brown, M. S., and Stone, N. J. (1977). Genetics of the LDL receptor:

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