Photoaffinity labeling of β-adrenergic receptors

Pharmac. Ther. Vol. 31, pp. 57 to 77, 1985 Printed in Great Britain. All rights reserved

0163-7258/85 $0.00+0.50 Copyright © 1986 Pergamon Journals Ltd

Specialist Subject Editor: J. FEDAN

PHOTOAFFINITY

LABELING

OF fl-ADRENERGIC

RECEPTORS

JEFFREY M. STADEL Department of Molecular Pharmacology, Smith Kline and French Laboratories, P.O. Box 7929, Philadelphia, PA 19101, U.S.A.

1. INTRODUCTION The endogenous catecholamines epinephrine and norepinephrine exert their physiological effects on target tissues through direct binding interactions with receptors. These receptors are present in the plasma membranes of responsive cells. The biological specificity and mechanisms of action of the catecholamines have been extensively studied as these compounds regulate cellular metabolism in a wide variety of tissues. The ubiquitous nature of the adrenergic responses has made this system a prototype or model for studying receptor-effector coupling (Ross and Gilman, 1980; Stadel et al., 1982a; Lefkowitz et al., 1983). Since the concept of a 'receptive substance' was originally proposed to explain the specificity of drug action (Langley, 1905), our understanding of receptors has evolved. Pharmacological studies of the metabolic effects of adrenergic agents both in vivo and in vitro provided indirect evidence for the existence of receptor moieties. The evaluation of these data on the action of several catecholamines in different organs pointed to a logical division of their effects. Ahlquist (1948) proposed two distinct types of adrenergic responses. His classification was based on the relative potency of catecholamine agonists to evoke a characteristic response in several tissues. The first type of response, which he designated 'alpha', followed the agonist potency series epinephrine > norepinephrine > isoproterenol. The second or 'beta' type response follows the order isoproterenol > epinephrine > norepinephrine. A typical ~-adrenergic response is the contraction of smooth muscle, fl-Adrenergic receptors mediate smooth muscle relaxation, inotropic and chronotropic regulation in the heart, and metabolic effects such as lipolysis. The concept of two discrete types of adrenergic responses was further supported by the development of highly potent antagonist compounds. ~t-Adrenergic responses are competitively inhibited by drugs such as phentolamine and ergot alkaloids, fl-Adrenergic responses are blocked by a different set of compounds, e.g. propranolol, alprenolol, and pindoiol. The antagonist compounds are highly specific and this characteristic along with their high potency makes these drugs particularly useful in the classification of adrenergic responses and in the direct study of adrenergic receptors (see below). Extensive studies of adrenergic pharmacology since the original definition of 0t- and fl-adrenergic responses pointed to a further division of each response into subtypes. Lands and coworkers (1967) proposed two subtypes of fl-adrenergic responses which they called fl~ and f12- This subclassification was based on the relative affinities of epinephrine and norepinephrine. These two catecholamines are approximately equipotent in evoking a fit-response, whereas fl2-adrenergic responses display a preference for epinephrine over norepinephrine. Similarly, ~-adrenergic responses have been divided into ~- and ~2-subtypes (Bethelsen and Pettinger, 1977; Hoffman and Lefkowitz, 1980). 0t-Adrenergic responses are differentiated by the relative potency of subtype selective antagonists. For example, ~j-responses are potently inhibited by prazosin, whereas ~t2-type responses are selectively blocked by a different antagonist, yohimbine. 57

58

J.M. STADEL

The responses to a- and f-adrenergic agonists also differ in the biochemical events necessary to produce the physiological changes that accompany adrenergic stimulation. f-Adrenergic receptors are directly linked to the activation of adenylate cyclase in the plasma membranes of target cells (Robison et aL, 1971). The cyclase catalyzes the conversion of ATP to the second messenger, adenosine 3',5' cyclic monophosphate (cAMP), which appears to mediate the intracellular effects of f-agonists. This is true of the responses evoked through either the fit- or f2-receptor subtype. The biochemistry of ~-adrenergic responses, on the other hand, is not so well defined. Increased hydrolysis of membrane phosphatidylinositol and alterations in calcium fluxes have been implicated as mediating cq-type responses (Exton, 1979). The ct2-receptor is also coupled to adenylate cyclase, however, agonist occupancy of this receptor results in inhibition of the enzyme's activity (Jakobs et al., 1976). Because f-adrenergic stimulation results in the modulation of physiological processes in a wide variety of tissues, and because f-receptors are closely coupled to a well-defined effector system, i.e. adenylate cyclase, the f-adrenergic receptor complex has become the premier model system in which to study transmembrane signaling. During the past decade, much progress has been made in understanding the molecular mechanism of f-adrenergic stimulation of adenylate cyclase (Ross and Gilman, 1980; Stadel et al., 1982a). In order to define the molecular events of receptor-effector coupling, it is necessary to biochemically characterize the individual components. Recent advances in receptor technology have provided new methods to assess the structure of f-receptors. One of the tools primarily responsible for these advances is photoaffinity probes. The development and application of photoaffinity labels to help define the structure and regulation of f-adrenergic receptors is the subject of the present review. 2. DEVELOPMENT OF PHOTOAFFINITY LABELS FOR f - A D R E N E R G I C RECEPTORS The investigation of receptors attained a new level of sophistication with the development of radioligand binding techniques. The consequences of these new innovations were very significant. First, by having a direct-assay method, the perception of receptors changed from a pharmacological concept to one of discrete physical entities. Second, the development of a reliable assay allowed a more biochemical approach to the characterization of receptors. Radioligand binding techniques were first applied to peptide hormones such as ACTH (Lefkowitz et al., 1970) and angiotensin (Lyn and Goodfriend, 1970), where it was shown that radionuclides could be covalently incorporated into the structure of the polypeptides with little or no alteration in the biological activity or specificity of the hormones. In 1974, radioligands specific for the fl-adrenergic receptor were first reported. These first radioligands were based on antagonist structures because of their high affinity and specificity. Three radioligands for the/3-adrenergic receptor were developed concurrently. These were ( - ) [3H]dihydroalprenolol (Lefkowitz et al., 1974), ( + / - )[3H]propranolol (Atlas et al., 1974), and ( + / - ) [125I]-hydroxybenzylpindolol (Aurbach et al., 1974). More recently, a number of additional radioligands for the fl-receptor have been synthesized including two widely-used antagonist ligands, ( - ) [t25I]cyanopindolol (Engle et al., 1981) and ( - ) [125I]pindolol (Barovsky and Brooker, 1980), as well as an agonist radioligand ( + / - ) [3H]hydroxybenzylisoproterenol (Lefkowitz and Williams, 1977). For each of the radioligands, the binding specificity, stereoselectivity, and saturability were all as expected for binding to physiologically relevant fl-adrenergic receptors. To characterize receptors, radioligands have been used in two ways. In 'saturation' type experiments, it is possible to determine the affinity of the receptor for the radioligand and the total number of receptor sites in the biological preparation. These experiments are valuable in identifying specific receptor binding sites, in determining subcellular localization of receptors, and in evaluating changes in receptor density that may accompany physiological and patho-physiological regulation (Stiles et al., 1984b). In an alternative

fl-Adrenergicreceptors

59

approach, the radioligand concentration is held constant while determining the dosedependent inhibition of radioligand binding by increasing concentrations of unlabeled compounds. These 'competition' binding studies provide information concerning the pharmacological binding properties of receptors for a wide spectrum of drugs and hormones. Radioligand binding techniques have been instrumental in the elucidation of the molecular mechanisms of receptor-effector coupling and also have provided the assay necessary for purification of the fl-adrenergic receptor (Lefkowitz et al., 1983). Radioligand binding studies have confirmed and extended the concept of receptor subtypes. The current radiolabeled antagonist ligands for fl-adrenergic receptors appear to have equal affinity for both fl-receptor subtypes. By utilizing these radioligands in conjunction with unlabeled subtype selective drugs, it is possible to characterize receptor subtypes in competition binding experiments. Detailed analysis of competition binding data by computer-assisted non-linear least-squares curve fitting procedures permits the quantification of receptor subtypes in various tissues (Hancock et al., 1980). These binding experiments and subsequent data analyses support pharmacological data which show that both ill- and fl2-receptor subtypes often coexist in a single tissue. To understand better the action of hormones and drugs at the molecular level, it will be necessary to characterize the structure and biochemical properties of receptors. The development of radioligand binding techniques has made possible more biochemical approaches to the study of receptors. Two approaches that have provided data concerning the structure of the fl-adrenergic receptor are photoaffinity labeling and purification. These two techniques are complimentary and when applied together they reinforce structural conclusions concerning a specific receptor. The synthesis of photoaffinity labels for the fl-adrenergic receptor is a logical next step following the development and validation of radioligand binding techniques. The utility of such labeling reagents can be judged by several criteria. The compound must: (1) possess high affinity for the binding site to be labeled; (2) contain in its structure a 'recorder' moiety such as a high-specific activity radionuclide; and (3) possess a reactive group that can readily be covalently incorporated into the receptor of interest. To obtain high affinity and selectivity, fl-adrenergic photoaffinity labels have again been based on the structures of antagonist drugs. As has been shown for reversible radioligands, it is possible to incorporate 3H or t25I into the photoaffinity reagent without perturbing its biological activity. In most cases, the fl-adrenergic antagonist has been modified to contain an arylazide moiety that upon irradiation forms a highly reactive nitrene intermediate which can insert covalently into the receptor. The advantages of photoaffinity labels over conventional affinity labeling techniques, the experimental application of photoaffinity reagents, and the chemistry of the photolysis reactions have all been reviewed elsewhere (Knowles, 1972; Bayley and Knowles, 1977) and will not be discussed here. A number of photoaffinity probes for fl-adrenergic receptors have been synthesized and are listed in Table l, but not all of them meet the criteria listed above. One of the first attempts to photoaffinity label the fl-adrenergic receptor was by direct photolysis of isoproternol (Takayanagi et al., 1976). The agonist was pre-equilibrated with guinea-pig taenia prior to irradiation by a u.v. light source. This photolysis procedure resulted in a parallel shift to lower affinity of the agonist dose-response curve for adenylate cyclase activation when compared to non-irradiated controls. The effect of the photolysis on adenylate cyclase could be blocked if the antagonist propranolol was included in the incubation along with isoproterenol. These data were interpreted as evidence for receptor reserve in this tissue, however, no direct radioligand binding data was reported to support the hypothesis that isoproterenol had covalently labeled the receptor. Two photoactive derivatives of propranolol have been reported, N-(2-hydroxy-3 naphthoxypropyl)-N'(2-nitro-5-azidophenyl)ethylenediamine (NAP-propranolol) (Darfler and Martinetti, 1977) and l[l-(p-nitrophenoxy),2-methyl, 2-propylamino],3-(~naphtyloxy),2-propanol (PNP) (Gozlan et al., 1982). NAP-propranolol was shown to competitively inhibit both isoproterenol-stimulated cAMP accumulation (Ki = 19 riM) and the binding of [3H]dihydroalprenolol ([3H]DHA) to fl-adrenergic receptors (K~ = 100 nM)

60

J. M. STADEL TABLE 1. Photoaffinity Labels f o r fl-Adrenergic Receptors Structure OH

I

Apparent K o (p~)

IOCHz-CH-CHe-NIH

Compound

R~

R,

N-(2-hydroxy-3-naphthoxypropyl)N'(2-nitro-5-azidophenyl) ethylene- R, diamine [NAP-propranolol]

20,000-100,000 ,o. x

Rz =

Reference Darfler and Marinetti, 1977

_

CHz--CHI--NH~ ( ~ ) N=

l[1-(p-Nitrophenoxy), 2-methyl, 2-propylamino],3-(ct-naphtyloxy), 2-propanol [PNP]

55,000

Gozlan et al., 1982

R~ =

/

CH,-- O - ' ( ( ~ CH=

R= = - - d - I

Acebutalol-azide RI =

NOz

400,000

O II

~

Wrenn and Homcy, 1980

C\cH:

N= /CH= Rz =

15-(4'-Azidobenzyl) carazolol [pABC]

CH \ CH:

5-100

R~= H

CH=

Lavin et al., 1981 Heald et al., 1983

Xl

R2 = - C - CHz

N=

CH= X2 [=H]pABC : Xl=X2=Zfl ['leq]pABC : Xl=lSq; X2=H

p-Azidobenzylpindolol [pABP]

300-900

Rashidbaigi and Ruoho, 1981

CHJ R= = - C - C H z ~ N =

CH=

1-(4-Azidobenzimidyl-3,3dimethyl-6-hydroxy-7-., - - - - --, ,. (2-cvano-3-iodoindol-4-vloxv~-

"I

I _ ~

Burgermeister et al.. 1982

40-50

Burgermeister et al., 1982

60

Burgermeister et al., 1983

R, = Nc

1,4-diazaheptane

H

[CYP-azide]

CH, I

R= = --C-CHI--NH-C -"(( CH= NH

l-(4-Azidobenzoyl)-3,3dimethyl-6-hydroxy-7(2-cyano-3-iodoindol-4-yloxy)-

40-50

)~-N=

i R, = NC

1,4-diazaheptane

H

[CYP-azide]

CH=

.,=

CH=

1-[4-(3-Trifluoromethyldiazirino) benzoyl]-3,3-dimethyl-6-hydroxy7-(2-cyano-3-iodoindol-4yloxy)- 1,4-diazehaptane [CYP-diazirine]

_ ~ R, =

i H ell,

N= N

CH=

15-(4'-Aminobenzyl) carazolol [pAMBC]

O

0

CF

Shorr et al., 1982a

R~ = ~ _ ~ 1

CH=

i H

R,= --i--CH. ~ CH:=

I NH.

fl-Adrenergic receptors

61

in turkey erythrocytes. Although an arylazide is part of the structure of NAP-propranolol, no data on the effect of this compound after photolysis was given. PNP inhibited with a Ki = 55 nr,l the binding of [3H]DHA to membranes from C6 glioma cells. Upon photolysis of PNP, a decrease in the maximal stimulation of fl-adrenergic sensitive adenylate cyclase, as well as a decrease in the maximal number of [3H]DHA binding sites was observed. The inhibition of adenylate cyclase and receptor binding activities persisted after extensive washing following photolysis, but both effects were attenuated if alprenolol was included with PNP during the irradiation. These data are consistent with covalent blockade of the fl-adrenergic receptor by PNP but a radioactive form of the reagent has not been reported. Although PNP appeared to photoaffinity label the fl-receptor in C6 glioma cell membranes, it could not be used for structural analysis of the receptor since it did not contain a 'recorder' moiety. A number of photoaffinity reagents specific for the fl-adrenergic receptor have been synthesized and radiolabeled to a high specific activity. Acebutalol-azide behaves as a competitive antagonist for isoproterenol-stimulated adenylate cyclase activity in rat reticulocyte membranes (Kd = 0.1 #M) prior to photolysis (Wrenn and Homcy, 1980). Upon irradiation of membranes pre-incubated with acebutalol-azide, the inhibition of the cyclase remained persistent following several washes. The inhibition of the cyclase was specific for fl-receptors as no inhibition of NaF or guanyl-5'-ylimidodiphosphate (Gpp(NH)p) stimulation was observed. The effect could be stereospecifically blocked by propranolol. The same characteristics of adenylate cyclase inhibition were reported when rat liver membranes were used and an additional control showed that another adenylate cyclase-coupled cell surface receptor, the glucagon receptor, was unaffected by the photolysis experiments (Wrenn and Homcy, 1980). A tritiated form of acebutalol-azide was synthesized (8 Ci/mmoi) and used to label a partially purified preparation of fl-adrenergic receptors from canine lung (Homcy et al., 1983). The relatively low affinity of acebutalol-azide and the low specific activity of the radiolabeled form have limited the utility of this photoaffinity label. A second tritiated photoaffinity label for the fl-receptor is [3H]p-azidobenzylcarazolol ([3H]pABC) which can be radiolabeled to a specific activity of 40 Ci/mmol (Lavin et al., 1981). This compound was shown to interact directly with the fl-adrenergic receptor in radioligand binding experiments employing frog erythrocyte membranes. The binding of [3H]pABC was of high affinity ( K d = 100 pM) and displayed the correct pharmacological specificity to insure its binding to fl-receptors. Following photolysis [3H]pABC appeared to label the frog erythrocyte fl-receptor; however, the receptor preparation needed to be enriched by solubilization and affinity chromatography over alprenolol-Sepharose to obtain consistent labeling. Due to the low density of fl-adrenergic receptors in plasma membranes, photoaffinity labels with higher specific activity were necessary for these reagents to be of more general use. The photoaffinity labeling of the fl-adrenergic receptor in its plasma membrane environment is feasible if radioiodine is used as the recorder molecule. Several radioiodinated photoaffinity labels (specific activity = 2200 Ci/mmol) have been synthesized: [125I]p-azidobenzylpindolol ([12~I]ABP) (Rashidbaigi and Ruoho, 1981), [~25I]cyanopindolol-azide ([125I]CYP-azide) (Burgermeister et al., 1982), [~25I]cyanopindolol-diazirine ([125I]CYP-diazirine) (Burgermeister et al., 1983), and [~25I]p-azidobenzylcarazolol ([125I]pABC) (Lavin et aL, 1982; Heald et al., 1983). All of these photoaffinity compounds have been shown to bind specifically and with high affinity (Kd= 10-200pM) to fl-adrenergic receptors prior to photolysis. With such high affinity, it is possible to equilibrate fl-receptors with relatively low concentrations of the photoaffinity label and wash away the non-associated excess ligand before irradiating the sample and thus increase the specificity of the labeling. One additional structural probe for the fl-receptor is [125I]p-aminobenzylcarazolol (Shorr et aL, 1982a). This compound has a recorder moiety but does not contain a functional group which can form a covalent bond with the receptor when exposed to light. [t25I]p-aminobenzylcarazolol has been used successfully to label the fl-adrenergic receptor in membranes prepared from frog erythrocytes when employed with a hetero-bifunctional

62

J.M. STADEL

crosslinking reagent such as N-succinimidyl-6-(4'-azido-2'-nitro-phenylamino)hexanoate (SANAH). In these experiments, the [~2SI]p-aminobenzylcarazolol was pre-incubated with the erythrocyte membranes and the free ligand then washed away. The membranes were next exposed to SANAH to allow the activated ester portion of this crosslinking agent to react with the amino group of the radioligand. Finally, the reaction mixture was photolyzed to promote the insertion of the arylazido portion of SANAH into the fl-receptor. These experiments demonstrate the feasibility of labeling the fl-adrenergic receptor by photo-crosslinking of a high affinity radioligand. However, the efficiency of labeling appears to be better when the photo-sensitive functional group is directly incorporated into the structure of the radioligand. 3. IDENTIFICATION OF fl-ADRENERGIC RECEPTORS BY PHOTOAFFINITY LABELING The [~25I]-labeled photoaffinity reagents have been successfully used to identify fl-adrenergic receptor structures in membranes prepared from a variety of tissues and species as shown in Table 2. In each case, control experiments showed that the irradiation alone did not affect receptor binding activity. Most experiments were carried out using a medium or high pressure mercury lamp equipped with a filter to screen out wavelengths below 300 nm. Photolysis times ranged from 3 to 120 sec in order to complete the reaction. The incorporation of the photoaffinity label into the receptor presumably occurs through receptor insertion of the photo-generated nitrene intermediates except for [~25I]CYP-diazirine in which case a reactive carbene intermediate is produced. The critical criterion which allows the identification of a macromolecule containing the ligand binding site of the receptor is the ability of well-characterized agonist and antagonist ligands to inhibit the covalent labeling of the receptor. Thus, the pharmacological specificity which defines receptors in bioassays or radioligand binding studies can be applied to validate photoaffinity labeling experiments. By quantifying the amount of the photoaffinity probe incorporated into the r-receptor in the absence and presence of competing ligands, it is also possible to calculate the efficiency of incorporation of the photolabel into the receptor. For the photoaffinity labels of the fl-receptor, this efficiency is between 5 and 17%. This range of efficiency for the photolysis reaction compares favorably to that measured for other photoaffinity labels with similar functional groups (Bayley and Knowles, 1977). It is interesting to note that the two [~25I]CYP-azides which differ in structure only slightly (see Table 1), show markedly different efficiencies of incorporation into the same fl-receptor, 8.9 vs 17% (Burgermeister et al., 1982). These results indicate that the covalent reaction is sensitive to small structural differences in the two compounds and the orientation of functional groups within the ligand binding site of the receptor may be critical for efficient labeling. Photoaffinity labels for the fl-adrenergic receptor were first applied to well-characterized model systems. Adenylate cyclase coupled fl-receptors on avian and amphibian erythrocytes have been studied in detail using radioligand binding techniques and these investigations contributed to our understanding of how receptors communicate with the cyclase enzyme (Stadel et aL, 1982a; Stadel and Lefkowitz, 1983). Plasma membranes prepared from these erythrocytes were the first source of the fl-receptors to be labeled. Following the incorporation of the photolabel, the membranes were solubilized in sodium dodecyl sulfate (SDS) and the proteins separated by polyacrylamide gel electrophoresis. The proteins tagged by the photoaffinity label were subsequently detected by autoradiography. In the case of the frog erythrocyte fl-adrenergic receptor, [~25I]pABC (Lavin et al., 1982; Stadel et al., 1983b) and [~25I]ABP (Rashidbaigi and Ruoho, 1982; Ruoho et al., 1983) both labeled a single polypeptide of Mr ~- 58,000-62,000. This is the same molecular weight as that obtained when the fl-receptor from this source was purified (Shorr et al., 1981). In avian erythrocytes, two fl-receptor polypeptides were specifically photoaffinity labeled: Mr ~ 38--40,000, and Mr ~ 45-50,000 (Short et al., 1982b; Lavin et al., 1982;

/3-Adrenergic receptors

63

TABLE 2. fl-Adrenergic R e c e p t o r Polypeptides Identified by Photoaffinity Labeling Tissue Erythrocyte

Reticulocyte Lung

Species Frog

fl-Receptor subtype /32

fl-Receptor protein identified (M,) 58,000

Reference

63,000

Lavin et al., 1982 Shorr et al., 1982a Stadel et al., 1983b Rashidbaigi and Ruoho, 1982

Rat

/32

65,000; 53,000; 39,000 62,500

Lavin et al., 1982 Rashidbaigi and Ruoho, 1982

Turkey

/31

45,000; 40,000 50,000; 40,000 49,000; 38,000 52,000; 39,000

Lavin et al., 1982 Shorr et al., 1982b Burgermeister et al., 1982 Stadel et al., 1983a Kelleher et al., 1984

48,000; 45,000 48,000; 40,000

Rashidbaigi and Ruoho, 1982 Sibley et al., 1984a

Duck

/3t

Pigeon

fll

52,500; 46,000; 45,000

Rashidbaigi and Ruoho, 1982

/32 /3~(20%) f12(80%)

65,000; 53,000; 39,000 62,000; 47,000, 36,000

Lavin et al., 1982 Lavin et al., 1982 Nambi et al., 1984a Benovic et al., 1983 Strasser et al., 1984 Benovic et al., 1984

Rat Rat

64,000; 53,000; 39,000 64,000; 54,000; 30,000 64,000 Rabbit

/3~(80%) /32(20%)

65,000; 45,000; 38,000

Lavin et al., 1982 Nambi et al., 1984a

Guinea pig

/31(20%) f12(80%)

63,000; 52,000; 49,000 64,000

Nambi et al., 1984a Benovic et al., 1984

/31(20%)

64,000

Benovic et al., 1984

Hamster

/32(80%)

Skeletal Heart

Cultured ceils

Dog

f11(20%) /32(80%)

52,500 52,000; 40,000

Homcy et al., 1983 Nambi et al., 1984a

' Rabbit muscle

fit(20%) f12(80%)

54,000; 48,000; 44,000

Nambi et al., 1984a Stiles Stiles Stiles Stiles Stiles

Rat Rabbit Pig Human Frog

fl~ fl~ fl~ fl~ //2

62,000; 62,000; 62,000; 62,000; 62,000;

55,000 55,000 55,000 55,000 55,000

$49 Lymphoma

f12

65,000; 55,000

et et et et et

al., al., al., al., al.,

1983b 1983b 1983b 1983b 1983b

Rashidbaigi et al., 1983

Rashidbaigi and Ruoho, 1981, 1982; Burgermeister et al., 1982). As shown in Fig. 1, the labeling of the turkey erythrocyte fl-receptor proteins by [~25I]pABC was dependent on photolysis and could be specifically blocked by both the agonist isoproterenol and the antagonist alprenolol. The same two polypeptides have been purified and pharmacologically shown to contain/~-adrenergic binding sites (Shorr et al., 1982b). Moreover, the proteins obtained by purification could also be photoaffinity labeled, providing strong evidence that the purified macromolecules were indeed fl-receptor proteins (Shorr et al., 1982b). The relationship between these two receptor proteins is not yet clear. Recent evidence suggests that the two forms may be closely related and that the larger polypeptide may be converted to the lower molecular weight form by proteolysis (Sibley et al., 1984c; Jurss et al., 1985). The fact that the photoaffinity labeling and purification techniques identified identical proteins as fl-adrenergic receptors in erythrocytes validated the photoaffinity labeling approach. These labeling techniques could then be applied with confidence to the

64

J.M. STADEL

MW 94 K 67 K

43 K

30K

I

2

3

4

FIG. I. SDS-Polyacrylamide gel electrophoresis of [~25I]pABC-labeled fl-adrenergic receptor proteins from turkey erythrocyte membranes. Turkey erythrocyte membranes were incubated in the dark with [~25I]pABCfor 60-90 min at 25°C under conditions such that the concentrations of [125I]pABC and #-adrenergic receptor were approximately equal (20-50pM). The incubation mixture was then diluted 1:1 (v/v) with cold (4°C) 75 mM Tris-HC1 (pH 7.4), 25 mM MgCI2 containing 0.5% fatty acid-free bovine serum albumin and the membranes pelleted by centrifugation for 10 min at 40,000 x g. The membranes were washed once more with this wash buffer and then washed a final time in cold 75 mM Tris-HCl, 25 mM MgC12.The membranes were resuspended in 5ml of 75mM Tris-HC1, 25mM MgC12 and irradiated in a Petri dish placed 12cm from a Hanovia 450 W medium pressure mercury lamp for 90 sec. The lamp was equipped with a 6-mm Pyrex glass filter and was air cooled. The irradiated membranes were collected by centrifugation (10min, 40,000 x g) and then washed once with 25mM Tri~HC1 (pH 6.8), 2mM MgC12. The membrane pellets were finally dissolved in SDS sample buffer: 25 mu Tris-HC1 (pH 6.8), 10% SDS, 10% glycerol, 5% #-mercaptoethanol for 30 min at 23°C. Samples 1 and 2 were incubated with [t25I]pABC alone, Sample 3 was incubated with [125I]pABCplus 10 4M isoproterenol, and Sample 4 was incubated with [t25I]pABCin the presence of 10 5Malprenolol. Sample 2 was not irradiated and served as a 'dark control'. The four samples were electrophoresed on a 12.5% polyacrylamide gel. The molecular weight standards are shown on the left. (From Stadel et al., 1982b, with permission.)

identification a n d c h a r a c t e r i z a t i o n of fl-receptors in p l a s m a m e m b r a n e s derived from a variety o f m a m m a l i a n tissues. F r o m a n u m b e r o f these studies, it was d e t e r m i n e d that the photoaffinity labels covalently tagged two or three proteins specifically, i.e. these proteins were specifically protected when excess agonist or a n t a g o n i s t ligands were i n c l u d e d d u r i n g the photolysis e x p e r i m e n t (Lavin et al., 1982; N a m b i et al., 1984a; Stiles et al., 1983b). The largest receptor p r o t e i n identified usually r a n g e d between M, -~ 62,000-67,000 (see T a b l e 2). The relationship a m o n g the multiples b a n d s was n o t immediately k n o w n . However, the ratio of the radioactivity i n c o r p o r a t e d into the various receptor proteins could be m o d u l a t e d by i n c l u d i n g a c o m b i n a t i o n o f protease inhibitors d u r i n g the m e m b r a n e p r e p a r a t i o n a n d s u b s e q u e n t photolysis experiments. U s i n g a m i x t u r e o f protease inhibitors, o f which E D T A a p p e a r e d to be the m o s t effective, the largest m o l e c u l a r weight receptor polypeptide became the p r e d o m i n a n t l y labeled m a c r o m o l e c u l e (Benovic et al.,

fl-Adrenergicreceptors

65

1983; Nambi et al., 1984a). These experiments suggest that mammalian fi-adrenergic receptors are very similar in molecular weight, between Mr,= 62,000-67,000. The fi-receptors have now been purified from rat, hamster, guinea-pig, and dog lung and have been shown to be single polypeptides identical to the largest molecular weight band identified by photoaffinity labeling techniques (Benovic et al., 1984; Homcy et al., 1983). For the first three species of lung, the MW is 64,000, but for canine lung both techniques identified a fi-receptor protein of Mr ~ 52,000 (Benovic et al., 1984; Homcy et al., 1983; Nambi et al., 1984a). Whether the dog lung fi-adrenergic receptor is indeed smaller or if proteolysis is still occurring in this preparation remains to be determined. It should be noted that although photoaffinity labeling has successfully identified fi-adrenergic receptor polypeptides in many tissues from a variety of species, the interpretation of these experiments is not always straightforward. The problem of proteolytic cleavage of the receptor has already been discussed above. In addition, a number of proteins may be non-specifically labeled during photolysis even after extensive washing of the preparation before irradiation. It has even been pointed out that a particular labeled protein may be protected by an antagonist but not an agonist ligand indicating it is a non-specific site (Stiles et al., 1983b). In certain tissues such as rat fat, attempts to photoaffinity label the fi-receptor in membranes and in purified preparations have not been successful (Cubero and Malbon, 1984). One of the first applications of photoaffinity labeling following the validation of the technique was to address the question of fi-receptor subtypes. The subtype specificity of the f-receptor had been shown to be an intrinsic property of the receptor protein and not determined by coupling elements distal to the receptor (Pike et al., 1979; Kaslow et al., 1979). The same pharmacological criteria used to characterize fi-receptor subtypes in radioligand binding experiments have been applied to photoaffinity labeling studies. In several studies, the inhibition of incorporation of the photoaffinity labels into receptor proteins was shown to be related to the concentration of competing ligand and followed the expected rank orders of potency when subtype specific agonist or antagonist ligands were employed (Lavin et al., 1982; Stiles et al., 1983b; Nambi et al., 1984a). This is illustrated in Fig. 2. The labeling of the fi-receptors from rat lung by [t25I]pABC was inhibited by both epinephrine and norepinephrine, but epinephrine was a more potent blocker, indicating the receptor subtype to be fi2. This subtype identification was supported by the use of subtype-selective antagonists to inhibit the incorporation of [~25I]pABC into the receptors. The fi~-selective antagonist ICI 118.551 was more potent than the f/it-selective antagonist betaxolol in inhibiting the photoaffinity labeling. In tissues known to possess heterogeneous populations of fi-receptors, only the predominant subtype appeared to be labeled (Lavin et al., 1982; Nambi et al., 1984a). Is the subtype specificity of the receptors reflected in structural differences? The earliest photoaffinity labeling experiments using fl-receptors from avian and amphibian erythrocytes suggested that this may be the case (Lavin et al., 1982; Rashidbaigi and Ruoho, 1982). The frog erythrocyte fi-receptor has a fi2-specificity and a single receptor polypeptide of Mr ~ 58,000 was identified by both purification and photoaffinity labeling techniques. In contrast, two receptor proteins of smaller molecular weight were identified by these same two procedures for the turkey erythrocyte which possesses a homogeneous population of fi~-receptors. However, when these techniques were applied to mammalian fi-receptors a different story emerged. Comparing rabbit lung preparations which are 80% fi~ to rat lung which is 80% fi2, the photoaffinity labeling patterns appeared to be nearly identical even though the subtype specificity could be definitely demonstrated in the labeling study (Lavin et al., 1982; Nambi et al., 1984a). Photoaflinity labeling of cardic fi~- and fi2-receptors identified a predominant Mr---62,000 receptor protein (Stiles et al., 1983b). These data indicated that fl-receptor subtypes can not be discriminated based on molecular weight alone. More extensive investigations involved the comparison of limited digestion peptide maps ofphotoaffinity labeled fi-receptor subtypes. Lefkowitz and coworkers (Stiles et al., 1983a; Benovic et al., 1984) have used these techniques to compare fi-receptor subtypes from JPT

31/I-2---E

66

J.M. STADEL

various tissues of a single species as well as to compare the structure of a receptor subtype in the same tissue from different species. They concluded that the structure of the B-adrenergic receptor appears to be conserved. The peptide maps of a single receptor subtype from the same tissue of different species were very similar with only a few notable differences indicating evolutionary homology. The comparison of the peptide maps of the Bit- and B2-receptor subtypes from tissues of a single species, again, showed a high degree of homology but also some distinct differences suggesting that receptor primary structure could influence receptor subtype specificity. These results are in good agreement with peptide mapping data of B-receptor subtypes using purified BI- and B2-receptor proteins (Graziano et al., 1985). 4. PHOTOAFFINITY LABELS AS PROBES OF B-ADRENERGIC RECEPTOR STRUCTURE

Photoaffinity labels in combination with other reagents can be used to explore the structure of #-adrenergic receptors in greater detail. The photoaffinity label assures that the protein being monitored is, indeed, the fl-receptor. Two studies have investigated the effect of thiol-reducing agents on photoaffinity-labeled fl-receptors. Using [125I]ABP, two fl-receptor polypeptides have been identified in membranes prepared from $49 murine lymphoma cells (Rashidbaigi et al., 1983). These two receptor proteins are of MW 65,000 and 55,000 and are labeled to the same extent. Treatment of $49 membranes or cells with dithiothreitol inhibited both agonist-stimulated adenylate cyclase activity as well as

(a)

94K 67 K

43K

30 K

.6"_

W.#

.4"

W .o° FIG. 2(a)

q

W.#

%

#-Adrenergic receptors

67

(b)

94 K 67K

43K

30K

FIG. 2. Photoal~nity labeling and pharmacologicalspecificityof incorporation of [125I]pABCin rat lung membranes. Rat lung membranes were photoatiinity labeled with [~25I]pABCalone (control) or in the presence of the indicated concentrations of agonists (a) or antagonists (b) as described in legend to Fig. I. ISO, isoproterenol;NE, norepinephrine;EPI, epinephrine; ICI, ICI 118.551; Betax, betaxolol. The samples were electrophoresed on an 8% polyacrylamidegel. Molecular weight standards are indicated at the left of each figure. (From Nambi et al., 1984a, with permission.)

radioligand binding to r-receptors (Clark et al., 1983). Photoaffinity labeling of membranes following dithiothreitol treatment decreased labeling in both receptor bands, although the lower molecular weight protein was more sensitive to the reducing agent. Additional experimentation will be necessary to define the relationship between the two receptor proteins and the differential sensitivity to dithiothreitol. Thiol-reducing agents have also been used to determine if r-receptors are composed of polypeptides linked together by disulfide bonds, as described for other hormone receptors, e.g. the insulin receptor (Stiles and Strasser, 1983). In these experiments, the photoaffinity labeled r-receptors were dissolved in SDS sample buffer without reducing agent prior to analysis by electrophoresis. The samples were then compared on reducing and nonreducing polyacrylamide gels. The molecular weights of the receptors differed only slightly when compared on the two gel systems indicating that the r-receptors may contain intrabut not intermolecular disulfide bridges. These data with thiol-reducing agents provided additional support for earlier reports indicating that photoaffinity-labeled or purified fl-adrenergic receptors exist as single polypeptide chains (Lefkowitz et aL, 1983; Cubero and Malbon, 1984; Benovic et al., 1984). These results also implied that a single protein contained both the ligand binding site and an additional domain required to communicate or couple the binding event to adenylate cyclase. Recent reconstitution studies proved that the purified receptor protein

68

J . M . STADEL

HAMSTER LUNG ,,BAR (1200 fmol)

~- >, I-- o O

5.0

........

o

E W

ci

_J r7

67K

2.0 0

O

W W 0

1.0

0

L9

o

:~,{ 30 K

~:

// ~.

ZO_ E~ U

X&X

X & xgAR

FIG. 3. Adenylate cyclase activity in fused hybrids of reconstituted pure fl-adrenergic receptor (flABR) from hamster lung and X. laevis erythrocytes. Pure fl-receptor (1200 fmol) was reconstituted into phosphatidylcholine lipid vesicles. Excess detergent was removed by eluting the mixture through an Extracti-gel (Pierce) column. The receptor containing vesicles were fused with X. laevis erythrocytes (X & flAR) at 30°C in the presence of polyethyleneglycol. As a control X. larvis erythrocytes were fused with vesicles which did not contain flAR (X & X). X. laevis erythrocytes were chosen because they have a very low number of fl-receptors but do possess adenylate cyclase coupled to PGE~ receptors. The vesicle-erythrocyte fusion hybrids were lysed by freeze-thaw and then assayed for adenylate cyclase activity. B, basal; ISO, isoproterenol (0.05 mu); PRO, propranolol (0.05mM); PGE, prostaglandin E I (3 #M). Inset: autoradiogram of SDS polyacrylamide gel electrophoresis of photoatfinity-labeled ([12SI]pABC) and iodinated (12sI) purified fl-receptor used in the reconstitution experiments. (From Cerione et al., 1983, with permission.)

is sufficient to functionally couple agonist binding to the other components of the adenylate cyclase system (Cerione et al., 1983; Kelleher et al., 1983). Such a reconstitution experiment is shown in Fig. 3. A number of membrane bound receptors including the fl-adrenergic receptor have been shown to be glycoproteins (Hedo et al., 1981; Stadel et al., 1981b). Information on the carbohydrate components of these receptors has begun to emerge over the past several years. Stiles and coworkers (1984a) investigated the carbohydrate structure of the mammalian fl-receptor using photoaffinity labeling. They labeled the fl-receptors in hamster lung and rat erythrocytes with [125I]pABC, and then treated the receptor preparations with both exo- and endoglycosidases; the effects of these enzymes were monitored as changes in apparent molecular weight of the receptors by SDS gel electrophoresis. The exoglycosidase neuraminidase and ~-mannosidase, when used individually, both slightly enhanced the electrophoretic mobility of the labeled fl-receptors. These data indicated the existence of sialic acid containing complex carbohydrates as well as high mannose carbohydates as part of the receptor structure (Kornfeld and Kornfeld, 1980). However, ~-mannoside treatment produced two receptor populations and sequential exposure of the receptors to both enzymes did not result in an additive effect. These results were interpreted as indicating discrete populations of fl-receptors containing either complex or high mannose chains. This interpretation is supported by elution profiles from immobilized lectin columns. The ['25I]pABC-labeled receptors containing complex carbo-

fl-Adrenergic receptors

69

hydrates could be adsorbed and specifically eluted from wheat germ agglutinin-Sepharose while those containing high mannose bound selectively to immobilized concanavalin-A. Both types of carbohydrate chains appear to be attached to the B-receptor through an N-linkage to asparagine (Stiles et al., 1984a). Treatment of either the hamster lung or rat erythrocyte B-receptors with endoglycosidase F to deglycosidate the receptors resulted in a single polypeptide of M r - 49,000 suggesting that the protein portions of these B-receptors are similar. The carbohydrate structure of the turkey erythrocyte B-receptor has also been investigated. As discussed above, both photoaffinity labeling and purification techniques have identified two receptor proteins in this system; Mr ~- 50,000 and M r - 40,000. By labeling these receptor proteins with [~25I]CYP-azide, it was then possible to investigate their interaction with immobilized wheat germ agglutinin (Jurss et al., 1985). Only the larger Mr -~ 50,000 receptor protein was adsorbed to the resin and subsequently eluted with N-acetylglucosamine; the protein of Mr ~- 40,000 was unretarded by the lectin-resin. Treatment of the larger molecular weight polypeptide with endoglycosidase F inhibited its binding to the wheat germ agglutinin. Exposure to the endoglycosidase did not alter the apparent molecular weight of the Mr = 40,000 receptor protein, but the enzyme did convert the M r - 50,000 protein to a Mr = 45,000 form. These data indicate that only the Mr ~ 50,000 form of the turkey eythrocyte B-receptor is glycosidated with N-linked complex carbohydrate chains accounting for about 10% of its apparent molecular weight. The M, - 40,000 form of the receptor arises from proteolytic cleavage of the larger protein. The proteolytic processing must clip off a peptide which contains the receptor carbohydrate. The physiological relevence of this processing event is unknown. These biochemical investigations indicate that as much as one quarter of the apparent molecular weight of B-receptors may be attributed to the carbohydrate portion of these glycoproteins. The carbohydrate chains may be different on subpopulations of B-receptors within a single tissue. Microheterogeneity of the carbohydrate structure may contribute to the broad banding pattern on SDS gels of some B-receptor preparations (Shorr et al., 1981; Lavin et al., 1982). However, the pharmacological properties of the B-receptor are unaltered by deglycosidation procedures (Stiles, 1985). The exact role of carbohydrates, if any, in the function of the B-adrenergic receptor remains to be determined. 5. PHOTOAFFINITY LABELING ILLUMINATES THE MECHANISMS OF B-ADRENERGIC RECEPTOR REGULATION It is widely recognized that the specificity of hormone or drug action is determined by the receptors of target tissues. A second fundamental property of these receptor-effector systems is that the sensitivity of tissues to agonist agents is dynamically regulated. Cells and tissues are able to maintain homeostasis by modulating their hormonal responsiveness. These regulatory mechanisms allow tissues to respond rapidly to an agonist while at the same time protecting them against extreme or sustained alterations in the concentration of external stimuli. Prolonged exposure of a tissue to an agonist drug or hormone leads to an attenuated responsiveness of the tissue to that agent. Clinically, this process is termed refractoriness or desensitization. Desensitization has long been recognized as a pharmacological consequence of drug therapy, but recent research indicates that these regulatory mechanisms are sensitive enough to adjust to normal fluctuations in circulating hormone levels (Fraser et al., 1981). The phenomenology of densensitization has been described for a wide variety of receptor systems including the B-adrenergic receptor-adenylate cyclase complex (Lefkowitz et al., 1980, 1983; Harden, 1983). The molecular mechanisms underlying desensitization remain to be fully elucidated; however, photoaffinity labeling has been instrumental in investigations of this subject. Desensitization has been extensively investigated in tissues containing receptors coupled to adenylate cyclase and these studies have led to a proposed division of the patterns of desensitization (Su et al., 1976a,b; Lefkowitz et al., 1980; Harden, 1983). 'Heterologous'

70

J . M . STADEL

desensitization results in attenuated responsiveness of adenylate cyclase to all hormonal activators as well as to other activators such as NaF. In contrast, 'homologous' desensitization is characterized by diminished responsiveness of the cyclase to the desensitizing agonist only, without alteration in enzyme sensitivity to other hormones or NaF. The heterologous form of desensitization was studied using the fl-adrenergic receptor-adenylate cyclase complex of avian erythrocytes (Hoffman et al., 1979; Stadel et al., 1981a; Simpson and Pfeuffer, 1980). Pre-incubation of turkey or pigeon erythrocytes with agonist, e.g. isoproterenol, induced a 50 to 60% decrease in subsequent agonist stimulation of adenylate cyclase as well as a significant inhibition of activation by NaF. The desensitization does not result from a loss of receptors from the plasma membrane but, apparently, uncouples the receptor from the other components of the adenylate cyclase complex. This uncoupling was documented in radioligand binding experiments which showed a decreased ability of agonist to promote a high affinity binding state of the receptor in membranes prepared from desensitized cells (Stadel et al., 1981a). Moreover, these alterations in the receptor-cyclase complex were partially mimicked by preincubation of these erythrocytes with analogs of cAMP (Simpson and Pfeuffer, 1980; Stadel et al., 1981a). These investigations suggested that the fl-receptor itself might be a site of modification during the desensitization process. In order to examine this possibility, the receptors in membranes from control and desensitized turkey erythrocytes were photoaffinity labeled with [125I]pABC. The photoaffinity labeling revealed that the fl-receptor proteins had an altered mobility when analyzed by SDS polyacrylamide gel electrophoresis (Fig. 4). The apparent molecular weights of the fl-receptor polypeptides from the desensitized preparation were 53,000 and 42,000, compared to 50,000 and 38,000 for the control (Stadel et al., 1982b, 1983a). Although the changes in electrophoretic mobility are small, they were reproducible and, in fact, a correlation was established between the alteration in mobility of photoaffinity labeled receptor and inhibition of agonist-stimulated adenylate cylase activity (Stadel et al., 1983a). The difference in apparent molecular weight was observed following photoaffinity labeling of the purified receptors from control and desensitized turkey erythrocytes indicating a direct modification of the fl-receptor during desensitization (Stadel et al., 1983a; Strulovici et al., 1984). This is further supported by reconstitution experiments in which the partially purified fl-receptor from desensitized cells was unable to stimulate adenylate cyclase as well as control receptor (Strulovici et al., 1984). The altered mobility of the fl-receptor from desensitized turkey erythrocytes, as determined by SDS polyacrylamide gel electrophoresis, appears to be due to an agonistinduced conformational change in the receptor (Stadel et al., 1986). This proposal is supported by two lines of evidence. The mobility of the photoaffinity-labeled receptor from desensitized erythrocytes increased to run coincident with the control receptor preparation when 5 M urea was included with SDS during the electrophoresis. Moreover, comparison of limited-digestion peptide maps of [~25I]pABC-labeled fl-receptor from control and desensitized cells revealed distinctly different sensitivities of the two fl-receptors to cleavage by chymotrypsin and Staphylococcus aureus protease. These results suggest that prolonged occupancy of the fl-receptor by agonist induces a conformational change that may be stabilized by covalent modification of the receptor protein. The 'desensitized' form of the fl-receptor is not able to communicate with the other components of adenylate cyclase. Desensitization of the turkey erythrocyte fl-receptor-adenylate cyclase complex by incubating the cells with the cAMP analog, 8Br-cAMP, also promoted an alteration in the electrophoretic mobility of the fl-receptor protein labeled with [~25I]pABC (Stadel et al., 1983a). The only known mechanism of action of cAMP is to activate cAMP-dependent protein kinase (Krebs and Beavo, 1979). Since reversible phosphorylation has been shown to be an important regulatory mechanism in biological systems, the possibility that the fl-adrenergic receptor is phosphorylated during desensitization was explored. Stadel and coworkers (1983a) were able to demonstrate that pre-incubation of turkey erythrocytes

fl-Adrenergic receptors

71

MW

94 K 67K

45K

30K

C C + ISO

D

D + ISO

F]O. 4. SDS polyacrylamide gel electrophoresis of [1251]pABC-labeled fl-adrenergic receptor proteins from turkey erythrocytemembranesprepared from control and desensitizedevils. Turkey erythrocyte membranes prepared from control (C) and desensitized (D) evils were photoatfmity labeled with [125I]pABCin the presence and absence of 10-4M isoproterenol (ISO) as described in legend to Fig. 1. Electrophoresiswas carried out on an 12.5% polyacrylamidegel. The molecular weight standards are shown at the left. (From Stadel et aL, 1982b, with permission.)

with agonist promoted a 2-3-fold increase in the phosphorylation of the fl-receptor proteins. Moreover, the change in electrophoretic mobility of the fl-receptor following desensitization was observed after endogenous 32p-labeling (Fig. 5, right) as had been previously demonstrated by [~25I]pABC-labeling (Fig. 5, left). The increase in phosphate incorporation and the alteration in receptor mobility were both shown to correlate with the desensitization of the cyclase enzyme (Stadel et al., 1983a; Sibley et al., 1984b). These data suggest that agonist promoted phosphorylation may be responsible, at least in part, for the uncoupling of the fl-receptor from adenylate cyclase. In addition to catecholamines and cAMP, the avian erythrocyte-fl-adrenergic receptor complex can also be desensitized by pre-incubating the cells with phorbol esters (Sibley et al., 1984a; Kelleher et al., 1984). These compounds also promote increased phosphorylation of the fl-receptor proteins presumably through the activation of protein kinase C. Whether agonist promoted phosphorylation of the fl-receptor also involves the

72

J.M. STADEL

94 67

43

30

C

D

C

D

FIG. 5. SDS polyacrylamide gel electrophoresis of [12SI]pABC- (left) and 32p. (right) labeled fl-adrenergic receptors prepared from control (C) and catecholamine desensitized (D) turkey erythrocytes. (Left) Turkey erythrocyte membranes prepared from control and desensitized cells were labeled with [125I]pABCas described in the legend to Fig. 1. (Right) Turkey erythrocytes were precincubated with [32p]PO~-3for 20 hr. The cells were divided into two groups and the incubation continued for an additional 4 hr in buffer alone as control or in the presence of 1 #M isoproterenol. The fl-receptors were subsequently solubilized from the plasma membrane by the non-ionic detergent digitonin and partially purified by affinity chromatography over alprenolol-Sepharose. Equal amounts of ~8-receptorprotein were then solubitized in SDS sample buffer and loaded onto the gel. Electrophoresis was carried out on a 12.5% polyacrylamide gel. The molecular weight standards are given on the left of the figure. (From Stadel et al., 1983a, with permission.)

a c t i v a t i o n o f p r o t e i n k i n a s e C r e m a i n s to be established. T h e d e v e l o p m e n t o f a b r o k e n cell system in which the effects o f p r o t e i n kinases on the f l - r e c e p t o r - a d e n y l a t e cyclase c o m p l e x can be e x a m i n e d directly p r o m i s e s to help elucidate the m e c h a n i s m s o f desensitization ( N a m b i et al., 1984b, 1985). P h o t o a f f i n i t y labeling has also been utilized to p r o b e a d e n y l a t e c y c l a s e - c o u p l e d f l - a d r e n e r g i c r e c e p t o r s t h a t u n d e r g o h o m o l o g o u s desensitization. This type o f desensit i z a t i o n has been extensively s t u d i e d in the frog e r y t h r o c y t e m o d e l system a n d in c u l t u r e d cells ( L e f k o w i t z et al., 1980; S u e t al., 1980; H a r d e n , 1983). H o m o l o g o u s desensitization also involves an u n c o u p l i n g o f the f l - r e c e p t o r f r o m the a d e n y l a t e cyclase but, in a d d i t i o n ,

fl-Adrenergicreceptors

73

the receptors are down-regulated or apparently lost from the cell surface. The uncoupled fl-receptors were found to be associated with a light vesicle fraction (Harden et al., 1980; Stadel et al., 1983b). The sequestered fl-receptors may be internalized, or remain part of the plasma membrane (Harden et al., 1980; Stadel et al., 1983b; Strader et al., 1984). The structure of the fl-receptors in the vesicles from desensitized frog erythrocytes was compared to that of fl-receptors in the plasma membrane by photoaffinity labeling with [t25I]pABC (Stadel et al., 1983b). The apparent molecular weight of both receptors was 58,000 indicating that the fl-receptor was not extensively processed during sequestration. When assayed in a reconstitution system, sequestered fl-receptors were shown to retain their functiorial ability to bind ligands and to translate the binding event into the stimulation of adenylate cyclase (Strulovici et al., 1983). Sequestration of fl-receptors has also been observed following catecholamine-induced desensitization of rat lung (Strasser et al., 1984). These studies suggest that the uncoupling of the fl-receptor during homologous desensitization may be due to the physical separation of the receptor from the other components of the adenylate cyclase complex. In addition, phosphorylation of the frog erythrocyte fl-adrenergic receptor during desensitization has recently been reported suggesting common elements in the mechanisms of heterologous and homologous desensitization (Sibley et al., 1985). The fl-receptors down-regulated by prolonged exposure to isoproterenol appear to be recycled to the plasma membrane once agonist is removed from the environment of the cells. During resensitization, normal functioning receptors reappear in the plasma membrane fraction even in the presence of protein synthesis inhibitors (Doss et al., 1981; Strulovici and Lefkowitz, 1984). The notion of recycling is further supported by studies using the protease trypsin in conjunction with the desensitization protocols. Treatment of frog erythrocytes with trypsin reduced the molecular weight of the fl-receptor from 58,000 to 40,000 as determined by photoaffinity labeling with [125I]pABC (Strulovici and Lefkowitz, 1984). Interestingly, the proteolysis did not alter the ligand binding properties nor the coupling efficiency of the fl-receptors to adenylate cyclase. Both control and trypsin-treated receptors could be down-regulated by incubating the cells with agonist, but only in control preparations did the receptor reappear on the cell surface during resensitization. The trypsinized receptors appear to be degraded within the cell. These experiments support the contention that fl-receptors recycle within the cell in much the same way as described for polypeptide hormone receptors (Brown and Goldstein, 1979). In addition to the erythrocyte model systems, catecholamine induced desensitization of the fl-adrenergic receptor complex has also been investigated using cultured mammalian cells. The $49 murine lymphoma cell line is one such system. Incubation of these cells with fl-adrenergic agonist promoted an uncoupling of the //-receptors from the adenylate cyclase followed by a gradual, but nearly complete, down-regulation of the receptors (Su et al., 1980). Sucrose density gradient analysis of the //-receptors following short-term desensitization showed that uncoupled receptors had moved to a light vesicle fraction as had been described for other cell types (Clark et al., 1985). Photoaffinity labeling of plasma membranes of $49 cells with [~25I]ABP identified two //-receptor proteins: M r - 65,000 and 55,000. After desensitization of the $49 cells, the same two polypeptides were labeled, but the labeling of the lower molecular weight protein was disproportionately diminished compared to the M ; ~ 65,000 receptor protein (Rashidbaigi et al., 1983). Fractionation of the fl-receptor population by sucrose density gradients subsequent to exposure of the cells to epinephrine revealed that the Mr ~ 65,000 receptor protein was selectively sequestered into the light vesicle fraction (Clark et al., 1985). The uncoupled light vesicle receptor was shown to retain its functional capacity in reconstitution experiments. These studies support earlier investigations in the frog erythrocyte which suggested that the uncoupling of the fl-receptor during desensitization may be due to a physical sequestration of the receptors from the adenylate cyclase. In the $49 cells, the selective incorporation of only one of the receptor proteins into the light vesicles during desensitization suggests the possibility of different functions for the two receptor proteins identified by [~25I]ABP.

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J . M . STADEL

6. CONCLUSIONS New technologies in receptor research have significantly advanced our understanding of both the structure and function of the fl-adrenergic receptor. Photoaffinity labeling is one of these important new tools. Labeling experiments, in conjunction with purification procedures, have identified both fl: and fl2-receptor proteins. Reconstitution studies have shown that the single polypeptide identified by photoaffinity labeling and purification is bifunctional in that it both ligands with the appropriate pharmacological specificity and communicates agonist binding to the effector, adenylate cyclase. Once validated, photoaffinity labeling has found a wider application than purification due to the fact that it can be used in readily obtainable membrane preparations. However, a cautionary note must be added. The pharmacological specificity of a photoaffinity reagent must be defined with both agonist and antagonist ligands, and precautions against proteolysis must be employed to insure the specific labeling of the fl-receptor. In tissues containing a mixture of fl-receptor subtypes, it appears that only the predominant subtype is labeled. Future studies with photoaffinity labels will see the application of these probes to examine fl-adrenergic receptors in intact cells, particularly under the controlled conditions of tissue culture. With the existence now of a variety of high specific activity labels, it will be possible to begin mapping the ligand binding site of the receptor. The enhanced sensitivity of micro-sequencing techniques along with the availability of substantial quantities of purified fl-receptor preparations suggests this mapping approach may soon be reality. An additional new area of investigation will be the development of high specific activity agonist photoaffinity labels. This type of labeling compound could provide new insights into the mechanisms of receptor-cyclase coupling and agonist-induced desensitization. Finally, photoaffinity labeling will find a broader application in attempts to define the molecular mechanisms of physiological and patho-physiological regulation of the fl-adrenergic receptor. REFERENCES AHLQUIST, R. P. (1948) A study of the adrenotropic receptors. Am. J. Physiol. 153: 585--600. ATLAS, D., SaXER, M. L. and LEVlTZKI, A. (1974) Stereospecific binding of propranolol and catecholamines to the fl-adrenergic receptor. Proc. natn. Acad. Sci. U.S.A. 71: 4246-4248. AURBACH, G. D., FEDAK, S. A., WOODARD, C. J., PALMER, J. S., HAUSER D. and TROXLER, F. (1974) The fl-adrenergic receptor: stereospecific interaction of an iodinated fl-blocking agent with a high-affinity site. Science 186: 1223-1224. BAROVSKY, K. and BROOKER, G. ~25I-Iodopindolol, a new highly selective radioiodinated fl-adrenergic receptor antagonist: measurement of fl-receptors on intact rat astrocytoma cells. J. Cyclic Nucl. Res. 6: 297-307. BAYLEY, H. and KNOWLES, J. R. (1977) Photoaffinity labeling. Meth. En2ymol. 46: 69-114. BENOVIC, J. L., STILES, G. L., LEFKOWITZ, R. J. and CARON, M. G. (1983) Photoaffinity labeling of mammalian fl-adrenergic receptors: metal dependent proteolysis explains apparent heterogeneity. Biochem. biophys. Res. Commun. 110:504-51 I. BENOVlC, J. L., SHORR, R. G. L., CARON, M. G. and LEFKOWITZ, R. J. (1984) The mammalian fl2-adrenergic receptor: purification and characterization. Biochemistry 23: 4510-4518. BETHELSON, S. and PET'rINGER, W. A. (1977) A functional basis for the classification of ct-adrenergic receptors. Life Sci. 21: 595-606. BROWN, M. S. and GOLDSTEIN,J. L. (1979) Receptor-mediated endocytosis: insights from the lipoprotein receptor system. Proc. natn. Acad. Sci. U.S.A. 76: 3330-3337. BURGERMEISTER, W., HEKMAN, M. and HELMREICH, E. J. M. (1982) Photoaffinity labeling of the fl-adrenergic receptor with azide derivatives of iodacyanopindolol. J. biol. Chem. 257: 5306-5311. BURGERMEISTER, W., NASSAL, M., WIELAND, T. and HELMREICH, E. J. i . (1983) A carbene-generating photoaffinity probe for fl-adrenergic receptors. Biochim. Biophys. Acta 729: 219-228. CERIONE, R. A., STRULOVICI, B., BENOVIC, J. L., LEFKOWITZ, R. J. and CARON, M. G. (1983) Pure fl-adrenergic receptor: the single polypeptide confers catecholamine responsiveness to adenylate cyclase. Nature 306: 562-567. CLARK, R. B., GREEN, D. A., RASHIDBAIGI,A. and RUOHO, A. E. (1983) Effect of dithiothreitol on the fl-adrenergic receptor of $49 wild type and cyc lymphoma cells: decreased affinity of the ligand-receptor interaction. J. Cyclic" Nucl. Prot. Phos. Res, 9: 203-220. CLARK, R. B., FRIEDMAN, J., PRASHAD, N. and Ruorlo, A. E. (1985) Epinephrine-induced sequestration of the fl-adrenergic receptor in cultured $49 WT and cyc- lymphoma cells. J. Cyclic Nucl. Prot. Phos. Res. 10: 97-119. CUBERO, A. and MALBON, C. C. (1984) The fat cell fl-adrenergic receptor: purification and characterization of a mammalian fl:adrenergic receptor. J. biol. Chem. 259: 1344-1350.

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NAMBI, P., SIBLEY, D. R., STADEL, J. M., MICHEL, T., PETERS, J. R. and LEFKOWITZ, R. J. (1984b) Cell-free desensitization of catecholamine sensitive adenylate cyclase: agonist and cAMP-promoted alterations in turkey erythrocyte fl-adrenergic receptors. J. biol. Chem. 259: 4629-4633. NAMBI, P., PETERS, J., SIBLEY, D. R. and LEEKOWITZ, R. J. (1985) Desensitization of the turkey erythrocyte fl-adrenergic receptor in a cell-free system: evidence that multiple protein kinases can phosphorylate and desensitize the receptor. J. biol. Chem. 260:2165 2171. PIKE, L. J., LIMBIRD, L. E. and LEFKOWlTZ, R. J. (1979)/~-Adrenoreceptors determine affinity but not intrinsic activity of adenylate cyclase stimulants. Nature 280: 502-504. RASHIDBAIGI, A. and RUOHO, A. E. (1981) Iodoazidobenzylpindolol, a photoaffinity probe for the fl-adrenergic receptor. Proe. hath. Acad. Sci. U.S.A. 78:1609 1613. RASHIDBAIGI, A. and RUOHO, A. E. (1982) Photoaffinity labeling of fl-adrenergic receptors: identification of the fl-receptor binding site(s) from turkey, pigeon, and frog erythrocytes. Biochem. biophys. Res. Commun. 106: 139 148. RASHIDBAIG1,A., RUOHO, A. E., GREEN, D. A. and CLARK, R. B. (1983) Photoaffinity labeling of the fl-adrenergic receptor from cultured lymphoma cells with [~251]iodoazidobenzylpindolol: loss of the label with desensitization. Proc. natn. Acad. Sci. U.S.A. 80: 2849-2853. RomsoY, G. A., Bu'rCHER, R. W. and SUTHERLAND, E. W. (1971) Cyclic A M P . Academic Press, New York. Ross, E. M. and GILMAN, A. G. (1980) Biochemical properties of hormone-sensitive adenylate cyclase. Ann. Rev. Biochem. 49:533 564. RUOHO, A. E., HALL, C. C. and RASHIDBAIGI, A. (1983) Use of photolabels to probe the Na, K-ATPase and the fl-adrenergic receptor. Fedn. Proc. 42:2837 2841. SHORR, R. G. L., LEEKOWITZ, R. J. and CARON, M. G. (1981) Purification of the fl-adrenergic receptor: identification of the hormonal binding subunit. J. biol. Chem. 256: 5820-5826. SHORR, R. G. L., HEALD, S. L., JEFFS,P. W., LAVIN,T. N., STROHSACKER,M. W., LEFKOWITZ,R. J. and CARON, M. G. (1982a) The fl-adrenergic receptor: rapid purification and covalent labeling by photoaffinity crosslinking. Proc. natn. Acad. Sci. U.S.A. 79:2778 2782. SHORR, R. G, L., STROHSACKER, M. W., LAVIN, T. N., LEFKOWITZ, R. J. and CARON, M. G. (1982b) The fl-adrenergic receptor of the turkey erythrocyte: molecular heterogeneity revealed by purification and photoaffinity labeling. J. biol. Chem. 257:12341 12350. S1BLEY, D. R., NAMm, P., PETERS, J. R. and LEFKOWlTZ, R. J. (1984a) Phorbol diesters promote fl-adrenergic receptor phosphorylation and adenylate cyclase desensitization in duck erythrocytes. Biochem. biophys. Res. Commun. 121:973 979. SIBLEY, D. R., PETERS,J. R., NAMBI, P., CARON, M. G. and LEFKOWITZ, R. J. (1984b) Desensitization of turkey eythrocyte adenylate cyclase: fl-adrenergic receptor phosphorylation is correlated with attenuation of adenylate cyclase activity. J. biol. Chem. 259: 9742-9749. SIBLEY, D. R., PETERS, J. R., NAMBI, P., CARON, M. G. and LEFKOWlTZ, R. J. (1984c) Photoaffinity labeling of turkey erythrocyte fl-adrenergic receptors: degradation of the M r = 49,000 protein explains apparent heterogeneity. Biochem. biophys. Res. Commun. ll9: 458-464. SIBLEY, D. R., STRASSER, R. H., CARON, M. G. and LEEKOWITZ, R. J. (1985) Homologous desensitization of adenylate cyclase is associated with phosphorylation of the fl-adrenergic receptor. J. biol. Chem. 260: 3883-3886. SIMPSON, A. and PFEUEEER,T. (1980) Functional desensitization of fl-adrenergic receptors of avian erythrocytes by catecholamines and adenosine Y,5' phosphate. Eur. J. Biochem. I l l : I 11 ll6. STADEL,J. M. and LEEKOWITZ,R. J. (1983) The fl-adrenergic receptor: ligand binding studies illuminate the mechanism of receptor adenylate cyclase coupling. Curt. Top. Memb. Transp. 18: 45-66. STADEL, J. M., MULLIKIN-KILPATRICK, D., DUKES, D. and LEFKOWITZ, R. J. (1981a) Catecholamine-induced desensitization in turkey erythrocytes: cAMP-mediated impairment of high affinity agonist binding without alteration in receptor number. J. Cyclic Nucl. Res. 7: 37-47. STADEL, J. M., SHORR, R. G. L., LIMBIRD, L. E. and LEFKOWITZ, R. J. (1981b) Evidence that a fl-adrenergic receptor-associated guanine nucleotide regulatory protein conveys guanosine 5'-O-(3-thiotriphosphate)dependent adenylate cyclase activity. J. biol. Chem. 256: 8718-8723. STADEL, J. M., DELEAN, A. and LEFKOWITZ, R. J. (1982a) Molecular mechanisms of coupling in hormone receptor adenylate cyclase systems. Adv. Enzymol. 53: 1-43. STADEL, J. M., NAMBI, P., LAVIN, T. N., HEALD, S. L., CARON, M. G. and LEFKOWITZ, R. J. (1982b) Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase: structural alterations in the fl-adrenergic receptor revealed by photoaffinity labeling. J. biol. Chem. 257: 924~9245. STADEL, J. M., NAMBI, P., SHORR, R. G. L., SAWYER,D. F., CARON, M. G. and LEFKOWITZ, R. J. (1983a) Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase is associated with phosphorylation of the fl-adrenergic receptor. Proc. num. Acad. Sci. U.S.A. 80: 3173-3177. STADEL,J. M., STRULOVICI, B., NAMBI, P., LAVIN,T. N., BRIGGS,M. M., CARON,M. G. and LEFKOWITZ,R. J. (1983b) Desensitization of the fl-adrenergic receptor of frog erythrocytes: recovery and characterization of the down-regulated receptors in sequestered vesicles. J. biol. Chem. 258: 3032-3038. STADEL, J. M., REBAR, R., SHORR, R. G. L., NAMBI, P. and CROOKE, S. T. (1986) Biochemical characterization ofphosphorylated fl-adrenergic receptors from catecholamine-desensitized turkey erythrocytes. Biochemistry 2 5 : 3 7 1 9 3724. STILES, G. L. (1985) Deglycosylated mammalian fl-adrenergic receptors: effects on radioligand binding and peptide mapping. Archs Biochem. Biophys. 237: 65-71. STILES, G. L. and STRASSER, R. H. (1983) The effects of reducing agents on the structure of mammalian fl-adrenergic receptors. Biochem. biophys. Res. Commun. 116: 777-782. STILES, G. L., STRASSER, R. H., CARON, M. G. and LEFKOWITZ, R. J. (1983a) Mammalian fl-adrenergic receptors: structural differences in fll and f12 subtypes revealed by peptide maps. J. biol. Chem. 258: 10689-10695. STILES, G. L., STRASSER, R. H., LAV1N, T. N., JONES, L. R., CARON, M. G. and LEFKOWITZ, R. J. (1983b) The

fl-Adrenergic receptors

77

cardiac fl-adrenergic receptor: structural similarities of fir and fl2-receptor subtypes demonstrated by photoailinity labeling. J. biol. Chem. 258: 8443-8449. STmES, G. L., BENOVIC,J. L., CARON, M. G. and LEFKOWlTZ,R. J. (1984a) Mammalian fl-adrenergic receptors: distinct glycoprotein populations contain high mannose or complex type carbohydrate chains. J. biol. Chem. 259: 8655-8663. STILES, G. L., CARON, M. G. and LEFKOWITZ,R. J. (1984b) fl-Adrenergic receptors: biochemical mechanisms of physiological regulation. Pharmacol. Rev. 64: 661-743. STRADER,C. O., SIBLEY,O. R. and LEFKOWlTZ,R. J. (1984) Association of sequestered fl-adrenergic receptors with the plasma membrane: a novel mechanism for receptor down-regulation. Life Sci. 35: 1601-1610. STRASSER,R. H., STILES,G. L. and LEFKOWITZ,R. J. (1984) Translocation and uncoupling of the fl-adrenergic receptor in rat lung after catecholamine promoted desensitization /n L,it'o. Endocrinology 115: 1392-1400. STRULOVICL B. and LEFKOWITZ, R. J. (1984) Activation, desensitization, and recycling of frog erythrocyte fl-adrenergic receptors: differential perturbation by in situ trypsinization. J. biol. Chem. 259: 4389-4395. STRULOVICI, B., STADEL,J. M. and LEFKOWITZ,R. J. (1983) Functional integrity of desensitized fl-adrenergic receptors: internalized receptors reconstitute catecholamine-stimulated adenylate activity. J. biol. Chem. 258: 6410-6414. STRULOVICI, B., CERIONE, R. A., KILPATRICK, B. F., CARON, M. G. and LEFKOWITZ, R. J. (1984) Direct demonstration of impaired function of a purified desensitized fl-adrenergic receptor in a reconstituted system. Science 225: 837-840. Su, Y. F., CUBEDDU-XIMENEZ,L. and PERKINS, J. P. (1976a) Regulation of adenosine 3'.5' monophosphate content of human astrocytoma cells: desensitization to catecholamines and prostaglandins. J. Cyclic Nucl. Res. 2: 257-270. Su, Y. F., JOHNSON,G. L., CUBEDDU-XIMENEZ,L., LEICHTLING,B. H., ORTMANN,R. and PERKINS,J. P. (1976b) Regulation of adenosine 3',5" monophosphate content of human astrocytoma cells: mechanism of agonist-specific desensitization. J. Cyclic Nucl. Res. 2: 271-285. Su, Y. F., HARDEN,T. K. and PERKINS,J. P. (1980) Catecholamine-specific desensitization of adenylate cyclase: evidence for a multi-step process. J. biol. Chem. 255: 741(b7419. TAKAYANAG1,I., YOSHIOKA,M., TAKAGI, K. and TAMURA,Z. (1976) Photoaffinity labeling of the fl-adrenergic receptors and receptor reserve for isoprenaline. Fur. J. Pharmacol. 35:121 125. WRENN, S. M. and HOMCY, C. J. (1980) Photoaffinity label for the fl-adrenergic receptor: synthesis and effects on isoproterenol-stimulated adenylate cyclase. Proc. nam. Acad. Sci. U.S.A. 77: 4449~,453.