The β-Adrenergic Receptor: Ligand Binding Studies Illuminate the Mechanism of Receptor-Adenylate Cyclase Coupling

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME i a

The p-Adrenergic Receptor: Ligand Binding Studies Illuminate the Mechanism of Recepto r-Adenylate Cyclase Coupling JEFFREY M. STADEL AND ROBERT J . L E F K O W I Z Department of Medicine (Cardio1og.v). Howard Hughes Medical Institute Duke University Medical Center Durhum, North Carolina

Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Development of Radioligands Specific for Adrenergic Receptors . . . . . . . . . . . . . . . . . 111. Study of Adrenergic Receptors in Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Characterization of Detergent-Solubilized Adrenergic Receptors. . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I.

11.

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45 47 49 57 63

INTRODUCTION

The concept of specific receptors or discriminators that recognize a particular hormone or drug and thereby initiate the biological action of these agents has been evolving for nearly a century (Dale, 1906). A wealth of information has been generated through the investigation of the metabolic effects of pharmacologically active agents, both in vivo and in vitro, and these studies have provided indirect evidence for the existence of discrete receptor moieties. Considerable investigative effort has been directed toward understanding the biological specificity as well as the mechanism of action of catecholamines, since these compounds regulate cellular metabolism in a wide variety of tissues. Evaluation of pharmacological data on the actions of catecholamines in several organs pointed to a logical division of their effects. Ahlquist (1948) proposed the existence of at least two types of adrenergic receptors. His studies, which utilized several catecholamine agonisfs, showed that organ responses could be grouped 45

Copyright 0 1983 by Academic RCW. Inc All right$ of reproduction in any form reserved. ISBN a-12-153xn-2

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JEFFREY M. STADEL AND ROBERT J. LEFKOWITZ

according to the order of potency of these agents in evoking a characteristic response. The first type of response, which he termed “alpha,” followed the agonist potency series epinephrine > norepinephrine > isoproterenol. The second or “beta” group of responses followed the order isoproterenol > epinephrine > norepinephrine. A typical a-adrenergic response is smooth muscle contraction. Smooth muscle relaxation, inotropic and chronotropic regulation in the heart, and metabolic effects such as lipolysis are mediated through P-adrenergic receptors. Further support for the concept of two discrete types of adrenergic responses was provided by the development of highly potent antagonist compounds. aAdrenergic responses to catecholamines are competitively blocked by drugs such as phentolamine, phenoxybenzamine, and the ergot-alkaloids. P-Adrenergic responses are inhibited by a different set of drugs which has little affinity for the areceptors, e.g., propranolol, alprenolol, and pindolol. The characteristics of high potency and biological specificity make antagonist compounds particularly useful in the classification of adrenergic responses and in the direct study of adrenergic receptors (see below). Recent, extensive studies using a large arsenal of synthetic compounds specific for adrenergic receptors have pointed to a further division of both a-(Berthelson and Pettinger, 1977) and P- (Lands et al., 1964) adrenergic responses into pharmacologically defined subtypes. The subclassification of P-adrenergic receptors is based on the relative potency of epinephrine and norepinephrine (Lands et al., 1964). p I-Receptors demonstrate approximately equal affinity for epinephrine and norepinephrine, whereas P,-receptors recognize epinephrine with higher affinity than norepinephrine. More recently it has been realized that a-adrenergic receptors can also be divided into receptor subtypes (Berthelson and Pettinger, 1977; Hoffman and Lefkowitz, 1980). a,and a,-Adrenergic receptors are differentiated by their affinities for a variety of subtype selective agents. For example, a,-receptors have very high affinity for the antagonist prazosin, whereas a,-receptors have high affinity for the antagonist yohimbine. It is also possible to distinguish between a-and P-adrenergic responses based on the biochemical events necessary to produce the physiological changes that accompany adrenergic stimulation. (3-Adrenergic responses are directly linked to the activation of adenylate cyclase in the plasma membranes of target cells, suggesting that cyclic AMP (CAMP) mediates the regulatory effects of P-adrenergic agonists (Robison el a/., 197 1). This is true of the responses mediated by both PI- and p,-receptor subtypes. a-Adrenergic responses, on the other hand, are not always linked to adenylate cyclase. Agonist binding to a,-adrenergic receptors does not affect adenylate cyclase activity directly. However, alterations in Ca2 fluxes have been implicated in aI-adrenergic responses (Exton, 1979). a,-Adrenergic receptors do appear to be linked to adenylate cyclase activity, +

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which they inhibit (Jakobs et al., 1976). This is in contrast to P-adrenergic receptors, which always stimulate the production of CAMP. The purpose of this article is to relate the study of adrenergic receptors to the regulation of adenylate cyclase activity. The ubiquitous nature of the adrenergic responses has made these systems prototypes or models for the study of receptor-effector coupling in general. Progress is being made in the elucidation of the molecular mechanisms of transmembrane signaling via adrenergic receptors. We will focus primarily on studies of the P-adrenergic receptor-adenylate cyclase complex. Work on P-adrenergic stimulation of adenylate cyclase is now proceeding toward the purification and reconstitution of the molecular components of the receptor-cyclase complex. This will be necessary to define precisely the chain of events which links agonist occupancy of the receptors to enzyme activation. The components currently identified include the agonist binding sites or receptors, the enzyme catalytic unit, and the guanine nucleotide regulatory protein (for reviews see Ross and Gilman, 1980; Stadel et al., 1982). Although in this article we rely heavily on the work derived from investigations in our own laboratory, we also relate our observations to those of other investigators. In addition, we will point out, where appropriate, how understanding of receptor-cyclase coupling distilled from the investigations of the stimulatory p-adrenergic receptor-adenylate cyclase system may also be applicable in understanding inhibition of adenylate cyclase activity, as mediated, for example, by a,-adrenergic receptors.

II. DEVELOPMENT OF RADIOLIGANDS SPECIFIC FOR ADRENERGIC RECEPTORS Pharmacological studies using intact tissues or broken cell preparations have indicated the existence of adrenergic receptor subtypes. Although such pharmacological studies suggested the existence of discrete receptor moieties, more direct experimental approaches were required to document this fact. As so often occurs in science, technical innovations led the way to an explosion of information about the nature of hormone and drug receptors. In the late 1960s and early 1970s procedures were developed to incorporate radionuclides covalently into hormone structures with little or no alteration in the biological activity and specificity of these hormones (Roth, 1973). These procedures were first applied to peptide hormones such as ACTH (Lefkowitz el al., 1970) and angiotensin (Lyn and Goodfriend, 1970). In 1974 three laboratories reported the successful development of radioligands specific for the P-adrenergic receptor (Atlas et al., 1974.;Aurbach et al., 1974; Lefkowitz et al., 1974). All three groups utilized P-

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M. STADEL AND ROBERT J. LEFKOWITZ

adrenergic antagonists, albeit three different compounds, to take advantage of the high affinity and specificity of these agents. Of the three original radioligands, ( +)[3H]propranolol (Atlas er al., 1974), ( -)[3H]dihydroalprenolol (DHA) (Lefkowitz et al., 1974), and ( +)'2sI-labeled hydroxybenzylpindolol (HYP) (Aurbach et af., 1974), the latter two, [3H]DHA' and '2sI-labeled HYP, are still widely used to characterize P-adrenergic receptors in a variety of tissues. Recently two more antagonist radioligands have been developed, ( +-)'251-labeled cyanopindolol (Engle, 1980) and (*)I2Tlabeled pindolol (Barovsky and Brooker, 1980). The utility of all five of these radioligands rests in the fact that they are of very high specific radioactivity (50-2000 Ci/mmole) and high affinity ( K , < 10 nM). Although specific for P-adrenergic receptors, these radioligands do not discriminate between P I - and P,-receptor subtypes. Shortly after the development of P-adrenergic specific radioligands, Williams and Lefkowitz (1976) reported that a tritiated derivative of an ergot alkaloid, ( -+)[3H]dihydroergocryptine, could be used to identify a-adrenergic receptors. [3H]Dihydroergocryptineis a potent a-adrenergic antagonist as are many of the subsequently developed radioligands for characterization of a-adrenergic receptors such as (?)[3H]WB4101 (Greenberg et al., 1976), (*)['H]prazosin (Greengrass and Brenner, 19791, and (+)[3H]yohimbine (Tharp er al., 1981). Unlike [3H]dihydroergocryptine, which does not discriminate between a-adrenergic receptor subtypes, [3H]prazosin and ['Hlyohimbine are highly selective for a,and a,-receptors, respectively. Although radiolabeled antagonist ligands have been valuable tools for determining the properties of adrenergic receptors, an underlying goal of direct receptor studies is to understand how agonist binding to receptors initiates a biological response. The study of receptor binding characteristics by employing radiolabeled antagonists in competition with unlabeled adrenergic agonists has provided indirect information as to how agonist drugs might work to activate intracellular signaling mechanisms (see below). However, binding studies with radiolabeled agonists would clearly provide an extra dimension to the investigation of how agonists interact with their receptors. Lefkowitz and Williams (1977) described the successful labeling of P-adrenergic receptors in frog erythrocyte membranes with the agonist ligand (+-)[3H]hydroxybenzylisoproterenol(['HIHBI). This agonist radioligand has been an important tool for probing the properties of Padrenergic receptors and particularly P,-receptors since the ligand is more potent in binding to this receptor subtype. [3H]HBI appears to be virtually unique as an 'Abbreviations used: (3H]DHA, (-)~3H]dihydroalprenolol: [12sI]HYP, (2)1251-labeled hydroxybenzylpindolol; ['HIHBI, ( ~)[3H]hydroxybenzylisoproterenol;Gpp(NH)p, guanyl-5'-yl imidodiphosphate; GTPyS, guanosine S'-O-(3-thiotriphosphate): ACTH, adrenocorticotrophic hormone; NEM, N-ethylmaleimide, SDS, sodium dodecyl sulfate; H, hormone or agonist; R , receptor; N , guanine nucleotide regulatory protein; C, adenylate cyclase catalytic unit; X, an unspecified additional membrane component.

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49

agonist radioligand specific for P-adrenergic receptors, although [ 3H]epinephrine has been reported to identify f3-adrenergic receptors in brain (U’Prichard et a / ., 1978). [3HH]Epinephrinehas, in fact, been more extensively used to investigate aadrenergic receptors (U’Prichard and Snyder, 1977; U’Prichard et d.,1977). In addition to [3H]epinephrine, [3HH]clonidine(U’Prichard et at., 1977), a partial 01adrenergic agonist, can be used in some systems. These agonist ligands have been used to confirm and extend early indirect observations of receptor binding properties employing radiolabeled antagonists in competition with unlabeled adrenergic agonist agents. The development of radioligands that identify specific receptors for drugs and hormones has had a far reaching impact on the investigation of the mechanisms of action of these agents. Radiolabeled ligands have now been developed for nearly every type of receptor known to the pharmacologist. The concept of “receptor” now implies a specific molecular entity whose properties can be probed and evaluated. Many different receptors have been extensively characterized with direct binding studies and the development of these sensitive assay methods has made possible the goal of receptor purification and biochemical analysis.

111.

STUDY OF ADRENERGIC RECEPTORS IN MEMBRANES

Radioligands have been most extensively used to characterize receptors on the surface of target cells or in plasma membrane preparations derived from these cells. It is necessary that a radioligand fulfill several essential criteria for identification of physiologically relevant receptor sites (Williams and Leflcowitz, 1978). (1) The binding of the radioligand to receptor preparations should be saturable, reflecting binding to a finite number of receptor sites. ( 2 ) The concentration range over which the ligand binds to the receptor should be comparable to the concentration range over which the ligand initiates or inhibits a biological response. (3) The kinetics of binding should likewise reflect the kinetics of the biological response. (4)The receptor sites labeled should demonstrate the appropriate specificity of the biological response. In the case of the P-adrenergic receptor this last criterion means that the ability of different agonists to compete for the binding of the radioligand must follow the same potency series which is characteristic of the stimulation of adenylate cyclase by catecholamines, namely, isoproterenol > epinephrine > norepinephrine. In addition, P-adrenergic antagonists should potently compete for the binding of the radioligand, reflecting their ability to inhibit cyclase stimulation. Furthermore, the biologically active (-) stereoisomers of adrenergic agonists and antagonists should be more potent in competing for the binding sites than the less active (+) stereoisomers. The radioligands currently employed to identify adrenergic receptors (see above)

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JEFFREY M. STADEL AND ROBERT J. LEFKOWITZ

have been shown to fulfill these criteria and therefore label physiologically relevant receptors in a variety of tissues. One of the earliest applications of the radioligand binding technique to the study of P-adrenergic receptors was the investigation of the interaction of a wide variety of unlabeled P-adrenergic agonist and antagonist compounds with the receptors. This is accomplished through competition binding experiments in which the dose-dependent inhibition of binding of a fixed concentration of radioligand is examined. By comparing the affinities of these unlabeled agents for the ligand binding sites with their abilities to modulate adenylate cyclase activity, it could be convincingly shown that agonists and antagonists compete for the same set of P-adrenergic receptor binding sites. The affinity of both agonists and antagonists for the P-receptors is primarily determined by their stereo configuration and the substitutions on the amino nitrogen. These studies also reinforced the notion that agonist “activity” is not simply related to binding affinity, but rather involves additional interactions not triggered by antagonists. Following the validation of the radioligand binding approach for studying adrenergic receptors, investigative emphasis shifted toward utilizing these new tools to explore the mechanism of agonist activation of adenylate cyclase. Agonist agents interact with P-adrenergic receptors in a fundamentally different way than do antagonists in order to initiate the chain of events leading to characteristic biological responses. Radioligand binding assays provided an opportunity to examine these differences at the receptor level. One of the first unique properties of agonist binding to (3-adrenergic receptors which was documented was that guanine nucleotides modulate receptor affinity for agonists but not antagonists (Maguire et al., 1976; Lefkowitz et al., 1976). The rationale for exploring the effects of guanine nucleotides on adrenergic receptor binding properties was based on the studies of Rodbell er al. (1971), who demonstrated that these nucleotides were essential regulators of the glucagon receptor-adenylate cyclase complex in rat liver membranes. For (3-adrenergic receptors, the initial demonstrations of guanine nucleotide regulation of agonist binding were accomplished using partially purified plasma membranes prepared from C6 glioma cells (Maguire et al., 1976) and frog erythrocytes (Lefkowitz et al., 1976). It was found that agonist competition binding curves vs radiolabeled antagonists were “shallow” in the absence of guanine nucleotides but became steeper and shifted toward the right, indicating a lower apparent affinity of the receptor for agonist, in the presence of exogenously added guanine nucleotides such as GTP or Gpp(NH)p (Fig. 1). The addition of guanine nucleotides to the binding assay did not affect the binding of the radiolabeled antagonist nor did it affect the shape or position of the competition binding curves generated by unlabeled antagonists (Fig. 2). These curves are “steep” and uniphasic under all experimental conditions. The guanine nucleotide-dependent ‘‘shift to the right” of the agonist binding curves, i.e., to lower receptor affinity, was observed for a series of p-

51

THE (3-ADRENERGICRECEPTOR: LIGAND BINDING STUDIES

9

8

7

-loglo

6

5

I-)Isoproterenol]

4

3

2

(M)

FIG. I . Computer modeling of competition binding data of isoproterenol for r3H]DHA in frog erythrocyte membranes. Competition of the agonist isoproterenol for ['HIDHA in the absence (0) and presence (0) of CTP. The curve in the absence of nucleotide was significantly ( p < 0.00 I ) better fit by a model for two binding states of the receptor. See text for details. (From Kent et a / ., 1979.)

25 5

5 100

s F

u

0 0

80-

(-1 Alprenolot

0

m

-

5

UL= KH * 12nM

60-

C

ea

0

40-

-s5

20-

I

0

e

D %

I

.-..

1

I

1

1

I

,. r

FIG. 2. Computer modeling of competition binding data of alprenolol for ['HIDHA in frog erythrocyte membranes. The competition curve of the antagonist alprenolol for ["IDHA is adequately modeled to a homogeneous class of binding sites. (From Kent er a / . . 1979.)

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JEFFREY M. STADEL AND ROBERT J. LEFKOWITZ

adrenergic agonists (Lefiowitz et a / ., 1976). Interestingly the magnitude of this “nucleotide-dependent shift” correlates with the intrinsic activity of the agonist for activation of the adenylate cyclase. Intrinsic activity is a term used to quantitate the maximum ability of an agonist to stimulate a biological response, such as adenylate cyclase activity. The correlation of intrinsic activity and nucleotide effects on agonist binding suggests a relationship between guanine nucleotide regulation of receptor affinity for an agonist and the drug’s ability to stimulate the cyclase enzyme. Thus agonists, but not antagonists, have the ability to form a high-affinity complex with the P-adrenergic receptor, and this agonist-receptor complex is modulated by guanine nucleotides. Computer-aided analysis of radioligand binding data has added a new quantitative dimension to our understanding of the differences between agonist and antagonist binding to P-adrenergic receptors. As described above, the competition binding curves of adrenergic agonists for a radiolabeled antagonist such as [3H]DHA are shallow (slope factors < 1) indicating complex binding interactions between agonists and the receptors. In contrast, the competition binding curve of unlabeled antagonists for [3H]DHA is always steep (slope factor = I ) indicating a uniform affinity of the receptors for antagonists. Computer modeling of the shallow agonist competition binding curves indicated a statistically significant improvement in the fit of the binding data by a model based on two binding states of the receptor (Fig. I ) (Kent er al., 1979). This two-state model was found to be appropriate for all agonists tested. The two affinity states of the receptor were characterized by specific dissociation constants (K,, KL) and the proportion of the total receptor population in each state was determined (RH, RL). Using a series of full and partial agonists, a significant correlation was shown to exist between the ability of an agonist to stimulate the adenylate cyclase (intrinsic activity) and the ratio of the dissociation constants of the agonist for the high- and low-affinity states of the receptor (KLIKH).A significant correlation was also found to exist between agonist intrinsic activity and the proportion of the receptors binding the agonist with high affinity (% R H ) (Kent et ai., 1979). Quantitative analysis of radioligand binding data thus provides additional evidence for the important role of agonist high-affinity binding in the process of transmembrane signaling by receptor-cyclase complexes. The ability of an agonist to form a high-affinity, nucleotide-sensitive complex with the P-adrenergic receptor is dependent upon the ionic environment of the membranes during the binding assay. Although neither receptor binding properties nor adenylate cyclase activity shows a significant dependence on ionic strength, divalent cations can be shown to be required for both of these activities. Concentrations of Mg2+ or Mn2+ in the millimolar range are necessary for adenylate cyclase activity and these same cations are also required for agonist high-affinity binding to receptors (Williams et al., 1978). Monovalent cations cannot substitute for these divalent metal ions. The regulation of receptor affinity

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by divalent cations is observed only with agonist binding; antagonist interactions with the P-adrenergic receptor are unperturbed by the presence or absence of metal ions in the binding medium. Subsequent radioligand binding studies have demonstrated several additional unique binding properties of agonists to P-adrenergic receptors. Pike and Lefkowitz (1978) reported that decreasing the temperature of the binding assay incubation increased the apparent affinity of turkey erythrocyte P-adrenergic receptors for agonists without a significant effect on receptor affinity for antagonists. This observation has recently been extended to show a similar specific temperature effect on agonist binding to (3-adrenergic receptors in a variety of mammalian tissues containing both p,- and @,-receptor subtypes (Weiland e l a / . , 1980). Briggs and Lefkowitz (1980) were able to show that when assayed below physiological temperatures, agonists are not able to induce the high-affinity, nucleotide-sensitive state of the P-adrenergic receptor in turkey erythrocyte membranes and that this observation correlates with an inhibition of the ability of agonists to stimulate the adenylate cyclase in these membranes. Treatment of the turkey erythrocyte membrane with the unsaturated fatty acid cis-vacennic acid, which increases the flujdity of these membranes (Rimon et al., 1978), led to reappearance of the ability of agonists to form a high-affinity complex with the receptor and concomitantly facilitated agonist activation of the adenylate cyclase at low temperature. These effects of temperature on agonist binding characteristics and activation of adenylate cyclase may reflect a specific agonist-induced conformational change in the receptor, specific receptor-lipid interactions, and/ or necessary lateral mobility of the receptor-cyclase components within the lipid matrix. Additional evidence supporting an agonist-induced conformational change in the p-adrenergic receptor comes from studies using N-ethylmaleimide (NEM) in turkey erythrocyte membranes. N-Ethylmaleimide reacts specifically and irreversibly with free sulfhydryl groups of proteins (Means and Feeney, 1971). Pretreatment of turkey erythrocyte membranes with NEM alone does not affect the ability of (3-adrenergic receptors to bind the radiolabeled antagonist [3H]DHA, but pretreatment of these membranes with NEM in the presence of a p-adrenergic agonist resulted in a loss of up to 50% of the (3-receptors as determined by a decrease in binding capacity for ["IDHA (Bottari et al., 1979). Simultaneous treatment of membranes with NEM and antagonist did not result in receptor loss, indicating that agonist binding induces a specific conformational change in the receptor which exposes a cysteine residue to NEM. This agonist-specific effect appears to be related to the mechanism by which agonist binding to receptor results in activation of adenylate cyclase. The rate at which NEM inactivates turkey erythrocyte (3-receptors correlates with the intrinsic activity of the agonist which occupies the receptor (Vauquelin et al., 1979). Guanine nucleotides which

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JEFFREY M. STADEL AND ROBERT J. LEFKOWITZ

regulate receptor affinity for agonists and are required for adenylate cyclase activation also inhibit the inactivation of receptors by NEM in the presence of agonists (Vauquelin et al., 1980). The effect of NEM in the presence of agonist is not restricted to the P ,-adrenergic receptor in turkey erythrocyte membranes. Similar results have been obtained by treating membranes from S49 mouse lymphoma cells (P,-receptor subtype) with NEM and agonist (Vauquelin and Maguire, 1980). It is noteworthy that membranes from S49 mutant cell clones that are functionally devoid of components necessary for guanine nucleotide regulation of receptor affinity failed to show an effect with NEM in the presence of agonists, suggesting that productive coupling between the P-adrenergic receptor and the guanine nucleotide regulatory protein of the adenylate cyclase complex may be necessary for the exposure of the critical sulfhydryl group (Vauquelin and Maguire, 1980). A perplexing question raised by these studies concerns the observation that the maximal effect of NEM and agonist reduces the receptor population only by 50%. Complete inactivation of the receptors was never achieved by this approach. “Heterogeneous” populations of receptors have been offered as a possible explanation, but further investigation will be required to clarify this observation. An important advance in the characterization of agonist binding to P-adrenergic receptors was achieved through the development of an effective radiolabeled agonist ligand. Lefiowitz and Williams (1977) reported the properties of the binding of (+)[ 3H]hydroxybenzylisoproterenolto the P-adrenergic receptor of frog erythrocyte membranes. It became evident that this radioligand would be useful in the direct investigation of P,-adrenergic receptors in purified plasma membrane preparations. Availability of this ligand permitted, for the first time, a direct examination of agonist binding to the P-adrenergic receptor. Previously such studies had been performed by examining agonist competition with radioligand antagonist binding. High-affinity [3H]HBI binding to the P-adrenergic receptor is characterized by a very slow rate of dissociation that is not affected by the addition of competing adrenergic ligands (Williams and Lefiowitz, 1977). In contrast, guanine nucleotides promote the rapid and complete dissociation of [3H]HBI from the membrane receptors. Thus [3H]HBI labels exclusively the high-affinity state of the P-adrenergic receptor in frog erythrocyte membranes. The effect of guanine nucleotides to lower receptor affinity for agonist appears to relate to their ability to destabilize the agonist high-affinity binding state resulting in rapid release of the agonist from the receptor. Chemical treatments of frog erythrocyte membranes which interfere with the ability of agonists to stimulate the adenylate cyclase generally prevent the ability of agonists to form a highaffinity, slowly dissociable complex with the receptor (Williams and Lefkowitz, 1977). Stimulation of adenylate cyclase by guanine nucleotides is therefore associated with a decrease in affinity of the receptor for agonists and rapid dissociation of the high-affinity agonist-receptor complex to free agonist and receptor. Formation of the tight complex between agonist and receptor thus

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appears in some way to facilitate activation of the cyclase enzyme by regulatory nucleotides. It is possible to demonstrate experimentally that the high-affinity, slowly dissociable agonist-receptor complex which is sensitive to guanine nucieotides is an intermediate state on the pathway of coupling between the receptor and adenylate cyclase activation (Stadel et al., 1980). The high-affinity state of the receptor can be isolated by preincubation of purified frog erythrocyte membranes with agonist. These membranes were then washed extensively to remove the free agonist and then assayed for adenylate cyclase activity in the presence of the antagonist propranolol. Basal and NaF-stimulated cyclase activity were unaffected by the preincubation procedures, but the ability of the nonhydrolyzable guanine nucleotide analog Gpp(NH)p to stimulate the enzyme directly was significantly enhanced in membranes preexposed to the agonist compared to membranes preincubated in buffer alone. Experiments of this type suggest that the increased stimulation of the adenylate cyclase by Gpp(NH)p in membranes pretreated with agonist is the result of high-affinity agonist binding to the receptor that persists through the washing procedures. Moreover, they demonstrate that this agonist-receptor complex is an intermediate for agonist activation of cyclase activity. These procedures may be repeated using turkey erythrocyte membranes with qualitatively similar results. Thus the mechanism by which agonists activate adenylate cyclase is similar for both a P I - and a P,-adrenergic receptor (Stadel er af., 1980). Additional insights into the molecular mechanisms of receptor-cyclase coupling can also be gained through the application of computer modeling techniques to radioligand binding data. Quantitative analysis of agonist competition binding curves for [”IDHA in the presence and absence of guanine nucleotides is compatible with the notion that nucleotides mediate a transition between highand low-affinity states of the receptors (Kent er al., 1979). Thus, the agonist competition binding curve is steep in the presence of guanine nucleotides (slope factor = 1) and the uniform dissociation constant for agonist binding to the receptor is identical to the low-affinity dissociation constant ( K J determined for the same agonist in the absence of the nucleotide (Fig. 1). The extent of the transition from high- to low-affinity state is dependent on the concentration of guanine nucleotide in the binding assay. The observations that guanine nucleotide mediates a transition of the agonist high-affinity state of the receptor to the low-affinity state without a similar effect in antagonist binding, and that partial agonists induce differing proportions of the receptor into the high- and low-affinity state at equilibrium (% R H ) ,represent strong evidence that the highand low-affinity states of the agonist-occupied receptors are interconvertible (Kent et al., 1979). A systematic comparison of the ability of several mechanistic models to fit and reproduce agonist competition binding data in the presence and absence of guanine nucleotides led De Lean er af. (1980) to propose a “ternary complex”

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JEFFREY M. STADEL AND ROBERT J. LEFKOWITZ

(precoupled)

FIG.3. Schematic diagram of the ternary complex model. The model involves the interaction of the hormone (H), the receptor (R), and an additional membrane component (X). Nucleotide-dependent coupling between the ternary complex (HRX) and activation of adenylate cyclase (E) is also shown. (From De Lean er al., 1980.)

model as an explanation for the agonist-specific binding properties of P-adrenergic receptors. This model involves the interaction of the receptor (R) with an additional membrane component (X) in the presence of agonist (H) to form a high-affinity ternary complex HRX (Fig. 3). Agonist initially binds to the receptor to form a low-affinity binary complex HR which precedes the ternary complex formation. The modeling indicates that the stoichiometry between the receptor and the component X is close to I:]. The intrinsic activity of an agonist correlates with the affinity constant (L) for the combinations of the agonistreceptor complex (HR) with the additional membrane component X: HR

+X

L

HRX

(Fig. 3). This correlation is entirely consistent with the relationships alluded to above between agonist intrinsic activity and other quantitative parameters of the agonist-promoted high-affinity state (KL/K,l, % RH). The computer-aided modeling of the binding data is independent of the nature of the additional membrane component X, but several lines of evidence suggest that X is a guanine nucleotide regulatory protein (N). The computer analyses of binding data indicate that the presence of guanine nucleotides in the binding assay specifically decreases the ability of an agonist to stabilize the ternary complex between HR and X (De Lean ef al., 1980). Biochemical experiments using high concentrations of Mn2+ ( > l o mM) to uncouple agonist binding to receptors from activation of adenylate cyclase (Limbird et al., 1979) or using the specific sulfhydryl reagent N-ethylmaleimide to inactivate adenylate cyclase catalytic activity (Howlett et al., 1978; Stadel and Lefkowitz, 1979) have demonstrated that a functional cyclase enzyme is not

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required for agonists to promote formation of a high-affinity, nucleotide-sensitive complex with the receptor. However, Stadel and Lefkowitz (1979) have shown that an additional membrane component necessary for agonist high-affinity binding was also sensitive to NEM, but at concentrations 100-fold greater than that necessary to inactivate cyclase catalytic activity. The effect of NEM on agonist high-affinity binding is distal to the ligand binding site of the receptor, since agonist low-affinity binding (in the presence of guanine nucleotide) and the binding of the competitive antagonist [ 3H]DHA were unaffected. Preformation of the agonist high-affinity state of the receptor protected the complex against the effects of NEM, and the complex was still fully sensitive to modulation by guanine nucleotides (Stadel and Lefkowitz, 1979). It is therefore unlikely that the ternary complex (HRX) contains the cyclase enzyme. However, the observation that agonist binding to the P-adrenergic receptor is uniquely modulated by guanine nucleotides is consistent with the notion that component X is a guanine nucleotide regulatory protein. It is of interest to note that a-adrenergic agonist binding to a,-receptors in platelets (Tsai and Lefkowitz, 1979) or neural cell lines (Haga and Haga, 1981) is also characterized by shallow competition binding curves with radiolabeled antagonists that steepen and shift to the right in the presence of guanine nucleotides (Hoffman et al., 1980). The guanine nucleotide sensitivity of the a,receptor is also an agonist-specific property since the affinity of antagonists appears to be unperturbed by the addition of nucleotides. The nucleotide sensitivity of agonist binding to a,-adrenergic receptors again provides clues to understanding the mode of action of a-adrenergic agonists in inhibiting adenylate cyclase, since guanine nucleotides are also stringently required for coupling of these receptors to the catalytic moiety of the enzyme (Jakobs ef al., 1978). Although computer modeling using the ternary complex model (De Lean el al., 1980) has not been applied to binding data for a-adrenergic agonists, it appears likely that such a complex is in fact an intermediate for inhibition as well as stimulation of adenylate cyclase.

IV. CHARACTERIZATION OF DETERGENT-SOLUBILIZED ADRENERGIC RECEPTORS A first step toward the biochemical characterization of membrane-bound hormone receptors is the solubilization of the binding activity from the lipid bilayer through the use of detergents. The P-adrenergic receptor was first solubilized from frog erythrocyte membranes using the plant glycoside digitonin (Caron and Lefkowitz, 1976). Although many different detergents were tested, digitonin was found to be uniquely capable of extracting the receptor in an active form. The binding properties of the soluble receptor sites were in most respects essen-

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tially the same as those of the membrane-bound receptors (Caron and Lefkowitz, 1976). The soluble receptors demonstrate the appropriate potency order for pzadrenergic receptors (isoproterenol > epinephrine > norepinephrine) and strict stereoselectivity for binding both agonist and antagonist ligands. The single difference in the properties of the soluble receptors is that agonist binding to soluble receptors is of uniformly low affinity. Thus agonists do not promote formation of the high-affinity state of the receptor in soluble preparations. This observation correlates with the inability of agonists to stimulate adenylate cyclase activity in these soluble preparations (Caron and Lefkowitz, 1976). Fractionation of detergent extracts by gel filtration (Limbird and Lefkowitz, 1977) or on sucrose gradients (Haga et al., 1977) has led to a clear resolution of receptor binding activity from adenylate cyclase activity, thus demonstrating that these two activities reside on different polypeptide chains. Studies of soluble receptor preparations have shed light on the unique interactions of P-adrenergic receptors with agonist agents. Soluble extracts from frog erythrocyte or rat reticulocyte membranes which were prelabeled with either the radiolabeled agonist [3H]HBI or the radiolabeled antagonist [3H]DHAwere fractionated by gel filtration over AcA34 resin (Limbird and Lefkowitz, 1978; Limbird et al., 1980b). The ['HIHBI prelabeled receptor was resolved from the antagonist-occupied receptor and appeared to elute with an apparent larger molecular size (Fig. 4A). Several explanations are consistent with this observation including an agonist-promoted asymmetric conformational change in the receptor, agonist-induced receptor aggregation, or the stable association of the agonist-occupied receptor with an additional membrane component. This latter explanation is, of course, consistent with the computer modeling described above. The proposed additional component of the agonist-receptor high-affinity state did not appear to be the cyclase enzyme itself, since the enzyme activity eluted several fractions removed from the receptor binding activity. Additional experiments using rat reticulocyte membranes implicated the nucleotide regulatory protein as a constituent of the agonist-receptor high-affinity complex (Fig. 4B). Prelabeling of rat reticulocyte membranes with [3H]HBI in the presence of guanine nucleotide allows agonist binding to the low-affinity form of the receptor. This low-affinity agonist-receptor complex survives the gel filtration procedures and now coelutes with the smaller antagonist prelabeled receptor. Thus guanine nucleotides which destabilize the high-affinity state of the receptor in the membrane also convert the larger molecular form of the agonist-receptor complex to a smaller species that coelutes with antagonistoccupied receptor. More direct evidence as to the molecular compositions of the agonist-promoted ternary complex was obtained by radioactively labeling of the nucleotide regulatory protein of the adenylate cyclase complex (Limbird et al., 1980a). Cholera toxin catalyzes the covalent transfer of ADP-ribose from NAD to the +

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59

FIG.4. Gel exclusion chromatography of P-adrenergic receptors solubilized from rat reticulocyte membranes with digitonin after prelabeling with agonist ['HIHBI or antagonist [ 3H]DHA. The column material was AcA34. (A) Prelabeling in the absence of nucleotides. (B) Prelabeling conducted in the presence of 0. I mM Gpp(NH)p. (From Limbird et a / . , 1980b.)

42,000 M , subunit of the guanine nucleotide regulatory protein (for reviews see Ross and Gilman, 1980; Stadel et al., 1982). If 32P-labeledNAD+ is used as the cofactor for the toxin, a radioactive tag is covalently incorporated into the nucleotide regulatory protein. Limbird et al. (1980a) were able to show that agonist pretreatment of rat reticulocyte membranes prior to solubilization resulted in the coelution of the 32P-labeled 42,000 M , protein in the [3H]HBI-receptor region from the gel filtration column. Similar pretreatment of these membranes with antagonist did not cause the labeled subunit of the nucleotide regulatory protein to associate with the receptor. These experiments provide biochemical evidence that agonist occupancy of the P-adrenergic receptor promotes the association of the receptor with the guanine nucleotide regulatory protein of the adenylate cyclase complex.

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Several recent reports provide additional evidence that the high-affinity, nucleotide-sensitive agonist-receptor complex represents a ternary complex of HRN. Cholate-solubilized extracts of membranes prepared from wild-type S49 lymphoma cells successfully reconstituted hormonally sensitive adenylate cyclase activity in membranes from S49 mutant cell clones that are functionally uncoupled (Sternweis and Gilman, 1979). The critical factor in the detergent extract that allows recoupling of the receptors to the cyclase is the guanine nucleotide regulatory protein (Ross et al., 1978). The P-adrenergic receptors of the mutant cell membranes reconstituted with the cholate extracts of S49 wildtype membranes also demonstrate nucleotide sensitivity of receptor affinity for agonists. It was not possible to separate the component necessary for recoupling of hormonal activation of the cyclase from the component required for restoration of nucleotide-sensitive agonist high-affinity binding to the P-adrenergic receptors. These experiments are consistent with the notion of a single membrane component regulating receptor affinity for agonists and for coupling agonist occupancy of the receptor to activation of the cyclase enzyme. Using reconstitution of lubrol-solubilized components, Stadel et al. (198 1) were able to isolate the nucleotide regulatory protein associated with the 6receptor as a result of agonist binding and subsequently show that this N protein conveyed nucleotide-dependent adenylate cyclase activity to a suitable catalytic unit acceptor. The high-affinity ternary complex HRN was solubilized from frog erythrocyte membranes in the nonionic detergent lubrol and then bound to wheat germ agglutinin immobilized on Sepharose. The ternary complex is bound to the lectin through the carbohydrate moieties of the receptor, which is a glycoprotein (Shorr et al., 1980). After extensive washing of the lectin gel the resin was eluted in the presence of GTPyS. The guanine nucleotide destabilizes the ternary complex HRN resulting in the release of an N-GTPyS complex. The GTPyS eluate from the lectin-Sepharose conveyed nucleotide-sensitive adenylate cyclase to a soluble catalytic unit acceptor. The ability of the GTPyS eluate of the lectin-resin to reconstitute adenylate cyclase activity was strictly dependent on the preformation of the agonist high-affinity state in frog erythrocyte membranes prior to solubilization. The notion that the nucleotide regulatory protein associated with the P-adrenergic receptor by the binding of agonist is the same N that modulates adenylate cyclase activity was supported by additional experimentation (Stadel et at., 1981). Radioactive labeling of the N protein by 32P-labeledNAD+ in the presence of cholera toxin allows the observation of the N protein throughout the solubilization, lectin chromatography, and elution procedures. The amount of 32P-labeled42,000 M, subunit associated with the lectin-resin in soluble extracts from membranes pretreated with agonist or antagonist correlated with the ability of these extracts to stimulate adenylate cyclase activity in the soluble reconstitution assay. These experiments bring together both structural and functional evi-

61

THE B-ADRENERGIC RECEPTOR: LIGAND BINDING STUDIES

p- Adrenergic Receptor Cycle

Nucleotide Regulatory

Adenylate Cyclase

Protein Cycle

Catalytic Moiety Cycle

FIG. 5 . Schematic model of hormonal activation of adenylate cyclase involving agonist (H), receptor (R), nucleotide regulatory protein (N), and enzyme catalytic unit ( C ) . See text for details.

dence that a single nucleotide regulatory protein acts as a “coupler” conveying information from the agonist-occupied receptors to the adenylate cyclase. The observations characterizing the unique binding properties of agonists to the P-adrenergic receptor in both membrane and soluble studies are consistent with a model for receptor-cyclase coupling shown in Fig. 5 . This model is based on the information contained in the experiments reviewed above as well as additional investigations of the properties of the enzyme adenylate cyclase reported by other investigators. In this model agonist occupancy of the receptor (Step 1) promotes or stabilizes the formation of the high-affinity HRN complex (Step 2). As a consequence, GDP is released from N, creating a vacant guanine nucleotide binding site (Step 3). The binding of a guanine nucleotide triphosphate to N (Step 4)results in dissociation of the HRN complex, and N-GTP now associates with C (Step 5 ) to stimulate catalytic activity (Pfeuffer, 1977, 1979). As shown by Cassel and Selinger (1976), hydrolysis of GTP by a GTPase associated with the NC complex (Step 6) is the “turn off” mechanism for adenylate cyclase activity and returns the system to the basal state (Step 7). Binding of agonist to the receptor reinitiates the cycle. The major features of the model are ( 1 ) agonist binding results in the stabilization of the high-affinity ternary complex HRN which facilitates the exchange of nucleotides bound to N; ( 2 ) the guanine nucleotide regulatory protein acts as a coupler between the receptor and the enzyme catalytic unit; (3) the GTPase activity associated with the NC complex deactivates enzyme catalytic activity and dissociates this complex. This model may also provide a starting point for investigating the mechanism of inhibition of adenylate cyclase mediated by qadrenergic receptors. As described above from radioligand binding studies it is apparent that a,-adrenergic receptors are capable of forming a high-affinity, nucleotide-sensitive complex with agonists but not antagonists. Recent studies (Michel er al., 1981; Smith and

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Limbird, 1981) also show that agonist occupancy of platelet a,-receptors similarly induces an increase in the apparent molecular size of the receptor compared to antagonist-occupied receptors as assessed by centrifugation through sucrose gradients. The soluble agonist-receptor complex is also sensitive to guanine nucleotides, consistent with the notion that the increased molecular size of the agonist-receptor complex is due to the agonist-promoted association of the receptor with a guanine nucleotide regulatory protein (Smith and Limbird, 1981). These data suggest that a-adrenergic inhibition of adenylate cyclase shows many features in common with, and may be analogous to, the mechanism of P-adrenergic stimulation of the cyclase. Further investigation will be necessary to determine how the formulations in the model shown in Fig. 5 for the stimulation of adenylate cyclase might apply to the mechanism of inhibition of the enzyme. A long-range goal of studies of the mechanism of receptor-cyclase coupling is the purification and reconstitution of the individual components of the systems in a functional way. In the past 2 years considerable progress has been made in this regard. The development of sensitive assays for detergent-solubilized components of the complex has allowed the application of biochemical techniques for purification. Purification of these components is a major undertaking since the constituents of the receptor-adenylate cyclase complex exist in very small quantities in the plasma membranes of target cells. Recently, the guanine nucleotide regulatory protein has been purified to apparent homogeneity by classic biochemical techniques (Northup et al., 1980). The purified protein is composed of three heterologous subunits with approximate molecular weights of 52,000, 45,000, and 35,000. The purified guanine nucleotide regulatory protein reconstitutes guanine nucleotide-, hormonal-, and sodium fluoride-dependent stimulation of adenylate cyclase activity in membranes prepared from mutant S49 lymphoma cells that lack a functional regulatory unit. All three subunits appear to be required for successful reconstitution. A key step in the purification of the P-adrenergic receptor was the development of an efficient affinity chromatography gel (Caron et a!., 1979). Alprenolol was immobilized on Sepharose 4B through a hydrophilic spacer arm. The biospecific nature of the interaction of the digitonin-solubilized P-adrenergic receptor with the affinity gel could be demonstrated. Both the adsorption of the soluble frog erythrocyte P-receptor to the resin and its subsequent elution demonstrated typical P-adrenergic specificity. For both processes (blocking adsorption and promoting elution), the agonist potency order was isoproterenol > epinephrine > norepinephrine and stereoselectivity was preserved for both agonist and antagonist agents. The resin adsorbed up to 95% of the receptor in the soluble preparations, and 60% was ultimately specifically eluted. By recycling the soluble receptor preparation through the affinity resin the purification was over 15,000fold from the original membranes (Caron et al., 1979). By coupling the affinity chromatography procedures to ion exchange chro-

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63

matography the ligand binding site of the P-adrenergic receptor has been purified 55,000-fold (Shorr et al., 1981). The purified receptor demonstrates a 58,000 M, band on SDS-polyacrylamide gel electrophoresis. The purified 58,000Mr protein comigrates with soluble radioligand antagonist prelabeled receptor on sucrose gradients and in isoelectric focusing procedures. Ligand binding experiments using I3H]DHA and the purified protein demonstrate the affinity, specificity, and stereoselectivity expected for the P-adrenergic receptor (Shorr et al., 1981). The purification of the P-adrenergic receptor and the nucleotide regulatory protein leaves the catalytic unit of adenylate cyclase as the only known component remaining to be purified. The rapid progress that has been made in the isolation and characterization of the molecular components of the P-adrenergic receptor-adenylate cyclase complex raises expectations that functional reconstitution of this system will be achieved in the not too distant future. REFERENCES Ahlquist, R. P. (1948). A study of the adrenotropic receptors. Am. J . Phvsiol. 153, 586-600. Atlas, D.. Steer, M. L., and Levitzki, A. (1974). Stereospecific binding of propranolol and catecholamines to the beta-adrenergic receptor. Proc. Narl. 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 beta-adrenergic receptor: Stereospecific interaction of an iodinated beta-blocking agent with a high affinity site. Science 186, 1223-1224. Barovsky, K..and Brooker, G . (1980). (-)('2sI]IodopindoloI, a new highly selective radioiodinated P-adrenergic receptor antagonist: Measurement of (3-receptors on intact rat astrocytoma cells. J. Cyclic Nucleoride Res. 6, 297-307. Berthelson, S., and Pettinger, W. A. (1977). A functional basis for the classification of alphaadrenergic receptors. Ljfe Sci. 21, 595-606. Bottari. S . , Vauquelin, 0.. Durien, O., Klutchko, C., and Strosberg, A. D. (1979). The P-adrenergic receptor of turkey erythrocyte membranes: Conformation modification by P-adrenergic agonists. Biochem. Biophys. Res. Commun. 86, 131 1-1318. Briggs, M. M., and Lefkowitz, R. J. (1980). Parallel modulation of catecholamine activation of adenylate cyclase and formation of the high-affinity agonist-receptor complex in turkey erythrocyte membranes by temperature and cis-vaccenic acid. Biochemisrv 19, 4461-4466. Caron, M. G . , and Lefkowitz, R . J. (1976). Solubilization and characterization of the P-adrenergic receptor binding sites of frog erythrocytes. J. B i d . Chem. 251, 2374-2384. Caron, M. G., Srinivasan, Y.,Pitha, J., Kociolek, K., and Letkowitz, R. J. (1979). Affinity chromatography of the P-adrenergic receptor. J. B i d . Chem. 254, 2923-2927. Cassel, D.,and Selinger, 2. (1976). Catecholamine-stimulated GTPase activity in turkey erythrocyte membrane. Biochim. Biophys. Acta 452, 538-55 I . Dale, H. H. (1906). On some physiological actions of ergot. J. Physiol. (London) 34, 165-206. De Lean. A . , Stadel, J. M., and Letkowitz, R. J . (1980). A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled P-adrenergic receptor. J. B i d . Chem. 255, 7108-71 17. Engle, G . ( 1 980). Identification of different subgroups of beta-receptors by means of binding studies in guinea-pig and human lung. Triangle 19, 69-76. Exton, J. H. (1979). Mechanisms involved in effects of catecholamines on liver carbohydrate metabolism. Biochem. Pharmacol. 28, 2237-2246.

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Greenberg, D. A , , U'Prichard, D. C., and Snyder, S. H. (1976). Alpha-noradrenergic receptor binding in mammalian brain: Differential labelling of agonist and antagonist states. Life Sci. 191, 69-76. Greengrass, P.. and Brenner, R. (1979). Binding characteristics of [3H]prazosin to rat brain alphaadrenergic receptors. Eur. J. Pharmacol. 55, 323-325. Haga, T., and Haga, K . (1981 ). Characterization by [3H]dihydroergocryptine binding of alphaadrenergic receptors in neuroblastomd X glioma hybrid cells. J. Neurochem. 36, 1152- 1159. Haga. T., Haga, K., and Gilman, A. G. (1977). Hydrodynamic properties of the f3-adrenergic receptor and adenylate cyclase from wild-type and variant S49 lymphoma cells. J . Biol.Chem. 252, 5776-5782. Hoffman, B. B., and Lefkowitz, R. J. (1980). Radioligand binding studies of adrenergic receptors. Annu. Rev. Pharmaml. Toxicol. 26, 581 -608. Hoffmann, B . B . , Mullikin-Kilpatrick, D., and Lefkowitz, R. J. (1980). Heterogeneity of radioligand binding to a-adrenergic receptors. J. B i d . Chem. 255, 4645-4652. Howlett, A. C., Van Arsdale, P. M., and Gilman, A. G . (1978). Efficiency of coupling between the beta-adrenergic receptor and adenylate cyclase. Mol. Pharmarol. 14, 53 1-539. Jakobs, K. H., Saur, W . , and Schultz, G . (1976). Reduction of adenylate cyclase activity in lysates of human platelets by alpha-adrenergic component of epinephrine. J. Cyclic. Nucleotide Res. 2, 381-392. Jakobs, K. H., Saur, W . , and Schultz, G. (1978). Inhibition of platelet adenylate cyclase by epinephrine requires GTP. FEBS Lett. 85, 167-170. Kent, R. S . , De Lean, A., and Lefkowitz, R. J. (1979). A quantitative analysis of beta-adrenergic receptor interactions: Resolution of high and low affinity states of the receptor by computer modeling of ligand binding data. Mol. Pharmacol. 17, 14-23. Lands, A. M., Arnold. A.. McAuliff, J. P., Luduena, F. P., and Braun, T. G . (1964). Differentiation of receptor systems activated by sympathomimetic amines. Nature (London) 214, 597-598. Lefkowitz, R. J., and Williams, L. T. (1977). Catecholamine binding to the beta-adrenergic receptor. Proc. Natl. Acad. Sci. U.S.A. 74, 515-519. Lefkowitz, R. J., Roth, I . , Pricer, W., and Pastan. I. (1970). ACTH receptors: Specific binding of ACTH-['2sI] and its relation to adenyl cyclase. Proc. Natl. Acad. Sci. U.S.A. 65, 745-752. Letkowitz. R . J . , Mukherjee, C., Coverstone, M . , and Caron, M. G. (1974). Stereospecific [3Hl(-)alprenolol binding sites, beta-adrenergic receptors and adenyl cyclase. Biochem. Biophys. Res. Commun. 60, 703-709. Lefkowitz, R. J., Mullikin, D.. and Caron, M. G . (1976). Regulation of beta-adrenergic receptors iw guanyl-5'-yl imidophosphate and other purine nucleotides. J. B i d . Chem. 251, 4680 4692. Limbird, L. E . , and Lefkowitz, R. J. (1977). Resolution of f3-adrenergic receptor lmiding and adenylate cyclase activity by gel exclusion chromatography. J. Biol. Chem. 252, 799-802. Limbird, L. E., and Lefkowitz, R. J. (1978). Agonist-induced increase in apparent P-adrenergic receptor size. Proc. Natl. Acad. Sci. U.S.A. 75, 228-232. Limbird, L. E., Hickey, A. R., and Lefkowitz, R. J. (1979). Unique uncoupling of the frog erythrocyte adenylate cyclase system by manganese. J. Biol. Chem. 254, 2677-2683. Limbird. L. E., Gill. D. M., and Lefkowitz, R. J . (1980a). Agonist-promoted coupling of the padrenergic receptor with the guanine nucleotide regulatory protein of the adenylate cyclase system. Proc. Nail. Acad. Sri. U.S.A. 77, 775-779. Limbird, L. E., Gill, D. M., Stadel, J. M., Hickey, A. R., and Lefkowitz, R. J . (1980b). Loss of padrenergic receptor-guanine nucleotide regulatory protein interactions accompanies decline in catecholamine responsiveness of adenylate cyclase in maturing rat erythrocytes. J . Biol. Chem. 255, 1854-1861. Lyn. S. Y., and Goodfriend, T. L. (1970). Angiotensin receptors. Am. J. Phvsiol. 218, 1319-1328.

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Maguire. M. E., Van Arsdale, P. M., and Gilman, A. G . (1976). An agonist-specific effect of guanine nucleotides on binding to the beta-adrenergic receptor. Mol. Pharmacol. 12, 335-339. Means, ti. E..and Feeney, R. E. (1971). “Chemical Modification of Proteins.” Holden-Day, San Francisco, California. Michel,T., Hoffman. B. B., Lefkowitz, R. J., andcaron, M. G . (1981). Differential sedimentation properties of agonist- and antagonist-labelled platelet alphaz-adrenergic receptors. Biochem. Biophys. Res. Cornmun. 100, 1 I3 I - 1 135. Northup, J. K.,Sternweis, P. C.. Smigel, M. D . , Schleifer, L. S., Ross, E. M., andGilman, A. G. (1980). Purification of the regulatory component of adenylate cyclase. Proc. Natl. Acad. Sci. U.S.A. 77, 6516-6520. Pfeuffer, T. (1977). GTP-binding proteins in membranes and the control of adenylate cyclase activity. J . Biol. Chem. 252, 7224-7234. Pfeuffer, T. ( 1979). Guanine nucleotide-controlled interactions between components of adenylate cyclase. FEBS Lett. 101, 85-89. Pike, L. J . , and Lefkowitz, R. I. (1978). Agonist specific alterations in receptor binding affinity associated with solubilization of turkey erythrocyte membrane beta-adrenergic receptors. Mol. Pharmacol. 14, 370-375. Rimon. G.. Hanski, E., Braun. S., and Levitzki, A. (1978). Mode of coupling between hormone receptors and adenylate cyclase elucidated by modulation of membrane fluidity. Nature (London) 276, 394-396. Robison, G. A,, Butcher, R . W . , and Sutherland, E. W . (1971). “Cyclic AMP.” Academic Press, New York. Rodbell, M., Birnbaumer, L., Pohl. S. L., and Krans. H. M. (1971). The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver: An obligatory role of guanyl nucleotides in glucagon action. J . Bio/. Chem. 246, 1877-1882. Ross, E. M., and Gilman, A. G. (1980). Biochemical properties of hormone-sensitive adenylate cyclase. Annu. Rev. Biochem. 49, 533-564. Ross. E. M., Howlett, A. C., Ferguson, K. M., and Gilman, A. 0 . (1978). Reconstitution of hormone-sensitive adenylate cyclase activity with resolved components of the enzyme. J . Biol. Chem. 253, 6406-6412. Roth. J. (1973). Peptide hormone binding to receptors: A review of direct studies in vitro. Merab. Clin. Exp. 22, 1059-1073. Shorr, R. G. L., Caron, M . G., and Lefkowitz, R. J . (1980). Isolation and characterization of betaadrenergic receptors from frog erythrocyte membranes. Fed. Proc., Fed. Am. Soc. Exp. Biol. 39, 1616. Shorr, R . G. L., Lefkowitz, R. 1 . . and Caron, M. G . (1981). Purification of the p-adrenergic receptor: Identification of the hormonal binding subunit. J . B i d . Chem. 256, 5820-5826. Smith, S. K.,and Limbird, L. E. (1981). Solubilization of human platelet a-adrenergic receptors: Evidence that agonist occupancy of the receptor stabilizes receptor-effector interactions. Proc. Nail. Acad. Sci. U.S.A. 78, 4026-4030. Stadel, I. M.. and Lefkowitz, R. J. (1979). Multiple reactive sulfhydryl groups modulate the functions of adenylate cyclase-coupled P-adrenergic receptors. Mol. Pharmacol. 16,709-71 8. Stadel, I. M.,De Lean, A . , and Lefkowitz, R. J . (1980). A high affinity agonist P-adrenergic receptor complex is an intermediate for catecholamine stimulation of adenylate cyclase in turkey and frog erythrocyte membranes. J . B i d . Chem. 255, 1436-1441. Stadel, J. M . , Shorr. R . G . L., Limbird. L. E., and Lefkowitz, R. I. (1981). Evidence that padrenergic receptor associated guanine nucleotide regulatory protein conveys guanosine 5’-O-(3-thiotriphosphate)dependent adenylate cyclase activity. J . Biol. Chem. 256, 8718-8723.

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Stadel, J. M..De Lean, A., and Lefkowitz, R. J. (1982). Molecular mechanisms of coupling in hormone receptor-adenylate cyclase systems. Adv. Enzyrtol. 53, 1-43. Sternweis, P. C., and Gilman, A. G. (1979). Reconstitution of catecholamine-sensitive adenylate cyclase. f . Biol. Chem. 254, 3333-3340. Tharp, D. M., Hoffman, B. B.. and Lefkowitz, R. J . (1981). a-Adrenergic receptors in human adipocyte membranes: Direct determination by ["]yohimbine binding. J. Clin. Endorrinol. Merah. 52, 709-714. Tsai. B. S., and Lefkowitz, R. J. (1979). Agonist-specific effects of guanine nucleotides on alphaadrenergic receptors in human platelets. Mol. Pharmacol. 16, 61-68. U'Prichard, D. C., and Snyder, S. H. (1977). 13H]Epinephrine and [3H]norepinephrine binding to alpha-noradrenergic receptors. L$e Sci. 20, 527-533. U'Prichard, D. C., Greenberg. D. A,, and Snyder, S. H. (1977). Binding characteristics of a radiolabelled agonist and antagonist at central nervous system alpha-noradrenergic receptors. Mol. Pharmacol. 13, 454-473. U'Prichard, D. C., Bylund, D. B., and Snyder. S . H. (1978). (+)-[3H]Epinephrine and ( -)-[7H]dihydroalprenolol binding to P I and Pz-noradrenergic receptors in brain, heart, and lung membranes. J. B i d . Chem. 253, 5090-5102. Vauquelin, G., and Maguire. M. E. (1980). Inactivation of 0-adrenergic receptors by N-ethylmaleimide in S49 lymphoma cells: Agonist induction of functional receptor heterogeneity. Mol. Pharmacol. 18, 362-369. Vauquelin, G., Bottari, S . , and Strosberg. A. D. (1979). Inactivation of P-adrenergic receptors by Nethylmaleimide: Permissive role of P-adrenergic agents in relation to adenylate cyclase activation. Mol. Pharmacol. 17, 163-171. Vauquelin, G., Bottari, S.. Andre, C.. Jacobson. B., and Strosberg, A. D. (1980). Interaction between P-adrenergic receptors and guanine nucleotide sites in turkey erythrocyte membranes. Proc. Nail. Acad. Sci. U.S.A. 77, 3801-3805. Weiland. G. A., Minneman, K. P., and Molinoff, P. B. (1980). Thermodynamics of agonist and antagonist interactions with mammalian P-adrenergic receptors. Mol. Pharmarol. IS, 34 1-347. Williams, L. T., and Lefkowitz, R. J . (1976). Alpha adrenergic receptor identification by 13H]dihydroergocryptine binding. Science 192, 791-793. Williams, L. T., and Letkowitz. R. J. (1977). Slowly reversible binding of catecholamine to a nucleotide-sensitive state of the beta-adrenergic receptor. J. Biol. Chem. 252, 7207-72 13. Williams, L. T., and Lefkowitz, R. J. (1978). "Receptor Binding Studies in Adrenergic Pharmacology." Raven, New York. Williams L. T., Mullikin. D., and Lefkowitz, R . J . (1978). Magnesium dependence of agonist binding to adenylate cyclase-coupled hormone receptors. J. Biol. Ckem. 253, 2984-2989.