Influence of the f8-adrenergic receptor concentration on functional coupling to the adenylate cyclase system

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 81, pp , August 1984 Biochemistry Influence of the f8-adrenergic receptor concentration on functional coupling to the adenylate cyclase system (receptor number/functional heterogeneity/agonist-n-ethylmaleimide sensitivity) YVONNE SEVERNE, DIRK COPPENS, SERGE BOTTARI, MICHELE RIVIEREt, RAPHAEL KRAMtt, AND GEORGES VAUQUELIN *Laboratorium Chemie der Proteinen, Instituut Moleculaire Biologie, Vrije Universiteit Brussel, 65 Paardenstraat, 1640 St. Genesius-Rode, Belgium; and tlaboratoire de Biologie Cellulaire et Moleculaire du Developpement, Departement de Biologie Moleculaire, Universite Libre de Bruxelles, 67 Rue des Chevaux, 1640 Rhode St. Genese, Belgium Communicated by J. Brachet, April 26, 1984 ABSTRACT Only part of the,b-adrenergic receptors can undergo functional coupling to the adenylate cyclase regulatory unit. This receptor subpopulation shows an increased affinity for agonists in the presence of Mg2' and undergoes rapid "inactivation" (locking-in of the agonist) by the alkylating reagent N-ethylmaleimide in the presence of agonists. Several experimental conditions, known to modify the total receptor concentration without alteration of the other components of the acenylate cyclase system, do not affect the percentage of receptors that can undergo functional coupling: (i) homologous regulation of B13 receptors in rat brain by noradrenaline (through antidepressive drug or reserpine injections); (it) upand down-regulation of the 182 receptors in Friend erythroleukemia cells by, respectively, sodium butyrate and cinnarizine treatment; and (iiw) dithiothreitol-mediated inactivation of receptors in turkey erythrocytes, Friend erythroleukemia cells, and rat brain. Our findings argue against a stoichiometric limitation in the number of regulatory components, genetically different receptor subpopulations, bound guanine nucleotides, or reduced accessibility of part of the receptors to the agonists as the cause for functional receptor heterogeneity. Differences in either the receptor conformation or its membrane microenvironment are more plausible explanations. Several recent studies have shed light on marked structural and functional differences between /-adrenergic agonists and antagonists in their interaction with their receptors (1-7). In particular, the receptors behave as a homogeneous population of sites with regard to antagonist binding, but two subpopulations can be discriminated for agonist binding. This has been established by two types of observations. First, magnesium ions cause an increase in agonist but not in antagonist affinity for binding to a well-defined proportion of the 8-adrenergic receptors in several tissues (5, 6). Second, agonist but not antagonist binding causes a conformational modification of only part of the B3-adrenergic receptors. This conformational modification can be monitored by the ability of a combination of agonist and the group-specific reagent N-ethylmaleimide (MalNEt) to impair subsequent radioligand binding (7-10). Interestingly, both Mg2+ and MalNEt affect the same receptor subpopulation (unpublished data; ref. 8). Moreover, the basis of receptor heterogeneity is functional rather than pharmacological. Experiments on turkey erythrocyte membranes, human adipose cell membranes (,81-adrenergic receptors), and S49 lymphoma cell membranes (132-adrenergic receptors) have shown that the agonist/malnet-sensitive and -resistant receptors have the same pharmacological specificity (7, 8, 11). In contrast, there is now ample evi- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. dence for the view that the action of both Mg2' and reagent requires the coupling between the receptor and the guanine nucleotide-binding regulatory component of the adenylate cyclase system, designated Ns (8, 12). In this context, current models concerning the MalNEt action mechanism are based on the fact that coupling of the agonist-bound /3-adrenergic receptor to Ns is accompanied by the exposure of sulfhydryl groups, probably at the surface of Ns itself (unpublished data; ref. 10). Alkylation of these groups by MalNEt results in the freezing of the receptors in an active, slow agonist-dissociating conformation (i.e., locking-in of the agonist) and, hence, in their apparent inactivation (9, 10). To investigate the possible basis for the restriction in receptor-ns coupling, we have tested whether various factors known to alter the total receptor number could also alter the proportion of coupling-prone receptors. Using the agonist/ MalNEt reaction, we show here that changes in the total receptor number by various factors (i.e., chemical inactivation, homologous hormonal regulation, and butyrate and cinnarizine treatment) do not necessarily imply an alteration of the Ns-coupled receptor fraction. Our data suggest that the limited receptor-ns coupling is probably related to limitations in the membrane or receptor structure. MATERIALS AND METHODS Materials. The following were obtained as kind gifts: (-)- isoproterenol bitartrate from Sterling Winthrop; fenoterol hydrobromide from Boehringer-Ingelheim; practolol hydrochloride from ICI; (+)-alprenolol hydrochloride, phentolamine hydrochloride, desipramine, and reserpine from CIBA-Geigy; doxepin from Pfizer; mianserin from Organon; pargyline from Abbott; and nisoxetine from Eli Lilly. Nialamide, MalNEt, and dithiothreitol were purchased from Sigma. GTP and GMP were from Boehringer Mannheim. (-)-[3H]Dihydroalprenolol hydrochloride ([3H]H2Alp, 91 Ci/mmol; 1 Ci = 37 GBq) was obtained from New England Nuclear. [3H]CGP (42 Ci/mmol) was from Amersham. All other chemicals were of analytical grade. Source and Preparation of Membranes. Turkey erythrocyte membranes were prepared as described (7). Male Wistar rats (-250 g) underwent either 14 daily intraperitoneal injections with saline solutions of antidepressant drugs (i.e., desipramine, doxepin, nialamide, nisoxetine, and mianserin at 10 mg/kg and pargyline at 25 mg/kg) or 4 daily injections with a reserpine solution at 2.5 mg/kg; they were then decapitated. All subsequent steps were performed at 4 C. The cerebral cortex was homogenized in 10 volumes of 10 mm Tris-HCl, ph 7.4/0.25 M sucrose in an Ultraturax Abbreviations: MalNEt, N-ethylmaleimide; [3H]H2Alp, (-)-[3H]dihydroalprenolol. tdeceased on May 25,

2 4638 Biochemistry: Severne et al. homogenizer and subsequently in a glass/teflon homogenizer (five strokes). The homogenate was centrifuged subsequently at 900, 10,000, and 40,000 x g for 15 min. The final pellet of cortical membranes was retained. Friend erythroleukemia cell culture and membrane preparation are described in ref. 13. Receptor induction was obtained by incubation of the culture with 2 mm sodium butyrate for 24 hr. Incubation with 10,M cinnarizine lasted 24 hr. All membrane preparations were suspended in 10 mm Tris HCl, ph 7.4, 10% (vol/vol) glycerol and stored in liquid nitrogen at a protein concentration of -10 mg/ml. Protein concentrations were determined according to Lowry et al. (14). Membrane Pretreatments. Membranes (1-4 mg of protein per ml) were preincubated for 10 min at 30'C in 75 mm Tris HCl, ph 7.4/25 mm MgCl2 containing 0.5 AuM (-)-isoproterenol/0.2 mm MalNEt in a final volume of 0.5 ml or 1 ml (for rat brain membranes). Preincubations were terminated as follows: turkey erythrocytes and rat brain, centrifugation (2 min at 12,000 rpm in an Eppendorf centrifuge at room temperature) and resuspension of the membranes in 1 ml of fresh buffer (three times); Friend erythroleukemia cells, centrifugation as above but in the presence of 4% (vol/vol) polyethylene glycol 6000 (final concentration). Polyethylene glycol 6000 allowed fast and quantitative precipitation of the membranes and affected neither [3H]H2Alp binding nor receptor-ns coupling. Preincubation of various membranes with 5 mm dithiothreitol was performed as described (15). Radioligand Binding. Binding of [3H]H2Alp and [3H]CGP to the membrane preparations was assayed by filtration on glass fiber filters. Membrane protein ( mg/ml) was incubated with the indicated concentrations of radioligand for 10 min at 30 C in 50 mm Tris HCl, ph 7.4/25 mm MgCl2 containing 10 AM phentolamine (to prevent radioligand binding to a-adrenergic receptors) in a final volume of ,l. Membranes were then filtered as described (7), and the radioactivity on the filters was assayed in a Packard liquid scintillation spectrometer. Specific binding was obtained by subtracting nonspecific binding [i.e., binding in the presence of 10,M (-)-isoproterenol] from total binding. In all figures and tables, bound radioligand refers to specific binding as defined above. The Scatchard plots (16) of the saturation binding curves were rectilinear for all of the membrane preparations, so that the total receptor number and equilibrium dissociation constant (Kd) for [ H]H2Alp and for [3H]CGP binding could be calculated by linear regression analysis. RESULTS We identified the 83-adrenergic receptors in membranes derived from rat brain frontal cortex, Friend erythroleukemia cells, and turkey erythrocytes by the specific binding of the radiolabeled antagonists [3H]H2Alp and [3H]CGP Pretreatment of these membranes with a combination of the 13-adrenergic agonist isoproterenol and the alkylating reagent MalNEt causes agonist locking (10) in only part of the receptors. This results in a decrease in the number of [3H]H2Alp binding sites without alteration of the binding characteristics to the remainder. The incomplete nature of the agonist/mal- NEt effect is clearly demonstrated 15y the kinetic experiment depicted in Fig. 1. For all three membrane preparations, there was an initial sharp fall in the number of residual [3H]H2Alp binding sites until a plateau level was reached, corresponding to 64% of the initial number for rat brain, 31% for Friend erythroleukemia cells, and 37% for turkey erythrocytes. Based on these kinetic data, we adopted agonist/ MalNEt preincubation conditions that caused maximal decline in the receptor number. Binding of the hydrophilic ra- 4-4 C 100i = 50 n 0 0 Proc. NatL Acad Sci. USA 81 (1984) 0 5 1* Preincubation time, min FIG. 1. Isoproterenol/MalNEt-mediated decrease in the 18-adrenergic receptor number in function of the preincubation time. Turkey erythrocyte membranes (A), rat brain membranes (U), and Friend erythroleukemia cell membranes (A) were pretreated at 30TC with buffer only (for control binding) or with 0.5 AtM isoproterenol/0.2 mm MalNEt for the indicated periods of time (abscissa), after which the membranes were washed and assayed for [3H]H2Alp binding. For all of the membrane preparations, control binding remained constant throughout the duration of preincubation. dioligand [3H]CGP (17) reached comparable plateau values-i.e., 60%, 40%, and 31%. Pretreatment of the three types of membranes for 15 min with 1 ttm (-)-isoproterenol/1 mm GMP, known to remove tightly bound guanine nucleotides (18), did not increase the subsequent agonist/mal- NEt-mediated decline in [3H]H2Alp binding either; plateau values were 71%, 39%, and 42%. Repeated intraperitoneal injections of antidepressive drugs and of reserpine in rats had opposite effects on the regulation of the number of f3-adrenergic receptors in the brain (Fig. 2)-i.e., from 71 to 98 fmol/mg of membrane protein after antidepressive drug treatment, 115 fmol/mg for control membranes, and 171 fmol/mg after reserpine treatment. The affinity for the radioligand (Kd = 1.1 x 10-9 M) was unaffected by these treatments. Fig. 2 shows also that there was a linear, proportional relationship between the total receptor number (abscissa) and the amount of agonist/ MalNEt-resistant receptors. The correlation was highly significant: r = Thus, despite the variation in total receptor number, the percentage of coupling-prone receptors (i.e., agonist/malnet-sensitive receptors) remained constant. Individual percentual values did not differ significantly from the mean value-i.e., 34.6 ± 5.5%. The concomitant presence of the P1 and P2 receptor subclasses in the brain was evidenced by the ability of the selective 831-adrenergic antagonist practolol and the f2-adrenergic antagonist fenoterol to form shallow competition binding curves with [3H]H2Alp. We calculated the amount of both receptor subclasses by the computer-assisted iterative analysis of these competition binding curves according to the method of Minneman et al. (19). Both fenoterol and practolol yielded quantitatively similar results. These results (Fig. 2 Inset) clearly show that desipramine and reserpine treatment only modify the Pi receptor number (abscissa). As expected, there was still a linear, proportional relationship between the total 813-receptor qum40lr and the amount of agonist/mal- NEt-resistant,1 receptor sites (ordinate). On the other hand, desipramine and reserpine treatment did not significantly affect the total 12 receptor number nor their degree of coupling to Ns (Fig. 2 Inset). The ability of sodium butyrate and cinnarizine to achieve an opposite modulation of the 832-adrenergic receptor number in Friend erythroleukemia cells has been documented (13, 20). Under the conditions used in our study, butyrate (2 mm, 24 hr) affected neither cellular cyclic AMP levels nor

3 Biochemistry: Severne et al Proc. NatL Acad. Sci. USA 81 (1984) 4639 I~~~22 Z Total receptor, fmol/mg FIG. 2. Effect of antidepressive drugs and reserpine treatment upon the coupling of 1- and,82-adrenergic receptors in rat brain. The total f3- adrenergic receptor density was determined by Scatchard analysis (16) of [3H]H2Alp saturation binding. Membranes were pretreated for 10 min with 0.5 AM isoproterenol (IPR)/0.2 mm MalNEt, after which residual [3H]H2Alp binding was measured. These parameters were determined in rat brain after chronic intraperitoneal injections of a 0.9% saline solution (designated 1) or antidepressive drugs [desipramine (designated 2), pargyline (3), nialamide (4), mianserin (5), doxepin (6)], or reserpine (designated 7). The amount of isoproterenol/malnet-resistant sites (ordinate) is expressed as a function of the amount of total receptors (both in fmol/mg of protein). (Inset) Practolol (,81-adrenergic receptorselective) and fenoterol (P2 selective) competition binding curves were analyzed by a computer-based iterative procedure derived from Minneman et al. (19) to yield the number of 13k- and.82-adrenergic receptors (abscissa) as well as their affinity for the considered drug. Competition binding also was performed on agonist/malnet-pretreated membranes to yield the number of resistant f3i- and 82-adrenergic receptor sites (ordinate). Experiments were carried out for control membranes (designated 1) and for membranes from desipramine-treated (designated 2) and reserpine-treated (designated 7) rats. A and A, Data derived respectively from fenoterol and practolol competition binding. the basal adenylate cyclase activity and fluoride stimulation in membranes (13). Cinnarizine (10,uM, 24 hr) caused a decline in the isoproterenol-stimulated adenylate cyclase activity without affecting the prostaglandin El stimulation (20). This suggests that the adenylate cyclase enzyme and Ns had not been affected by the two compounds. The treatment of these cells with sodium butyrate provoked a 3-fold increase in the 8-adrenergic receptor number, whereas cinnarizine treatment resulted in a 50% decline (Table 1; refs. 13 and 20). However, these treatments did not affect the receptor affinity for [3H]H2Alp (Kd = x 10-9 M) or the percentage of receptors that can undergo functional coupling to Ns. Both the agonist/mainet-sensitivity method and the computerized iterative determination of the number of high-agonist-affinity sites in the presence of Mg2+ gave a consistent percentage of coupling-prone receptors (Table 1). The lack Table 1. Isoproterenol competition binding characteristics and isoproterenol/mainet sensitivity of membranes from control and butyrate- and cinnarizine-pretreated Friend erythroleukemia cells IPR/[3H]H2AIp competition binding Receptor -..IPR/MalNEt-resistant Friend cell sites High affinity Low affinity sites, pretreatment per cell IC50, AM IC50,,AM % sites % of control Control Butyrate Cinnarizine 960 * 39 Cells were pretreated as described. Competition binding curves for 1 nm to 0.1 mm isoproterenol (IPR)/5 nm [3H]H2Alp were performed in the presence of 25 mm Mg2' and were analyzed by a computer-based iterative procedure derived from Minneman's procedure (19) to yield the number of high- and low-affinity sites and their IC50 for the agonist. Membranes were pretreated at 300C with 0.5 AuM isoproterenol/0.2 mm MaINEt for 15 min, after which the membranes were washed and assayed for [3H]H2AIp binding. *Experiment not performed due to the low concentration of receptor sites.

4 4640 Biochemistry: Severne et al. Proc. NatL Acad Sci. USA 81 (1984) of butyrate effect upon the percentage of coupling-prone receptors was also in full agreement with an earlier reported equal-fold increase in total 8-adrenergic receptor number and in catecholamine-stimulation of the adenylate cyclase activity (13). Turkey erythrocyte l31-adrenergic receptors and rat liver /32-adrenergic receptors have already been found to contain essential disulfide bonds at their ligand binding site (15, 21). Reduction of these bonds by the reagent dithiothreitol causes a time- and dose-dependent decrease in the total receptor number (15) without alteration of the other components pf the adenylate cyclase system (22, 23). In this study, we pretreated rat brain membranes, Friend erythroleukemia cell membranes, and turkey erythrocyte membranes with 5 mm of this reagent for periods of time for which there was a 50-67% decline in the total receptor number. This dithiothreitol pretreatment was not associated with a significant modification of the percentage of agonist/malnet-sensitive receptors in turkey erythrocyte membranes and Friend erythroleukemia. cell membrane and only a slight increase (from 39% to 55%) in rat brain membranes (Table 2). However, when considering the absolute amount of receptor sites in this latter tissue, dithiothreitol pretreatment caused a decrease to 1/2.3 in the amount of agonist/malnet-sensitive receptors, which is comparable to the decrease to 1/2.7 in total receptor number. DISCUSSION Several studies have already shown that only part of the /3- adrenergic receptors can undergo functional coupling to Ns (5, 6, 8). Only this receptor subpopulation shows an increased affinity for agonists in the presence of Mg2+ (5, 6) and locks agonists in the presence of the alkylating reagent MalNEt (8, 10). This receptor heterogeneity might be intrinsic to the use of purified membranes because they might contain inside-out vesicles, so that part of the receptors are located at the internal face, thereby slowing down the agonist/ MalNEt effect. Being more accessible to hydrophobic ligands such as [3H]H2Alp as compared to the hydrophilic agonist molecules, these receptors should be detected as agonist/malnet-resistant sites. Receptor identification by the hydrophilic,8-antagonist' [3H]CGP (which only binds to external receptors; ref. 17) revealed, however, no difference in the percentage of resistant sites as compared to [3H]H2Alp. Accordingly, shielding of receptors from the agonists cannot be retained as a main cause for the observed receptor heterogeneity. The receptor heterogeneity appears to be functional rather than the consequence of artefacts in the membrane preparation and might be related to a stoichiometric limitation of the number of Ns components (bound guanine nucleotides), to the concomitant presence of two or more genetically different receptor molecules (but having the same 1- or f32-adrenergic specificity), to differences in receptor conformation, or finally to membrane structural limitations in receptor-ns coupling. To investigate the possible basis for this functional receptor heterogeneity, we tested whether the proportion of coupling-prone receptors is affected by the total' receptor number. Using the agonist/malnet method, we showed'that experimental conditions known to modify the total (3-adrenergic receptor concentration without alteration of the other components of the adenylate cyclase system (there are no alterations of the basal and the fluoride-stimulated adenylate cyclase activity) do not affect the percentage of receptors that can undergo functional coupling to' Ns. The following experimental conditions were used. (i) Homologous receptor regulation by noradrenaline. When injected in rats, antidepressive drugs inhibit the presynaptic noradrenaline reuptake (doxepin and desipramine) and catabolism (pargyline and nialamide) or increase the noradrenaline release (mianserin). The resulting increase in synaptic noradrenaline is presumed to play a key role in the observed decrease of 81-adrenergic receptor'concentration in the brain (24). On the other hand, chronic reserpine treatment depletes the catecholamine stores, resulting in supersensitivity-i.e., an increase in 31-adrenergic receptor number (24). The other components of the adenylate cyclase system are apparently not affected by variations in the synaptic noradrenaline content, since its depletion by reserpine or by 6-hydroxydopamine treatment does not alter basal or fluoride stimulation of the enzyme (25, 26). (ii) Butyrate treatment of different cultured cells. This causes a marked increase in their B-adrenergic receptor number (13). The phenomenon is linked to butyrate stimulation of de novo synthesis of new receptor molecules (27). On the other hand, cinnarizine treatment causes a decline in the receptor number by a yet unidentified mechanism (20). (Mi) Dithiothreitol treatment. This causes the chemical inactivation of 1,i- as well as 82-adrenergic receptors by reducing an essential disulfide bond located at the ligand binding site of the receptor (15). Although the functional stoichiometry of the receptors, of the Ns component, and of the adenylate cyclase enzyme is still obscure, our findings argue against a limitation in the number of Ns components as a major source for receptor heterogeneity in the investigated membrane systems. Such a stoichiometric limitation should indeed imply that any alteration of the total receptor number should not influence the absolute number of coupled receptors. Accordingly, the percentage of coupling-prone receptors should have increased under conditions where the total receptor number declines (i.e., antidepressive drug, cinnarizine, and dithiothreitol treatment) and decreased under conditions mediating an'increase in the total receptor number (i.e., reserpine and butyrate treatments). The observed invariance of the agonist/ MalNEt sensitivity under these latter conditions would even suggest that the Ns components might be present in excess with respect to' the receptors. The observed ability of buty- Table 2. Isoproterenol/MalNEt sensitivity of 3-adrenergic receptors in dithiothreitolpretreated membranes Control membranes Dithiothreitol-pretreated membranes IPR/MalNEt-resistant % receptor IPR/MaINEt-resistant Source sites, % of total sites remaining sites, % of remaining sites Turkey erythrocyte Rat brain Friend erythroleukemia (butyrate treated) Membranes were pretreated with 5 mm dithiothreitol for 5 min at 300C, after which the membranes were washed. An aliquot was taken to determine the percentage of remaining (3H]H2Alp binding sites. Membranes were further submitted to isoproterenol (IPR)/MalNEt inactivation, washed, and assessed for [3H]H2Alp binding. Values are means of two to four experiments.

5 Biochemistry: Severne et al rate to produce an equal rise in the number of coupling-prone receptors and in the catecholamine stimulation of the adenylate cyclase activity in Friend erythroleukemia cells (13) pleads also in favor of the existence of an excess of Ns components in this system. Another possible cause for receptor heterogeneity is that part of them might form complexes with GDP-bound Ns (18). This bound GDP might decrease the Ns sensitivity to MalNEt (10) and, hence, prevent agonist locking-in in the receptors. However, this explanation is weakened by the fact that pretreatment of the membranes with high concentrations of isoproterenol and GMP, known to remove tightly bound GDP (18), had no effect on the percentage of agonist/ MalNEt-sensitive receptors in the three investigated tissues. The eventuality of genetically different receptor subpopulations as the cause of the functional receptor heterogeneity is unlikely too. Indeed this hypothesis cannot explain the fact that butyrate, which affects genomic expression [through histone hyperacetylation (27)] causes an equiproportional increase in both the coupled and uncoupled receptor populations. Along the same line, previous experiments on Pd variants of S49 lymphoma cells have shown that although the total receptor density is reduced by about 75% in the Pd clone, adenylate cyclase activation and agonist/mal- NEt-sensitivity ratio are unaltered (8, 28). The assumption of genetically different receptors is further weakened by the fact that these receptors have homogeneous physicochemical characteristics in most tissues (29). An alternative cause for the limitation in the agonist/mal- NEt effect could be that the resistant receptors cannot interact with Ns in a productive manner because of differences in either the receptor conformation or its membrane microenvironment. At the present level of investigation, it is not yet possible to discriminate between these two possibilities, especially since both might be related to the presence of different lipid microdomains in the membrane. The requirement for regions of facilitated coupling between /8 receptors and Ns results also from theoretical considerations indicating that random collisions are not sufficient to explain the high level of P-adrenergic stimulation of the adenylate cyclase system (30). Finally, it has already been demonstrated that factors that perturb the membrane, such as the incorporation of fillipin (31), hamper the Mg2+-dependent formation of high-agonist-affinity sites and, hence, receptor-ns coupling. In conclusion, we suggest that the basis for the functional,b3adrenergic receptor heterogeneity might result from the presence of only part of them in specialized membrane areas with facilitated coupling to Ns. Y.S. holds a Research Fellowship of the Instituut tot aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw, Belgium. G.V. is Bevoegdverklaard Navorser of the Nationaal Fonds voor Wetenschappelijk Onderzoek, Belgium. This work was supported by grants from the Fonds voor Geneeskundig en Wetenschappelijk Onderzoek and from the Fondation Universitaire A. et D. Van Buuren. Proc. NatL Acad. Sci. USA 81 (1984) Limbird, L. E. & Lefkowitz, R. J. (1978) Proc. Natl. Acad. Sci. USA 75, Lefkowitz, R. J. (1983) Annu. Rev. Biochem. 52, Weiland, G. A., Minneman, K. P. & Molinoff, P. B. (1979) Nature (London) 281, Bird, S. J. & Maguire, M. E. (1978) J. Biol. Chem. 254, Wessels, M. R., Mullikin, D. & Lefkowitz, R. J. (1979) Mol. Pharmacol. 16, Stadel, J. M., DeLean, A. & Lefkowitz, R. J. (1980) J. Biol. Chem. 255, Vauquelin, G., Bottari, S. & Strosberg, A. D. (1980) Mol. Pharmacol. 17, Vauquelin, G. & Maguire, M. E. (1980) Mol. Pharmacol. 18, Heidenreich, K. A., Weiland, G. 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