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Proc. Natl. Acad. Sci. USA Vol. 81, pp. 4637-4641, August 1984 Biochemistry

Influence of the f8- concentration on functional coupling to the adenylate cyclase system (receptor number/functional heterogeneity/-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 dence for the view that the action of both Mg2' and reagent undergo functional coupling to the adenylate cyclase regula- requires the coupling between the receptor and the guanine tory unit. This receptor subpopulation shows an increased af- nucleotide-binding regulatory component of the adenylate finity for in the presence of Mg2' and undergoes rap- cyclase system, designated Ns (8, 12). In this context, cur- id "inactivation" (locking-in of the agonist) by the alkylating rent models concerning the MalNEt action mechanism are reagent N-ethylmaleimide in the presence of agonists. Several based on the fact that coupling of the agonist-bound /3-adren- experimental conditions, known to modify the total receptor ergic receptor to Ns is accompanied by the exposure of sulf- concentration without alteration of the other components of hydryl groups, probably at the surface of Ns itself (unpub- the acenylate cyclase system, do not affect the percentage of lished data; ref. 10). Alkylation of these groups by MalNEt receptors that can undergo functional coupling: (i) homolo- results in the freezing of the receptors in an active, slow ago- gous regulation of B13 receptors in rat brain by noradrenaline nist-dissociating conformation (i.e., locking-in of the ago- (through antidepressive or reserpine injections); (it) up- nist) and, hence, in their apparent inactivation (9, 10). and down-regulation of the 182 receptors in Friend erythroleu- To investigate the possible basis for the restriction in re- kemia cells by, respectively, sodium butyrate and cinnarizine ceptor-Ns coupling, we have tested whether various factors treatment; and (iiW) dithiothreitol-mediated inactivation of re- known to alter the total receptor number could also alter the ceptors in turkey erythrocytes, Friend erythroleukemia cells, proportion of coupling-prone receptors. Using the agonist/ and rat brain. Our findings argue against a stoichiometric lim- MalNEt reaction, we show here that changes in the total re- itation in the number of regulatory components, genetically ceptor number by various factors (i.e., chemical inactiva- different receptor subpopulations, bound guanine nucleotides, tion, homologous hormonal regulation, and butyrate and cin- or reduced accessibility of part of the receptors to the agonists narizine treatment) do not necessarily imply an alteration of as the cause for functional receptor heterogeneity. Differences the Ns-coupled receptor fraction. Our data suggest that the in either the receptor conformation or its membrane microen- limited receptor-Ns coupling is probably related to limita- vironment are more plausible explanations. tions in the membrane or receptor structure. Several recent studies have shed light on marked structural MATERIALS AND METHODS and functional differences between /-adrenergic agonists and antagonists in their interaction with their receptors (1- Materials. The following were obtained as kind gifts: (-)- 7). In particular, the receptors behave as a homogeneous isoproterenol bitartrate from Sterling Winthrop; population of sites with regard to antagonist binding, but two hydrobromide from Boehringer-Ingelheim; hydro- subpopulations can be discriminated for agonist binding. chloride from ICI; (+)- hydrochloride, phentol- This has been established by two types of observations. hydrochloride, , and reserpine from First, magnesium ions cause an increase in agonist but not in CIBA-Geigy; from Pfizer; from Orga- antagonist affinity for binding to a well-defined proportion of non; pargyline from Abbott; and nisoxetine from Eli Lilly. the 8-adrenergic receptors in several tissues (5, 6). Second, Nialamide, MalNEt, and dithiothreitol were purchased from agonist but not antagonist binding causes a conformational Sigma. GTP and GMP were from Boehringer Mannheim. modification of only part of the B3-adrenergic receptors. This (-)-[3H] hydrochloride ([3H]H2Alp, 91 conformational modification can be monitored by the ability Ci/mmol; 1 Ci = 37 GBq) was obtained from New England of a combination of agonist and the group-specific reagent Nuclear. [3H]CGP 12177 (42 Ci/mmol) was from Amersham. N-ethylmaleimide (MalNEt) to impair subsequent radioli- All other chemicals were of analytical grade. gand binding (7-10). Source and Preparation of Membranes. Turkey erythro- Interestingly, both Mg2+ and MalNEt affect the same re- cyte membranes were prepared as described (7). ceptor subpopulation (unpublished data; ref. 8). Moreover, Male Wistar rats (-250 g) underwent either 14 daily intra- the basis of receptor heterogeneity is functional rather than peritoneal injections with saline solutions of pharmacological. Experiments on turkey erythrocyte mem- (i.e., desipramine, doxepin, nialamide, nisoxetine, and branes, human adipose cell membranes (,81-adrenergic re- mianserin at 10 mg/kg and pargyline at 25 mg/kg) or 4 daily ceptors), and S49 lymphoma cell membranes (132-adrenergic injections with a reserpine solution at 2.5 mg/kg; they were receptors) have shown that the agonist/MalNEt-sensitive then decapitated. All subsequent steps were performed at and -resistant receptors have the same pharmacological 4°C. The cerebral cortex was homogenized in 10 volumes of specificity (7, 8, 11). In contrast, there is now ample evi- 10 mM Tris-HCl, pH 7.4/0.25 M sucrose in an Ultraturax

The publication costs of this article were defrayed in part by page charge Abbreviations: MalNEt, N-ethylmaleimide; [3H]H2Alp, (-)-[3H]di- payment. This article must therefore be hereby marked "advertisement" hydroalprenolol. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tDeceased on May 25, 1983. 4637 Downloaded by guest on September 29, 2021 4638 Biochemistry: Severne et al. Proc. NatL Acad Sci. USA 81 (1984)

homogenizer and subsequently in a glass/Teflon homogeniz- er (five strokes). The homogenate was centrifuged subse- 100i quently at 900, 10,000, and 40,000 x g for 15 min. The final pellet of cortical membranes was retained. 4-4 Friend erythroleukemia cell culture and membrane prepa- C ration are described in ref. 13. Receptor induction was ob- tained by incubation of the culture with 2 mM sodium buty- =n 050 rate for 24 hr. Incubation with 10 ,M cinnarizine lasted 24 hr. All membrane preparations were suspended in 10 mM 0 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. 0 5 1*0 15 20 (14). Preincubation time, min Membrane Pretreatments. Membranes (1-4 mg of protein per ml) were preincubated for 10 min at 30'C in 75 mM FIG. 1. Isoproterenol/MalNEt-mediated decrease in the 18-ad- Tris HCl, pH 7.4/25 mM MgCl2 containing 0.5 AuM (-)-iso- renergic receptor number in function of the preincubation time. Tur- proterenol/0.2 mM MalNEt in a final volume of 0.5 ml or 1 key erythrocyte membranes (A), rat brain membranes (U), and ml (for rat brain membranes). Preincubations were terminat- Friend erythroleukemia cell membranes (A) were pretreated at 30TC ed as follows: turkey erythrocytes and rat brain, centrifuga- with buffer only (for control binding) or with 0.5 AtM isoprotere- tion (2 min at 12,000 rpm in an Eppendorf centrifuge at room nol/0.2 mM MalNEt for the indicated periods of time (abscissa), and of the membranes in 1 ml of after which the membranes were washed and assayed for [3H]H2Alp temperature) resuspension binding. For all of the membrane preparations, control binding re- fresh buffer (three times); Friend erythroleukemia cells, cen- mained constant throughout the duration of preincubation. trifugation as above but in the presence of 4% (vol/vol) poly- ethylene glycol 6000 (final concentration). Polyethylene gly- col 6000 allowed fast and quantitative precipitation of the dioligand [3H]CGP 12177 (17) reached comparable plateau membranes and affected neither [3H]H2Alp binding nor re- values-i.e., 60%, 40%, and 31%. Pretreatment of the three ceptor-Ns coupling. Preincubation of various membranes types of membranes for 15 min with 1 ttM (-)-isoprotere- with 5 mM dithiothreitol was performed as described (15). nol/1 mM GMP, known to remove tightly bound guanine nu- Radioligand Binding. Binding of [3H]H2Alp and [3H]CGP cleotides (18), did not increase the subsequent agonist/Mal- 12177 to the membrane preparations was assayed by filtra- NEt-mediated decline in [3H]H2Alp binding either; plateau tion on glass fiber filters. Membrane protein (0.75-2 mg/ml) values were 71%, 39%, and 42%. was incubated with the indicated concentrations of radiolig- Repeated intraperitoneal injections of antidepressive and for 10 min at 30°C in 50 mM Tris HCl, pH 7.4/25 mM drugs and of reserpine in rats had opposite effects on the MgCl2 containing 10 AM (to prevent radiolig- regulation of the number of f3-adrenergic receptors in the and binding to a-adrenergic receptors) in a final volume of brain (Fig. 2)-i.e., from 71 to 98 fmol/mg of membrane pro- 200-500-,l. Membranes were then filtered as described (7), tein after antidepressive drug treatment, 115 fmol/mg for and the radioactivity on the filters was assayed in a Packard control membranes, and 171 fmol/mg after reserpine treat- liquid scintillation spectrometer. Specific binding was ob- ment. The affinity for the radioligand (Kd = 1.1 x 10-9 M) tained by subtracting nonspecific binding [i.e., binding in the was unaffected by these treatments. Fig. 2 shows also that presence of 10 ,M (-)-isoproterenol] from total binding. In there was a linear, proportional relationship between the to- all figures and tables, bound radioligand refers to specific tal receptor number (abscissa) and the amount of agonist/ binding as defined above. The Scatchard plots (16) of the MalNEt-resistant receptors. The correlation was highly sig- saturation binding curves were rectilinear for all of the mem- nificant: r = 0.985. Thus, despite the variation in total recep- brane preparations, so that the total receptor number and tor number, the percentage of coupling-prone receptors (i.e., equilibrium dissociation constant (Kd) for [ H]H2Alp and for agonist/MalNEt-sensitive receptors) remained constant. In- [3H]CGP 12177 binding could be calculated by linear regres- dividual percentual values did not differ significantly from sion analysis. the mean value-i.e., 34.6 ± 5.5%. The concomitant presence of the P1 and P2 receptor sub- RESULTS classes in the brain was evidenced by the ability of the selec- tive 831- practolol and the f2-adrenergic We identified the 83-adrenergic receptors in membranes de- antagonist fenoterol to form shallow competition binding rived from rat brain frontal cortex, Friend erythroleukemia curves with [3H]H2Alp. We calculated the amount of both cells, and turkey erythrocytes by the specific binding of the receptor subclasses by the computer-assisted iterative analy- radiolabeled antagonists [3H]H2Alp and [3H]CGP 12177. sis of these competition binding curves according to the Pretreatment of these membranes with a combination of the method of Minneman et al. (19). Both fenoterol and practolol 13- isoproterenol and the alkylating reagent yielded quantitatively similar results. These results (Fig. 2 MalNEt causes agonist locking (10) in only part of the recep- Inset) clearly show that desipramine and reserpine treatment tors. This results in a decrease in the number of [3H]H2Alp only modify the Pi receptor number (abscissa). As expected, binding sites without alteration of the binding characteristics there was still a linear, proportional relationship between the to the remainder. The incomplete nature of the agonist/Mal- total 813-receptor quM40lr and the amount of agonist/Mal- NEt effect is clearly demonstrated 15y the kinetic experiment NEt-resistant ,1 receptor sites (ordinate). On the other depicted in Fig. 1. For all three membrane preparations, hand, desipramine and reserpine treatment did not signifi- there was an initial sharp fall in the number of residual cantly affect the total 12 receptor number nor their degree of [3H]H2Alp binding sites until a plateau level was reached, coupling to Ns (Fig. 2 Inset). corresponding to 64% of the initial number for rat brain, 31% The ability of sodium butyrate and cinnarizine to achieve for Friend erythroleukemia cells, and 37% for turkey eryth- an opposite modulation of the 832- num- rocytes. Based on these kinetic data, we adopted agonist/ ber in Friend erythroleukemia cells has been documented MalNEt preincubation conditions that caused maximal de- (13, 20). Under the conditions used in our study, butyrate (2 cline in the receptor number. Binding of the hydrophilic ra- mM, 24 hr) affected neither cellular cyclic AMP levels nor Downloaded by guest on September 29, 2021 Biochemistry: Severne et aL Proc. NatL Acad. Sci. USA 81 (1984) 4639

I~~~22

Z 501

0 50 100 150 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 receptor- selective) and fenoterol (P2 selective) competition binding curves were analyzed by a computer-based iterative procedure derived from Minne- man 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 treatment resulted in a 50% decline (Table 1; refs. 13 and 20). in membranes (13). Cinnarizine (10 ,uM, 24 hr) caused a de- However, these treatments did not affect the receptor affini- cline in the isoproterenol-stimulated adenylate cyclase activ- ty for [3H]H2Alp (Kd = 0.6-0.7 x 10-9 M) or the percentage ity without affecting the prostaglandin El stimulation (20). of receptors that can undergo functional coupling to Ns. This suggests that the adenylate cyclase enzyme and Ns had Both the agonist/MaINEt-sensitivity method and the com- not been affected by the two compounds. The treatment of puterized iterative determination of the number of high-ago- these cells with sodium butyrate provoked a 3-fold increase nist-affinity sites in the presence of Mg2+ gave a consistent in the 8-adrenergic receptor number, whereas cinnarizine 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 2100 0.022 0.78 42 35 Butyrate 5500 0.014 0.76 41 36 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. Downloaded by guest on September 29, 2021 4640 Biochemistry: Severne et al. Proc. NatL Acad Sci. USA 81 (1984)

of butyrate effect upon the percentage of coupling-prone re- finally to membrane structural limitations in receptor-Ns ceptors was also in full agreement with an earlier reported coupling. equal-fold increase in total 8-adrenergic receptor number To investigate the possible basis for this functional recep- and in catecholamine-stimulation of the adenylate cyclase tor heterogeneity, we tested whether the proportion of cou- activity (13). pling-prone receptors is affected by the total' receptor num- Turkey erythrocyte l31-adrenergic receptors and rat liver ber. Using the agonist/MalNEt method, we showed'that /32-adrenergic receptors have already been found to contain experimental conditions known to modify the total (3-adren- essential disulfide bonds at their ligand binding site (15, 21). ergic receptor concentration without alteration of the other Reduction of these bonds by the reagent dithiothreitol components of the adenylate cyclase system (there are no causes a time- and dose-dependent decrease in the total re- alterations of the basal and the fluoride-stimulated adenylate ceptor number (15) without alteration of the other compo- cyclase activity) do not affect the percentage of receptors nents pf the adenylate cyclase system (22, 23). In this study, that can undergo functional coupling to' Ns. The following we pretreated rat brain membranes, Friend erythroleukemia experimental conditions were used. cell membranes, and turkey erythrocyte membranes with 5 (i) Homologous receptor regulation by noradrenaline. mM of this reagent for periods of time for which there was a When injected in rats, antidepressive drugs inhibit the pre- 50-67% decline in the total receptor number. This dithio- synaptic noradrenaline (doxepin and desipramine) threitol pretreatment was not associated with a significant and catabolism (pargyline and nialamide) or increase the nor- modification of the percentage of agonist/MalNEt-sensitive release (mianserin). The resulting increase in syn- receptors in turkey erythrocyte membranes and Friend aptic noradrenaline is presumed to play a key role in the ob- erythroleukemia. cell membrane and only a slight increase served decrease of 81-adrenergic receptor'concentration in (from 39% to 55%) in rat brain membranes (Table 2). Howev- the brain (24). On the other hand, chronic reserpine treat- er, when considering the absolute amount of receptor sites in ment depletes the catecholamine stores, resulting in super- this latter tissue, dithiothreitol pretreatment caused a de- sensitivity-i.e., an increase in 31-adrenergic receptor num- crease to 1/2.3 in the amount of agonist/MalNEt-sensitive ber (24). The other components of the adenylate cyclase sys- receptors, which is comparable to the decrease to 1/2.7 in tem are apparently not affected by variations in the synaptic total receptor number. noradrenaline content, since its depletion by reserpine or by 6-hydroxydopamine treatment does not alter basal or fluo- DISCUSSION ride stimulation of the enzyme (25, 26). (ii) Butyrate treatment of different cultured cells. This Several studies have already shown that only part of the /3- causes a marked increase in their B-adrenergic receptor adrenergic receptors can undergo functional coupling to Ns number (13). The phenomenon is linked to butyrate stimula- (5, 6, 8). Only this receptor subpopulation shows an in- tion of de novo synthesis of new receptor molecules (27). On creased affinity for agonists in the presence of Mg2+ (5, 6) the other hand, cinnarizine treatment causes a decline in the and locks agonists in the presence of the alkylating reagent receptor number by a yet unidentified mechanism (20). MalNEt (8, 10). This receptor heterogeneity might be intrin- (Mi) Dithiothreitol treatment. This causes the chemical in- sic to the use of purified membranes because they might con- activation of 1,i- as well as 82-adrenergic receptors by reduc- tain inside-out vesicles, so that part of the receptors are lo- ing an essential disulfide bond located at the ligand binding cated at the internal face, thereby slowing down the agonist/ site of the receptor (15). MalNEt effect. Being more accessible to hydrophobic li- Although the functional stoichiometry of the receptors, of gands such as [3H]H2Alp as compared to the hydrophilic ag- the Ns component, and of the adenylate cyclase enzyme is onist molecules, these receptors should be detected as agon- still obscure, our findings argue against a limitation in the ist/MalNEt-resistant sites. Receptor identification by the number of Ns components as a major source for receptor hydrophilic ,8-antagonist' [3H]CGP 12177 (which only binds heterogeneity in the investigated membrane systems. Such a to external receptors; ref. 17) revealed, however, no differ- stoichiometric limitation should indeed imply that any alter- ence in the percentage of resistant sites as compared to ation of the total receptor number should not influence the [3H]H2Alp. Accordingly, shielding of receptors from the ag- absolute number of coupled receptors. Accordingly, the per- onists cannot be retained as a main cause for the observed centage of coupling-prone receptors should have increased receptor heterogeneity. under conditions where the total receptor number declines The receptor heterogeneity appears to be functional rather (i.e., antidepressive drug, cinnarizine, and dithiothreitol than the consequence of artefacts in the membrane prepara- treatment) and decreased under conditions mediating an'in- tion and might be related to a stoichiometric limitation of the crease in the total receptor number (i.e., reserpine and buty- number of Ns components (bound guanine nucleotides), to rate treatments). The observed invariance of the agonist/ the concomitant presence of two or more genetically differ- MalNEt sensitivity under these latter conditions would even ent receptor molecules (but having the same 1- or f32-adren- suggest that the Ns components might be present in excess ergic specificity), to differences in receptor conformation, or with respect to' the receptors. The observed ability of buty- Table 2. Isoproterenol/MalNEt sensitivity of 3-adrenergic receptors in dithiothreitol- pretreated 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 34 41 35 Rat brain 61 37 45 Friend erythroleukemia (butyrate treated) 34 45 39 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. Downloaded by guest on September 29, 2021 Biochemistry: Severne et aL Proc. NatL Acad. Sci. USA 81 (1984) 4641

rate to produce an equal rise in the number of coupling-prone 1. Limbird, L. E. & Lefkowitz, R. J. (1978) Proc. Natl. Acad. receptors and in the catecholamine stimulation of the adenyl- Sci. USA 75, 228-232. ate cyclase activity in Friend erythroleukemia cells (13) 2. Lefkowitz, R. J. (1983) Annu. Rev. Biochem. 52, 159-186. pleads also in favor of the existence of an excess of Ns com- 3. Weiland, G. A., Minneman, K. P. & Molinoff, P. B. (1979) ponents in this system. Nature (London) 281, 114-117. 4. Bird, S. J. & Maguire, M. E. (1978) J. Biol. Chem. 254, 8826- Another possible cause for receptor heterogeneity is that 8834. part of them might form complexes with GDP-bound Ns 5. Wessels, M. R., Mullikin, D. & Lefkowitz, R. J. (1979) Mol. (18). This bound GDP might decrease the Ns sensitivity to Pharmacol. 16, 10-20. MalNEt (10) and, hence, prevent agonist locking-in in the 6. Stadel, J. M., DeLean, A. & Lefkowitz, R. J. (1980) J. Biol. receptors. However, this explanation is weakened by the Chem. 255, 1436-1441. fact that pretreatment of the membranes with high concen- 7. Vauquelin, G., Bottari, S. & Strosberg, A. D. (1980) Mol. trations of isoproterenol and GMP, known to remove tightly Pharmacol. 17, 163-171. bound GDP (18), had no effect on the percentage of agonist/ 8. Vauquelin, G. & Maguire, M. E. (1980) Mol. Pharmacol. 18, MalNEt-sensitive receptors in the three tissues. 362-369. investigated 9. Heidenreich, K. A., Weiland, G. A. & Molinoff, P. B. (1982) The eventuality of genetically different receptor subpop- J. Biol. Chem. 257, 804-810. ulations as the cause ofthe functional receptor heterogeneity 10. Korner, M., Gilon, C. & Schramm, M. (1982) J. Biol. Chem. is unlikely too. Indeed this hypothesis cannot explain the 257, 3389-3396. fact that butyrate, which affects genomic expression 11. Jacobsson, B., Vauquelin, G., Wesslau, C., Smith, U. & Stros- [through histone hyperacetylation (27)] causes an equipro- berg, A. D. (1981) Eur. J. Biochem. 114, 349-354. portional increase in both the coupled and uncoupled recep- 12. Erdos, J. J., Vauquelin, G., Cech, S. Y., Broaddus, W. C., Ja- tor populations. Along the same line, previous experiments cobs, P. L. & Maguire, M. E. (1981) Adv. Cyclic Nucleotide on Pd variants of S49 lymphoma cells have shown that al- Res. 14, 69-81. 13. Kram, R., Guyaux, M., Riviere, M. & Schmitt, H. (1980) in though the total receptor density is reduced by about 75% in Hormones and Cell Regulation, eds. Dumont, J. and Nunez, J. the Pd clone, adenylate cyclase activation and agonist/Mal- (Elsevier/North-Holland, Amsterdam), Vol. 4, pp. 241-256. NEt-sensitivity ratio are unaltered (8, 28). The assumption of 14. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, genetically different receptors is further weakened by the R. J. (1951) J. Biol. Chem. 193, 265-275. fact that these receptors have homogeneous physicochemi- 15. Vauquelin, G., Bottari, S., Kanarek, L. & Strosberg, A. D. cal characteristics in most tissues (29). (1979) J. Biol. Chem. 254, 4462-4469. An alternative cause for the limitation in the agonist/Mal- 16. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51, 660-672. NEt effect could be that the resistant receptors cannot inter- 17. Staehelin, M. & Simons, P. (1982) EMBO J. 1, 187-190. act with Ns in a productive manner because of differences in 18. Lad, P. M., Nielsen, T. B., Preston, M. S. & Rodbell, M. (1980) J. Biol. Chem. 255, 988-995. either the receptor conformation or its membrane microenvi- 19. Minneman, K. P., Hegstrand, L. R. & Molinoff, P. B. (1979) ronment. At the present level of investigation, it is not yet Mol. Pharmacol. 16, 34-46. possible to discriminate between these two possibilities, es- 20. Goldberg, Y., Guyaux, M., Riviere, M. & Kram, R. (1982) pecially since both might be related to the presence of differ- Arch. Int. Physiol. Biochim. 90, B119-B120. ent lipid microdomains in the membrane. The requirement 21. Guellaen, G. & Hanoune, J. (1979) Biochim. Biophys. Acta for regions of facilitated coupling between /8 receptors and 587, 618-627. Ns results also from theoretical considerations indicating 22. Lucas, M., Hanoune, J. & Bockaert, J. (1978) Mol. Pharma- that random collisions are not sufficient to explain the high col. 14, 227-236. level of stimulation of the adenylate cyclase 23. Drummond, G. I. (1980) Arch. Biochem. Biophys. 221, 30-38. P-adrenergic 24. Baldessarini, R. J. (1980) in Pharmacological Basis of Thera- system (30). Finally, it has already been demonstrated that peutics, eds. Goodman, L. S. & Gilman, A. (MacMillan, New factors that perturb the membrane, such as the incorporation York), pp. 391-447. of fillipin (31), hamper the Mg2+-dependent formation of 25. Kalisker, A., Rutledge, C. 0. & Perkins, J. P. (1973) Mol. high-agonist-affinity sites and, hence, receptor-Ns coupling. Pharmacol. 9, 619-629. In conclusion, we suggest that the basis for the functional 26. Nahorski, S. R. (1977) Mol. Pharmacol. 13, 679-689. ,B3adrenergic receptor heterogeneity might result from the 27. Kruh, J. (1982) Mol. Cell. Biochem. 42, 65-82. presence of only part of them in specialized membrane areas 28. Johnson, G. L., Bourne, H. R., Gleason, M. K., Coffino, with facilitated coupling to Ns. P. A., Insel, P. & Melmon, K. L. (1979) Mol. Pharmacol. 15, 16-27. 29. Stiles, G. L., Strasser, R. H., Lavin, T. N., Jones, L. R., Y.S. holds a Research Fellowship of the Instituut tot aanmoedig- Caron, M. G. & Lefkowitz, R. J. (1983) J. Biol. Chem. 258, ing van het Wetenschappelijk Onderzoek in Nijverheid en Land- 8443-8449. bouw, Belgium. G.V. is Bevoegdverklaard Navorser of the Nation- 30. Sonenberg, M. & Schneider, A. S. (1977) in Receptors and aal Fonds voor Wetenschappelijk Onderzoek, Belgium. This work Recognition, eds. Cuatrecasas, P. & Greaves, M. F. (Chap- was supported by grants from the Fonds voor Geneeskundig en We- man & Hall, London), Series A, Vol. 4, pp. 1-74. tenschappelijk Onderzoek and from the Fondation Universitaire A. 31. Puchwein, G., Pfeuffer, T. & Helmreich, E. J. M. (1974) J. et D. Van Buuren. Biol. Chem. 249, 3232-3240. Downloaded by guest on September 29, 2021