Proc. Natl. Acad. Sci. USA Vol. 81, pp. 4637-4641, 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 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 agonists 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 drug 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; fenoterol population of sites with regard to antagonist binding, but two hydrobromide from Boehringer-Ingelheim; practolol hydro- subpopulations can be discriminated for agonist binding. chloride from ICI; (+)-alprenolol hydrochloride, phentol- This has been established by two types of observations. amine hydrochloride, desipramine, and reserpine from First, magnesium ions cause an increase in agonist but not in CIBA-Geigy; doxepin from Pfizer; mianserin 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]Dihydroalprenolol 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 antidepressant pharmacological. Experiments on turkey erythrocyte mem- drugs (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