Antibody Capture Assay Reveals Bell-Shaped Concentration- Response Isotherms for h5-HT1A Receptor-Mediated G i3 Activation: Conformational Selection by High-Efficacy Agonists, and Relationship to Trafficking of Receptor Signaling

Although serotonin 5-HT1A receptors couple to several Gi/o Gprotein subtypes, little is known concerning their differential activation patterns. In this study, in membranes of Chinese hamster ovary cells expressing h5-hydroxytryptamine1A receptors (CHOh5-HT1A), isotherms of 5-HT-stimulated guanosine-5 -O-(3[S]thio)-triphosphate ([S]GTP S) binding were biphasic, suggesting coupling to multiple G-protein subtypes. The high potency component was abolished by preincubation with an antibody recognizing G i3 subunits and was resistant to induction of [S]GTP S dissociation by unlabeled GTP S, thus yielding a bell-shaped concentration-response isotherm. To directly investigate G i3 activation, we adopted an antibody-capture/scintillation proximity assay. 5-HT and other high-efficacy agonists yielded bell-shaped [S]GTP S binding isotherms, with peaks at nanomolar concentrations. As drug concentrations increased, G i3 stimulation progressively returned to basal values. In contrast, the partial agonists ( )-pindolol and 4-(benzodioxan-5-yl)1-(indan-2yl)piperazine (S15535) displayed sigmoidal stimulation isotherms, whereas spiperone and other inverse agonists sigmoidally inhibited [S]GTP S binding. Agonist-induced stimulation and inverse agonist-induced inhibition of G i3 activation were i) abolished by pretreatment of CHO-h5-HT1A cells with pertussis toxin; ii) reversed by the selective 5-HT1A antagonist (N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)-cyclohexane-carboxamide) fumarate (WAY100,635), and iii) absent in nontransfected CHO cell membranes. 5-HT isotherms could be modified by altering sodium concentration; only stimulatory actions were observed at 300mM NaCl, whereas only inhibitory actions were seen at 10 mM NaCl. Furthermore, bell-shaped isotherms were not detected at short incubation times, suggesting time-dependent changes in receptor/G i3 coupling. Taken together, these data show that low but not high concentrations of high-efficacy 5-HT1A agonists direct receptor signaling to G i3. In contrast, partial agonists favor h5-HT1A receptor signaling to G i3 over a wide concentration range, whereas inverse agonists inhibit constitutive G i3 activation. Serotonin 5-HT1A receptors are therapeutic targets in the management of anxiety, depression, and schizophrenia (Barnes and Sharp, 1999; Millan, 2000). Cloning of human 5-HT1A receptors and their expression in recombinant cell lines has permitted the elucidation of their signal transduction pathways. They couple, thus, to multiple transduction pathways, including adenylyl cyclase, phospholipase C, sodium-dependent phosphate uptake, protein kinase C, Na /K ATPase, K channels, and mitogen-activated protein kinase (Fargin et al., 1989; for reviews, see Gerhardt and Heerikhuizen, 1997; Raymond et al., 1999). These cellular responses are abolished by pretreatment with Bordetella pertussis toxin (PTX), which ADP-ribosylates G subunits of the Gi/o family, indicating the coupling of 5-HT1A receptors to Gi/o-proteins. Subtypes of G subunits engaged by 5-HT1A receptors have been identified by coexpression studies in a variety of bacterial, insect, and mammalian expression systems (for review, see Raymond et al., 1999). Thus, reconstitution of h5-HT1A receptors expressed in Escherichia coli with recombinant G i3 subunits most markedly increased affinity for the agonist [H]( )-8-hydroxy-dipropyl-aminotetralin ([H]8OH-DPAT), followed by G i2 and G i1 subunits (Bertin et al., ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; PTX, Bordetella pertussis toxin; 8-OH-DPAT, ( )-8-hydroxy-dipropyl-aminotetralin; CHO, Chinese hamster ovary; CHO-h5-HT1A, Chinese hamster ovary cells expressing human 5-HT1A receptors; GPCR, G-protein coupled receptor; SPA, scintillation proximity assay; [S]GTP S, guanosine-5 -O-(3-[S]thio)-triphosphate; S15535, 4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine; WAY100,635, (N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)-cyclohexane-carboxamide) fumarate; 5-CT, 5-carboxyamidotryptamine; S16924, [(R)-2-{1-[2-(2,3-dihydro-benzo[1,4]dioxin-5-yloxy)-ethyl]-pyrrolidin-3-yl}-1-(4-fluoro-phenyl)-ethanone] HCl; S14506, (1-[2-(4fluorobenzoylamino)ethyl]-4-(7-methoxynaphtyl)piperazine) HCl; S14671, (1-(7-methoxynaphth-1-yl)-4-[2-(2-thenoylamino)-ethyl]piperazine) HCl; UK14,304, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; RGS, regulator of G-protein signaling; Ro318220, 3-[1-[3-(amidinothio) propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl)maleimide methanesulfonate; pERK, phosphorylated extracellular signal regulated kinase. 0026-895X/02/6203-590–601$7.00 MOLECULAR PHARMACOLOGY Vol. 62, No. 3 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1774/1003847 Mol Pharmacol 62:590–601, 2002 Printed in U.S.A. 590 at A PE T Jornals on Sptem er 0, 2017 m oharm .aspeurnals.org D ow nladed from 1992). A similar reconstitution strategy in insect Spodoptera frugiperda (Sf9) cells likewise showed coupling of 5-HT1A receptors to Gi/o G-protein subtypes, and particularly to G i3 (Butkerait et al., 1995; Clawges et al., 1997). Functional responses of 5-HT1A receptors have also been shown to be mediated by G i3 and G i2 subunits. Thus, microphysiometric measurement of medium acidification showed that PTXresistant mutants of G i3 or G i2 “rescued” agonist-induced Na /H exchange in h5-HT1A receptor-expressing Chinese hamster ovary (CHO) cells pretreated with PTX (Garnovskaya et al., 1997). Furthermore, antisera raised against G i3 and G i2 subunits attenuated h5-HT1A receptor-mediated inhibition of adenylyl cyclase activity in CHO and HeLa cells (Raymond et al., 1993). Taken together, these studies converge toward the conclusion that 5-HT1A receptors couple preferentially to G i3 subunits, followed by G i2 and, less strongly, G o and G i1 subunits (Clawges et al., 1997). On the other hand, coupling of 5-HT1A receptors to G q and G s is weak or absent (Raymond et al., 1993). Nevertheless, coupling to G-protein subtypes can be investigated when receptor/G-protein stoichiometry is constrained [e.g., in h5-HT1AG i1 fusion proteins (Kellett et al., 1999)], and a recent study reported stimulation of adenylyl cyclase by 5-HT1A receptors at high agonist concentrations, suggesting that Gs activation is, in fact, possible under certain conditions (Malmberg and Strange, 2000). The issue of differential coupling to G-proteins is of particular interest in view of accumulating reports describing agonist-directed trafficking of receptor signaling (Kenakin, 1995). Thus G-protein-coupled receptors (GPCRs) may exhibit differential coupling to intracellular G-proteins or second messenger systems depending on the agonist employed. For example, agonists at h5-HT2C receptors display differential orders of efficacy for activation of phospholipase C versus phospholipase A2 (Berg et al., 1998; for review, see Clarke and Bond, 1998), whereas agonists differentially induce coupling of h 2A-adrenoceptors to Gior Gs-mediated modulation of adenylyl cyclase activity (Brink et al., 2000). In the case of 5-HT1A receptors, Gettys et al. (1994a) undertook a limited study of the potency of different agonists in activating G i3 and G i2 subunits, as detected by 4-azidoanilido-[ P]GTP labeling, membrane solubilization, and immunoprecipitation of G subunits. They showed that, whereas some agonists (5-HT and 8-OH-DPAT) more potently activated G i3 than G i2, rauwolscine activated both subunits with similar potency (Gettys et al., 1994a). However, this technique is cumbersome and ill-adapted to broad investigation of numerous drugs and assay conditions. Furthermore, the molecular basis for these examples of agonist-directed trafficking are little characterized and are probably influenced by multiple factors, including receptor and G-protein expression levels, ionic conditions, etc. In view of the above considerations, the present study employed a recently developed antibody-capture technique coupled to scintillation proximity assay (SPA) detection (DeLapp et al., 1999) to characterize G i3 subunit activation at recombinant human 5-HT1A receptors stably expressed in Chinese hamster ovary cells (CHO-h5-HT1A cells; NewmanTancredi et al., 1998). CHO cells constitute a useful model to investigate 5-HT1A receptor coupling because they express G i3 and G i2 (Gerhardt and Neubig, 1991; Law et al., 1993; Gettys et al., 1994b). CHO cells also express low levels of G o, but G i1 is undetectable (Gerhardt and Neubig, 1991; Gettys et al., 1994b; Law et al., 1993; present study). Using antibody capture/SPA methods, we show here that highefficacy agonists, but not partial agonists or inverse agonists, exhibit bell-shaped isotherms for G i3 activation at h5-HT1A receptors. The data indicate that low concentrations of highefficacy agonists direct coupling of the receptor to G i3, whereas, at higher concentrations, this coupling is suppressed. A preliminary report of the present data was presented in abstract form (Newman-Tancredi et al., 2002). Materials and Methods Membranes Preparations from CHO-h5-HT1A cells. Membranes from transfected CHO cells stably expressing the human serotonin 5-HT1A (h5-HT1A) receptor ( 4 pmol/mg) were prepared as described previously (Newman-Tancredi et al., 1998). Briefly, cells were grown in RPMI 1640 medium containing 10% (v/v) fetal bovine serum, penicillin, and streptomycin until they reached confluence. Cells were harvested by centrifugation and homogenized using a Polytron homogenizer (Kinematica, Basel, Switzerland) in buffer A (20 mM HEPES, pH 7.4, and 3 mM MgCl2). The homogenate was centrifuged at 50,000g for 30 min, the membrane pellet resuspended in buffer A and stored at 80°C. Protein concentration was determined by use of a bicinchoninic acid kit (Sigma-Aldrich, St. Quentin-