Vol. 62, Issue 3, 590-601, September 2002

Antibody Capture Assay Reveals Bell-Shaped Concentration-Response Isotherms for h5-HT_1A Receptor-Mediated G_i3 Activation: Conformational Selection by High-Efficacy Agonists, and Relationship to Trafficking of Receptor Signaling

Adrian Newman-Tancredi, Didier Cussac, Laetitia Marini, and Mark J. Millan

Department of Psychopharmacology, Institut de Recherches Servier, Croissy-sur-Seine, France

Although serotonin 5-HT_1A receptors couple to several Gi/o G-protein subtypes, little is known concerning their differential activation patterns. In this study, in membranes of Chinese hamster ovary cells expressing h5-hydroxytryptamine_1A receptors (CHO-h5-HT_1A), isotherms of 5-HT-stimulated guanosine-5'-O-(3-[³⁵S]thio)-triphosphate ([³⁵S]GTPS) 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 [³⁵S]GTPS dissociation by unlabeled GTPS, 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 [³⁵S]GTPS 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-2-yl)piperazine (S15535) displayed sigmoidal stimulation isotherms, whereas spiperone and other inverse agonists sigmoidally inhibited [³⁵S]GTPS binding. Agonist-induced stimulation and inverse agonist-induced inhibition of G_i3 activation were i) abolished by pretreatment of CHO-h5-HT_1A cells with pertussis toxin; ii) reversed by the selective 5-HT_1A antagonist (N-{2-[4-(2-methoxy-phenyl)-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-HT_1A agonists direct receptor signaling to G_i3. In contrast, partial agonists favor h5-HT_1A receptor signaling to G_i3 over a wide concentration range, whereas inverse agonists inhibit constitutive G_i3 activation.