Antibody Capture Assay Reveals Bell-Shaped Concentration-Response Isotherms for h5-HT1A Receptor-Mediated Gαi3Activation: Conformational Selection by High-Efficacy Agonists, and Relationship to Trafficking of Receptor Signaling

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Abstract
Although serotonin 5-HT1A 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-hydroxytryptamine1A receptors (CHO-h5-HT1A), isotherms of 5-HT-stimulated guanosine-5′-O-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding were biphasic, suggesting coupling to multiple G-protein subtypes. The high potency component was abolished by preincubation with an antibody recognizing Gαi3 subunits and was resistant to induction of [35S]GTPγS dissociation by unlabeled GTPγS, thus yielding a bell-shaped concentration-response isotherm. To directly investigate Gαi3 activation, we adopted an antibody-capture/scintillation proximity assay. 5-HT and other high-efficacy agonists yielded bell-shaped [35S]GTPγS binding isotherms, with peaks at nanomolar concentrations. As drug concentrations increased, Gαi3 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]GTPγS binding. Agonist-induced stimulation and inverse agonist-induced inhibition of Gαi3 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-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/Gαi3coupling. Taken together, these data show that low but not high concentrations of high-efficacy 5-HT1A agonists direct receptor signaling to Gαi3. In contrast, partial agonists favor h5-HT1A receptor signaling to Gαi3 over a wide concentration range, whereas inverse agonists inhibit constitutive Gαi3 activation.