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

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-[ 35 S]thio)-triphosphate ([ 35 S]GTPγS) binding were biphasic, suggesting coupling to multiple G-protein subtypes. The high potency component was abolished by preincubation with an antibody recognizing Gα i3 subunits and was resistant to induction of [ 35 S]GTPγS dissociation by unlabeled GTPγS, thus yielding a bell-shaped concentration-response isotherm. To directly investigate Gα i3 activation, we adopted an antibody-capture/scintillation proximity assay. 5-HT and other high-efficacy agonists yielded bell-shaped [ 35 S]GTPγS binding isotherms, with peaks at nanomolar concentrations. As drug concentrations increased, Gα 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 [ 35 S]GTPγS binding. Agonist-induced stimulation and inverse agonist-induced inhibition of Gα 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/Gα i3 coupling. Taken together, these data show that low but not high concentrations of high-efficacy 5-HT 1A agonists direct receptor signaling to Gα i3 . In contrast, partial agonists favor h5-HT 1A receptor signaling to Gα i3 over a wide concentration range, whereas inverse agonists inhibit constitutive Gα i3 activation.