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Newman-Tancredi A, Cussac D, Marini L, Millan MJ: Antibody capture assay reveals bell-shaped concentration-response isotherms for h5-HT (1A) receptor-mediated Galpha (i3) activation: conformational selection by high-efficacy agonists, and relationship to trafficking of receptor signaling. Mol Pharmacol. 2002 Sep;62(3):590-601.
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] GTPgammaS) binding were biphasic, suggesting coupling to multiple G-protein subtypes. The high potency component was abolished by preincubation with an antibody recognizing Galpha (i3) subunits and was resistant to induction of [(35) S] GTPgammaS dissociation by unlabeled GTPgammaS, thus yielding a bell-shaped concentration-response isotherm. To directly investigate Galpha (i3) activation, we adopted an antibody-capture/scintillation proximity assay. 5-HT and other high-efficacy agonists yielded bell-shaped [(35) S] GTPgammaS binding isotherms, with peaks at nanomolar concentrations. As drug concentrations increased, Galpha (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] GTPgammaS binding. Agonist-induced stimulation and inverse agonist-induced inhibition of Galpha (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)-cyclohexa ne-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/Galpha (i3) coupling. Taken together, these data show that low but not high concentrations of high-efficacy 5-HT (1A) agonists direct receptor signaling to Galpha (i3). In contrast, partial agonists favor h5-HT (1A) receptor signaling to Galpha (i3) over a wide concentration range, whereas inverse agonists inhibit constitutive Galpha (i3) activation. |
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