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.
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)-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/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.
Department of Psychopharmacology, Institut de Recherches Servier, Croissy-sur-Seine, France.
Adrian
A
Newman-Tancredi
Didier
D
Cussac
Laetitia
L
Marini
Mark J
MJ
Millan
D000818:Animals; D002021:Buffers; D016466:CHO Cells; D006224:Cricetinae; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D006153:Guanosine Diphosphate / Q000494:pharmacology; D020962:Heterotrimeric GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D015636:Magnesium Chloride / Q000494:pharmacology; D010869:Pindolol / Q000494:pharmacology; D011487:Protein Conformation; D021122:Protein Subunits; D011985:Receptors, Serotonin / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D044263:Receptors, Serotonin, 5-HT1; D012701:Serotonin / Q000378:metabolism; D012702:Serotonin Antagonists / Q000494:pharmacology; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology*; D012965:Sodium Chloride / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D002021:Buffers; D021122:Protein Subunits; D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; D015636:Magnesium Chloride; D006153:Guanosine Diphosphate; D012701:Serotonin; D012965:Sodium Chloride; D010869:Pindolol; C498819:GNAI3 protein, human; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins