Constitutive activity at serotonin 5-HT(1D) receptors: detection by homologous GTPgammaS versus [(35)S]-GTPgammaS binding isotherms.
Although many G-protein-coupled receptors (GPCRs) may display constitutive activity, their detection has, to date, depended on the use of inverse agonists. The present study exploited a novel procedure to investigate constitutive activity at recombinant human (h) serotonin (5-HT) 5-HT(1D) receptors stably expressed in Chinese hamster ovary (CHO) cells. 5-HT modestly stimulated guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]-GTPgammaS) binding to CHO-h5-HT(1D) membranes whereas methiothepin and the 5-HT(1B/1D)-selective ligand, SB224,289, exerted robust inhibition of basal [(35)S]-GTPgammaS binding (inverse agonism). These actions were specific inasmuch as they were reversed by the novel, selective 5-HT(1B/1D) ligand, S18127. Constitutive activity was investigated by homologous inhibition of [(35)S]-GTPgammaS binding to CHO-h5-HT(1D) membranes with unlabelled GTPgammaS. Under 'basal' conditions (absence of receptor ligand), biphasic isotherms were observed. Most (80%) [(35)S]-GTPgammaS binding sites were in the high affinity (HA) versus low affinity (LA) component of the isotherms. HA binding was augmented by 5-HT (to 155%; relative to basal values=100%), but decreased by methiothepin (to 23%) and by SB224,289 (to 67%). In contrast, LA binding was not altered. Further, membranes of untransfected CHO cells exhibited only LA binding sites, indicating that the latter are not related to h5-HT(1D) receptor-G-protein coupling. Thus, at 5-HT(1D) receptors expressed in this CHO cell line, HA binding detected in homologous inhibition experiments (GTPgammaS versus [(35)S]-GTPgammaS) under basal conditions provides a measure of constitutive G-protein activation. Thus, it is suggested that for h5-HT(1D) receptors and, possibly, other GPCRs, inverse agonists will be detectable by [(35)S]-GTPgammaS binding if a HA component is present under basal conditions.
Audinot
V
V
Department of Psychopharmacology, Institut de Recherches Servier, 125 Chemin de Ronde, 78290 Croissy-sur-Seine, Paris, France.
Newman-Tancredi
A
A
Millan
M J
MJ
D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects; D016466:CHO Cells; D006224:Cricetinae; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006801:Humans; D008719:Methiothepin / Q000494:pharmacology; D010881:Piperidones / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D044346:Receptor, Serotonin, 5-HT1D; D011985:Receptors, Serotonin / Q000187:drug effects* / Q000235:genetics; D012701:Serotonin / Q000494:pharmacology; D012702:Serotonin Antagonists / Q000494:pharmacology; D013141:Spiro Compounds / Q000494:pharmacology; D013462:Sulfur Radioisotopes; D013816:Thermodynamics; D014162:Transfection
D001549:Benzamides; C107225:N-(4-methoxy-3-(4-methylpiperazin-1-yl)phenyl)-3-methyl-4-(4-pyridyl)benzamide; D010881:Piperidones; D011725:Pyridines; D044346:Receptor, Serotonin, 5-HT1D; D011985:Receptors, Serotonin; C108518:SB 22489G; D012702:Serotonin Antagonists; D013141:Spiro Compounds; D013462:Sulfur Radioisotopes; D012701:Serotonin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008719:Methiothepin