Constitutive activity at serotonin 5-HT1D receptors:: detection by homologous GTPγS versus [35S]-GTPγS 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-HT1D receptors stably expressed in Chinese hamster ovary (CHO) cells. 5-HT modestly stimulated guanosine-5'-0-(3-[S-35]thio)-triphosphate ([S-35]-GTP gammaS) binding to CHO-h5-HT1D membranes whereas methiothepin and the 5-HT1B/1D-selective ligand, SB224,289, exerted robust inhibition of basal [S-35]-GTP gammaS binding (inverse agonism). These actions were specific inasmuch as they were reversed by the novel, selective 5-HT1B/1D ligand, S18127. Constitutive activity was investigated by homologous inhibition of [S-35]-GTP gammaS binding to CHO-h5-HT1D membranes with unlabelled GTP gammaS. Under 'basal' conditions (absence of receptor ligand), biphasic isotherms were observed. Most (80%) [S-35]-GTP gammaS 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-HT1D receptor-G-protein coupling. Thus, at 5-HT1D receptors expressed in this CHO cell line, HA binding detected in homologous inhibition experiments (GTP gammaS versus [S-35]-GTP gammaS) under basal conditions provides a measure of constitutive G-protein activation. Thus, it is suggested that for h5-HT1D receptors and, possibly, other GPCRs, inverse agonists will be detectable by [S-35]-GTP gammaS binding if a HA component is present under basal conditions. (C) 2000 Elsevier Science Ltd. All rights reserved.