Vol. 58, Issue 5, 1042-1049, November 2000

Inverse Agonism and Constitutive Activity as Functional Correlates of Serotonin h5-HT_1B Receptor/G-Protein Stoichiometry

Adrian Newman-Tancredi, Valérie Audinot, Celia Moreira, Laurence Verrièle, and Mark J. Millan

Departments of Psychopharmacology (A.N.-T., C.M., L.V., M.J.M.) and Molecular and Cellular Pharmacology (V.A.), Institut de Recherches Servier, Croissy-sur-Seine (Paris), France

ABSTRACT

This study evaluated the influence of receptor/G-protein (R:G) stoichiometry on constitutive activity and the efficacy of agonists, partial agonists, and inverse agonists at human (h) 5-hydroxytryphamine 1B (5-HT_1B) receptors. Two Chinese hamster ovary cell lines were used; they expressed 8.5 versus 0.4 pmol h5-HT_1B receptors/mg (determined by [³H]GR125,743 saturation analysis) and 3.0 versus 1.5 pmol receptor-activated G-proteins/mg [determined by guanosine-5'-O-(3-[³⁵S]thio)-triphosphate ([³⁵S]GTPS) isotopic dilution], respectively. Thus, they displayed R:G ratios of ~3.0 (RGhigh) and ~0.3 (RGlow), respectively. In competition-binding experiments, the agonists, 5-HT and sumatriptan, displayed fewer high-affinity (HA)-binding sites and the partial agonists, BMS181,101 and L775,606, displayed decreased affinity in RGhigh versus RGlow membranes. In contrast, the inverse agonists, SB224,289 and, to a lesser extent, methiothepin, showed increased affinity. In G-protein activation experiments, both basal and 5-HT-activated [³⁵S]GTPS binding were higher in RGhigh than in RGlow membranes. Constitutive activity (determined by inhibition of basal [³⁵S]GTPS binding with GTPS in the absence of receptor ligands) was more pronounced in RGhigh versus RGlow membranes, as revealed by the >5-fold greater proportion of HA sites. Correspondingly, the negative efficacy of inverse agonists was strikingly augmented, inasmuch as they suppressed approximately two-thirds of HA [³⁵S]GTPS binding in RGhigh membranes, but only approximately one-third in RGlow membranes. Furthermore, the efficacy of partial agonists was greater at RGhigh versus RGlow membranes, as estimated by their ability to enhance [³⁵S]GTPS binding. In conclusion, an increase in R:G ratios at h5-HT_1B receptors was associated with an increase in relative efficacy of partial agonists and, most notably, an increase in both constitutive G-protein activation and negative efficacy of inverse agonists.