Vol. 57, Issue 6, 1132-1141, June 2000

Reconstitution of the Human 5-HT_1D Receptor-G-Protein Coupling: Evidence for Constitutive Activity and Multiple Receptor Conformations

Reginald Brys,¹ Katty Josson,¹ Maria Paola Castelli, Mirek Jurzak, Peter Lijnen, Walter Gommeren, and Josée E. Leysen

Janssen Research Foundation, Department of Biochemical Pharmacology, Beerse, Belgium (R.B., K.J., M.J., P.L., W.G., J.E.L.), University of Cagliari, Department of Neuroscience, Cagliari, Italy (M.P.C.)

The 5-hydroxytryptamine (5-HT) 1D/1B receptors have gained particular interest as potential targets for treatment of migraine and depression. G-protein coupling and other intrinsic properties of the human 5-HT_1D receptor were studied using a baculovirus-based expression system in Sf9 cells. Coexpression of the human 5-HT_1D receptor with G_i1, _i2, _i3, or G_o-proteins and G₁₂-subunits reconstituted a Gpp(NH)p-sensitive, high affinity binding of [³H]5-HT to this receptor, whereas the G_q12 heterotrimer was ineffective in this respect. Competition of [³H]5-HT binding by various compounds confirmed that coexpression of the human 5-HT_1D receptor with G_i/o12 reconstitutes the receptor in a high affinity agonist binding state, having the same pharmacological profile as the receptor expressed in mammalian cells. Binding of the antagonist ocaperidone to the human 5-HT_1D receptor in coupled or noncoupled state was analyzed. This compound competed with [³H]5-HT binding more potently on the human 5-HT_1D receptor in the noncoupled state, showing its inverse agonistic character. Ocaperidone acted as a competitive inhibitor of [³H]5-HT binding when tested with the coupled receptor form but not so when tested with the noncoupled receptor preparation. Finally, [³⁵S]GTPS binding experiments using the inverse agonist ocaperidone revealed a high level of constitutive activity of the human 5-HT_1D receptor. Taken together, the reconstitution of the human 5-HT_1D receptor-G-protein coupling using baculovirus-infected Sf9 cells made possible the assessment of coupling specificity and the detection of different binding states of the receptor induced by G-protein coupling or ligand binding.