TY - JOUR T1 - Reconstitution of the Human 5-HT<sub>1D</sub> Receptor-G-Protein Coupling: Evidence for Constitutive Activity and Multiple Receptor Conformations JF - Molecular Pharmacology JO - Mol Pharmacol SP - 1132 LP - 1141 VL - 57 IS - 6 AU - Reginald Brys AU - Katty Josson AU - Maria Paola Castelli AU - Mirek Jurzak AU - Peter Lijnen AU - Walter Gommeren AU - Josée E. Leysen Y1 - 2000/06/01 UR - http://molpharm.aspetjournals.org/content/57/6/1132.abstract N2 - 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-HT1D receptor were studied using a baculovirus-based expression system in Sf9 cells. Coexpression of the human 5-HT1D receptor with Gαi1, αi2, αi3, or Gαo-proteins and Gβ1γ2-subunits reconstituted a Gpp(NH)p-sensitive, high affinity binding of [3H]5-HT to this receptor, whereas the Gαqβ1γ2 heterotrimer was ineffective in this respect. Competition of [3H]5-HT binding by various compounds confirmed that coexpression of the human 5-HT1D receptor with Gαi/oβ1γ2 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-HT1Dreceptor in coupled or noncoupled state was analyzed. This compound competed with [3H]5-HT binding more potently on the human 5-HT1D 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]GTPγS binding experiments using the inverse agonist ocaperidone revealed a high level of constitutive activity of the human 5-HT1D receptor. Taken together, the reconstitution of the human 5-HT1Dreceptor-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. The American Society for Pharmacology and Experimental Therapeutics ER -