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

Abstract

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 Gα_i1, α_i2, α_i3, or Gα_o-proteins and Gβ₁γ₂-subunits reconstituted a Gpp(NH)p-sensitive, high affinity binding of [³H]5-HT to this receptor, whereas the Gα_qβ₁γ₂ 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 Gα_i/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-HT_1Dreceptor 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]GTPγS 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_1Dreceptor-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.