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Titolo: Reconstitution of the human 5-HT(1D) receptor-G-protein coupling: evidence for constitutive activity and multiple receptor conformations
Data di pubblicazione: 2000
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-HT1D receptor were studied using a baculovirus-based expression system in Sf9 cells. Coexpression of the human 5-HT1D receptor with G alpha(i1), alpha(i2), alpha(i3),or G alpha(o)-proteins and G beta(1)gamma(2)-subunits reconstituted a Gpp(NH)p-sensitive, high affinity binding of [H-3]5-HT to this receptor, whereas the G alpha(q)beta(1)gamma(2) heterotrimer was ineffective in this respect. Competition of [H-3]5-HT binding by various compounds confirmed that coexpression of the human 5-HT1D receptor with G alpha(i/o)beta(1)gamma(2) 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-HT1D receptor in coupled or noncoupled state was analyzed. This compound competed with [H-3]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 [H-3]5-HT binding when tested with the coupled receptor form but not so when tested with the noncoupled receptor preparation. Finally, [S-35]GTP gamma S 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-HT1D 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
Tipologia:1.1 Articolo in rivista

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