title: 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 Galpha(i1), alpha(i2), alpha(i3), or Galpha(o)-proteins and Gbeta(1)gamma(2)-subunits reconstituted a Gpp(NH)p-sensitive, high affinity binding of [(3)H]5-HT to this receptor, whereas the Galpha(q)beta(1)gamma(2) heterotrimer was ineffective in this respect. Competition of [(3)H]5-HT binding by various compounds confirmed that coexpression of the human 5-HT(1D) receptor with Galpha(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-HT(1D) receptor in coupled or noncoupled state was analyzed. This compound competed with [(3)H]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 [(3)H]5-HT binding when tested with the coupled receptor form but not so when tested with the noncoupled receptor preparation. Finally, [(35)S]GTPgammaS 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.
authors.affiliation: Janssen Research Foundation, Department of Biochemical Pharmacology, Beerse, Belgium.
authors.forename: R
authors.initials: R
authors.lastname: Brys
authors.forename: K
authors.initials: K
authors.lastname: Josson
authors.forename: M P
authors.initials: MP
authors.lastname: Castelli
authors.forename: M
authors.initials: M
authors.lastname: Jurzak
authors.forename: P
authors.initials: P
authors.lastname: Lijnen
authors.forename: W
authors.initials: W
authors.lastname: Gommeren
authors.forename: J E
authors.initials: JE
authors.lastname: Leysen
mesh_terms: D000818:Animals; D014150:Antipsychotic Agents / Q000494:pharmacology; D001667:Binding, Competitive; D002478:Cells, Cultured; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007313:Insecta; D010880:Piperidines / Q000494:pharmacology; D011487:Protein Conformation; D011744:Pyrimidinones / Q000494:pharmacology; D044302:Receptor, Serotonin, 5-HT1B; D044346:Receptor, Serotonin, 5-HT1D; D011985:Receptors, Serotonin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014316:Tritium
chemical_list: D014150:Antipsychotic Agents; C470302:HTR1B protein, human; D010880:Piperidines; D011744:Pyrimidinones; D044302:Receptor, Serotonin, 5-HT1B; D044346:Receptor, Serotonin, 5-HT1D; D011985:Receptors, Serotonin; D013462:Sulfur Radioisotopes; D014316:Tritium; C072259:ocaperidone; D012701:Serotonin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins