title: A 5-HT4 receptor transmembrane network implicated in the activity of inverse agonists but not agonists.
abstract: Activation of G protein-coupled receptors is thought to involve disruption of intramolecular interactions that stabilize their inactive conformation. Such disruptions are induced by agonists or by constitutively active mutations. In the present study, novel potent inverse agonists are described to inhibit the constitutive activity of 5-HT(4) receptors. Using these compounds and specific receptor mutations, we investigated the mechanisms by which inverse agonists may reverse the disruption of intramolecular interactions that causes constitutive activation. Two mutations (D100(3.32)A in transmembrane domain (TMD)-III and F275(6.51)A in TMD-VI) were found to completely block inverse agonist effects without impairing their binding properties nor the molecular activation switches induced by agonists. Based on the rhodopsin model, we propose that these mutated receptors are in equilibrium between two states R and R* but are unable to reach a third "silent" state stabilized by inverse agonists. We also found another mutation in TMD-VI (W272(6.48)A) that stabilized this silent state. This mutant remained fully activated by agonists. Molecular modeling indicated that Asp-100, Phe-275, and Trp-272 might constitute a network required for stabilization of the silent state by the described inverse agonists. However, this network is not necessary for agonist activity.
authors.affiliation: Centre National de la Recherche Scientifique, UPR 9023, Montpellier, France.
authors.forename: Lara
authors.initials: L
authors.lastname: Joubert
authors.forename: Sylvie
authors.initials: S
authors.lastname: Claeysen
authors.forename: Michèle
authors.initials: M
authors.lastname: Sebben
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Bessis
authors.forename: Robin D
authors.initials: RD
authors.lastname: Clark
authors.forename: Renee S
authors.initials: RS
authors.lastname: Martin
authors.forename: Joël
authors.initials: J
authors.lastname: Bockaert
authors.forename: Aline
authors.initials: A
authors.lastname: Dumuis
mesh_terms: D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D017931:DNA Primers; D008958:Models, Molecular; D009154:Mutation; D011985:Receptors, Serotonin / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D044422:Receptors, Serotonin, 5-HT4; D012702:Serotonin Antagonists / Q000494:pharmacology*; D017366:Serotonin Receptor Agonists / Q000494:pharmacology*
chemical_list: D017931:DNA Primers; D011985:Receptors, Serotonin; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; D044422:Receptors, Serotonin, 5-HT4