title: Conformational toggle switches implicated in basal constitutive and agonist-induced activated states of 5-hydroxytryptamine-4 receptors.
abstract: The extended classic ternary complex model predicts that a G protein-coupled receptor (GPCR) exists in only two interconvertible states: an inactive R, and an active R(*). However, different structural active R(*) complexes may exist in addition to a silent inactive R ground state (Rg). Here we demonstrate, in a cellular context, that several R(*) states of 5-hydroxytryptamine-4 (5-HT(4)) receptors involve different side-chain conformational toggle switches. Using site-directed mutagenesis and molecular modeling approaches, we show that the basal constitutive receptor (R(*)basal) results from stabilization of an obligatory double toggle switch (Thr3.36 from inactive g- to active g+ and Trp6.48 from inactive g+ to active t). Mutation of either threonine or tryptophan to alanine resulted in a lowering of the activity of the R(*)basal similar to the Rg. The T3.36A mutation shows that the Thr3.36 toggle switch plays a minor role in the stabilization of R(*) induced by 5-HT (R(*)-5-HT) and BIMU8 (R(*)-BIMU8) and is fully required in the stabilization of R(*) induced by (S)-zacopride, cisapride, and 1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1-butyl-4-piperidinyl)-1-propanone (RS 67333) (R(*)-benzamides). Thus, benzamides stabilize R(*)-benzamides by forming a specific hydrogen bond with Thr3.36 in the active g+ conformation. Conversely, R(*)-BIMU8 was probably the result of a direct conformational transition of Trp6.48 from inactive g+ to active t by hydrogen bonding of this residue to a carboxyl group of BIMU8. We were surprised that the Trp6.48 toggle switch was not necessary for receptor activation by the natural agonist 5-HT. R(*)-5-HT is probably attained through other routes of activation. Thus, different conformational arrangements occur during stabilization of R(*)basal, R(*)-5-HT, R(*)-benzamides, and R(*)-BIMU8.
authors.affiliation: Centre National de Recherche Scientifique Unité Mixte de Recherche 5203, Institut de Génomique Fonctionnelle, Institut National de Sant et de Recherche Médicale U661, Universités Montpellier 1 and 2, Montpellier, France.
authors.forename: Lucie P
authors.initials: LP
authors.lastname: Pellissier
authors.forename: Jessica
authors.initials: J
authors.lastname: Sallander
authors.forename: Mercedes
authors.initials: M
authors.lastname: Campillo
authors.forename: Florence
authors.initials: F
authors.lastname: Gaven
authors.forename: Emilie
authors.initials: E
authors.lastname: Queffeulou
authors.forename: Marion
authors.initials: M
authors.lastname: Pillot
authors.forename: Aline
authors.initials: A
authors.lastname: Dumuis
authors.forename: Sylvie
authors.initials: S
authors.lastname: Claeysen
authors.forename: Joël
authors.initials: J
authors.lastname: Bockaert
authors.forename: Leonardo
authors.initials: L
authors.lastname: Pardo
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation / Q000187:drug effects; D017433:Protein Structure, Secondary; D044422:Receptors, Serotonin, 5-HT4 / Q000737:chemistry* / Q000502:physiology; D058828:Serotonin 5-HT4 Receptor Agonists; D017366:Serotonin Receptor Agonists / Q000494:pharmacology*
chemical_list: D058828:Serotonin 5-HT4 Receptor Agonists; D017366:Serotonin Receptor Agonists; D044422:Receptors, Serotonin, 5-HT4