title: Involvement of tryptophan W276 and of two surrounding amino acid residues in the high constitutive activity of the ghrelin receptor GHS-R1a.
abstract: The human ghrelin receptor (GHS-R1a) is known to display a high level of signaling in the absence of ligand. The Trp276, located in the fully conserved CWXP motif of G protein-coupled receptors, is believed to function as a rotameric switch in these receptors. A comparative modelling of GHS-R1a with the motilin receptor, the most related G protein-coupled receptor to GHS-R1a known to date, but characterized by a very low ligand-independent signaling level, revealed that only two surrounding residues of Trp276, that are Val131 and Ile134, were different from these receptors. We mutated them at once in GHS-R1a to create a "motilin receptor-like" environment of Trp276 in order to study the consequences on GHS-R1a activation. We studied the pharmacological properties of the W276A, V131L-I134M GHS-R1a mutants. Basal as well as maximal ghrelin-induced signaling was assessed both by inositol-phosphate accumulation and SRE pathways. As compared to the wild type receptor, the SRE-luciferase assay displayed a markedly impaired basal activity for W276A whereas that of V131L-I134M was, strikingly, two fold increased. Nevertheless, the efficacy of ghrelin to bind or to stimulate mutant receptors remained unchanged. It is concluded that Trp276, Val131 and Ile134 have a significant impact on constitutive signaling of GHS-R1a, V131L-I134M being the first example of a GHS-R1a mutant with a higher basal activity than the wild type receptor.
authors.affiliation: Institut des Biomolécules Max Mousseron, UMR, CNRS, Universités Montpellier, France. catherine.goze@univ-montp1.fr
authors.forename: Catherine
authors.initials: C
authors.lastname: Gozé
authors.forename: Gilbert
authors.initials: G
authors.lastname: Bergé
authors.forename: Céline
authors.initials: C
authors.lastname: M'Kadmi
authors.forename: Nicolas
authors.initials: N
authors.lastname: Floquet
authors.forename: Didier
authors.initials: D
authors.lastname: Gagne
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Galleyrand
authors.forename: Jean-Alain
authors.initials: JA
authors.lastname: Fehrentz
authors.forename: Jean
authors.initials: J
authors.lastname: Martinez
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D017124:Conserved Sequence; D054439:Ghrelin / Q000378:metabolism; D006801:Humans; D007532:Isoleucine / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D011964:Receptors, Gastrointestinal Hormone / Q000737:chemistry; D054440:Receptors, Ghrelin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018013:Receptors, Neuropeptide / Q000737:chemistry; D015398:Signal Transduction; D014364:Tryptophan / Q000737:chemistry / Q000502:physiology*; D014633:Valine / Q000378:metabolism
chemical_list: D054439:Ghrelin; D011964:Receptors, Gastrointestinal Hormone; D054440:Receptors, Ghrelin; D018013:Receptors, Neuropeptide; C050802:motilin receptor; D007532:Isoleucine; D014364:Tryptophan; D014633:Valine