title: SET protein interacts with intracellular domains of the gonadotropin-releasing hormone receptor and differentially regulates receptor signaling to cAMP and calcium in gonadotrope cells.
abstract: In mammals, the receptor of the neuropeptide gonadotropin-releasing hormone (GnRHR) is unique among the G protein-coupled receptor (GPCR) family because it lacks the carboxyl-terminal tail involved in GPCR desensitization. Therefore, mechanisms involved in the regulation of GnRHR signaling are currently poorly known. Here, using immunoprecipitation and GST pull-down experiments, we demonstrated that SET interacts with GnRHR and targets the first and third intracellular loops. We delineated, by site-directed mutagenesis, SET binding sites to the basic amino acids (66)KRKK(69) and (246)RK(247), located next to sequences required for receptor signaling. The impact of SET on GnRHR signaling was assessed by decreasing endogenous expression of SET with siRNA in gonadotrope cells. Using cAMP and calcium biosensors in gonadotrope living cells, we showed that SET knockdown specifically decreases GnRHR-mediated mobilization of intracellular cAMP, whereas it increases its intracellular calcium signaling. This suggests that SET influences signal transfer between GnRHR and G proteins to enhance GnRHR signaling to cAMP. Accordingly, complexing endogenous SET by introduction of the first intracellular loop of GnRHR in αT3-1 cells significantly reduced GnRHR activation of the cAMP pathway. Furthermore, decreasing SET expression prevented cAMP-mediated GnRH stimulation of Gnrhr promoter activity, highlighting a role of SET in gonadotropin-releasing hormone regulation of gene expression. In conclusion, we identified SET as the first direct interacting partner of mammalian GnRHR and showed that SET contributes to a switch of GnRHR signaling toward the cAMP pathway.
authors.affiliation: Université Paris Diderot, Sorbonne Paris Cité, Biologie Fonctionnelle et Adaptative, Equipe Physiologie de l'Axe Gonadotrope, Case Courrier 7007, CNRS-EAC 4413, 4 Rue MA Lagroua Weill Hallé, 75013 Paris, France.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Avet
authors.forename: Ghislaine
authors.initials: G
authors.lastname: Garrel
authors.forename: Chantal
authors.initials: C
authors.lastname: Denoyelle
authors.forename: Jean-Noël
authors.initials: JN
authors.lastname: Laverrière
authors.forename: Raymond
authors.initials: R
authors.lastname: Counis
authors.forename: Joëlle
authors.initials: J
authors.lastname: Cohen-Tannoudji
authors.forename: Violaine
authors.initials: V
authors.lastname: Simon
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D000242:Cyclic AMP / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D052681:Gonadotrophs / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D011966:Receptors, LHRH / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D034741:RNA, Small Interfering; D011966:Receptors, LHRH; D011994:Recombinant Proteins; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D002118:Calcium