title: Influence of the accessory protein SET on M3 muscarinic receptor phosphorylation and G protein coupling.
abstract: The proto-oncogene and inhibitor of protein phosphatase 2A (PP2A), SET, interacts with the third intracellular loop of the M3 muscarinic receptor (M3-MR), and SET knockdown with small interfering RNA (siRNA) in Chinese hamster ovary (CHO) cells augments M3-MR signaling. However, the mechanism of this action of SET on receptor signaling has not been defined, and we initiated studies to address this question. Knockdown of SET by siRNA in CHO cells stably expressing the M3-MR did not alter agonist-induced receptor phosphorylation or receptor internalization. Instead, it increased the extent of receptor dephosphorylation after agonist removal by ∼60%. In competition binding assays, SET knockdown increased high-affinity binding of agonist in intact cells and membrane preparations. Glutathione transferase pull-down assays and site-directed mutagenesis revealed a SET binding site adjacent to and perhaps overlapping the G protein-binding site within the third intracellular loop of the receptor. Mutation of this region in the M3-MR altered receptor coupling to G protein. These data indicate that SET decreases M3-MR dephosphorylation and regulates receptor engagement with G protein, both of which may contribute to the inhibitory action of SET on M3-MR signaling.
authors.affiliation: University Paris Diderot, Sorbonne Paris Cité, Biologie Fonctionnelle et Adaptative, Centre National de la Recherche Scientifique-Equipe d’Accueil Conventionée 4413, Paris, France.
authors.forename: Violaine
authors.initials: V
authors.lastname: Simon
authors.forename: Sukru S
authors.initials: SS
authors.lastname: Oner
authors.forename: Joelle
authors.initials: J
authors.lastname: Cohen-Tannoudji
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Tobin
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Lanier
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D004268:DNA-Binding Proteins; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D056488:Histone Chaperones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation; D010766:Phosphorylation; D054648:Protein Phosphatase 2 / Q000235:genetics / Q000378:metabolism; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D043587:Receptor, Muscarinic M3 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D056488:Histone Chaperones; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D043587:Receptor, Muscarinic M3; C513473:SET protein, human; D014157:Transcription Factors; D054648:Protein Phosphatase 2; D019204:GTP-Binding Proteins