PT  - JOURNAL ARTICLE
AU  - Violaine Simon
AU  - Sukru S Oner
AU  - Joelle Cohen-Tannoudji
AU  - Andrew B Tobin
AU  - Stephen M Lanier
TI  - Influence of the Accessory Protein Set on M3 Muscarinic Receptor Phosphorylation and G Protein Coupling
AID  - 10.1124/mol.111.075523
DP  - 2012 Jan 01
TA  - Molecular Pharmacology
PG  - mol.111.075523
4099  - http://molpharm.aspetjournals.org/content/early/2012/03/30/mol.111.075523.short
4100  - http://molpharm.aspetjournals.org/content/early/2012/03/30/mol.111.075523.full
AB  - 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 CHO cells augments M3-MR signaling. However, the mechanism of this action of SET on receptor signaling is not 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. GST 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.