PHOSPHORYLATION OF HUMAN M1 MUSCARINIC ACETYLCHOLINE-RECEPTORS BY G-PROTEIN-COUPLED RECEPTOR KINASE-2 AND PROTEIN-KINASE-C

Human muscarinic acetylcholine receptor m1 subtypes (m1 receptors) wer e expressed in and purified from insect Sf9 cells and then subjected t o phosphorylation by G protein-coupled receptor kinase 2 (GRK2) expres sed in and purified from Sf9 cells and by protein kinase C purified fr om rat brain (a mixture of alpha, beta, and gamma types, PKC). The m1 receptor was phosphorylated by either GRK2 or PKC in an agonist-depend ent or independent manner, respectively, G protein beta gamma subunits stimulated the phosphorylation by GRK2 but did not affect the phospho rylation by PKC. The number of incorporated phosphates was 4.6 and 2.8 mol/mol of receptor for phosphorylation by GRK2 and PKC, respectively , The number of incorporated phosphates was 7.5 mol/mol receptor for p hosphorylation by GRK2 followed by PKC, but was 5.8 mol/mol of recepto r for the phosphorylation by PKC followed by GRK2. Major sites phospho rylated by GRK2 and PKC were located in the third intracellular loop a nd the carboxyl-terminal tail, respectively, These results indicate th at GRK2 and PKC phosphorylate different sites of mi receptors and that the phosphorylation by PKC partially inhibits the phosphorylation by GRK2, probably by affecting activation of GRK2 by agonist-bound recept ors.