Phosphorylation of the Gq/11-coupled M3-muscarinic receptor is involved in receptor activation of the ERK-1/2 mitogen-activated protein kinase pathway

We investigated the role played by agonist-mediated phosphorylation of the G(q/11)-coupled M-3-muscarinic receptor in the mechanism of activation of the mitogen-activated protein kinase pathway, ERK-1/2, in transfected Chinese hamster ovary cells. A mutant of the M-3-muscarinic receptor, where residues Lys(370)-Ser(425) Of the third intracellular loop had been deleted, showed a reduced ability to activate the ERK-1/2 pathway. This reduction was evident despite the fact that the receptor was able to couple efficiently to the phospholipase C second messenger pathway. Importantly, the ERK-1/2 responses to both the wild-type M-3-muscarinic receptor and Delta Lys(370)-Ser(425) receptor mutant were dependent on the activity of protein kinase C. Our results, therefore, indicate the existence of two mechanistic components to the ERK-1/2 response, which appear to act in concert. First, the activation of protein kinase C through the diacylglycerol arm of the phospholipase C signaling pathway and a second component, absent in the Delta Lys(370)-Ser(425) receptor mutant, that is independent of the phospholipase C signaling pathway. The reduced ability of the Delta Lys(370)-Ser(425) receptor mutant to activate the ERK-1/2 pathway correlated with an similar to 80% decrease in the ability of the receptor to undergo agonist-mediated phosphorylation. Furthermore, we have previously shown that M-3-muscarinic receptor phosphorylation can be inhibited by a dominant negative mutant of casein kinase 1 alpha and by expression of a peptide corresponding to the third intracellular loop of the M-3-muscarinic receptor. Expression of these inhibitors of receptor phosphorylation reduced the wild-type M-3-muscarinic receptor ERK-1/2 response. We conclude that phosphorylation of the M-3-muscarinic receptor on sites in the third intracellular loop by casein kinase 1 alpha contributes to the mechanism of receptor activation of ERK-1/2 by working. in concert with the diacylglycerol/ PKC arm of the phospholipase C signaling pathway.