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 DeltaLys(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 DeltaLys(370)-Ser(425) receptor mutant, that is independent of the phospholipase C signaling pathway. The reduced ability of the DeltaLys(370)-Ser(425) receptor mutant to activate the ERK-1/2 pathway correlated with an approximately 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 1alpha 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 1alpha 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.
Budd
D C
DC
Department of Cell Physiology and Pharmacology, University of Leicester, P. O. Box 138, Medical Sciences Building, University Road, Leicester, LE1 9HN, United Kingdom.
Willars
G B
GB
McDonald
J E
JE
Tobin
A B
AB
D000818:Animals; D016466:CHO Cells; D047388:Casein Kinases; D006224:Cricetinae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D010455:Peptides; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D011494:Protein Kinases; D047388:Casein Kinases; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins