TYROSINE PHOSPHORYLATION OF PAXILLIN AND FOCAL ADHESION KINASE BY ACTIVATION OF MUSCARINIC M3 RECEPTORS IS DEPENDENT ON INTEGRIN ENGAGEMENTBY THE EXTRACELLULAR-MATRIX

The G protein coupled mi and m3 muscarinic acetylcholine receptors inc rease tyrosine phosphorylation of several proteins, including the foca l adhesion-associated proteins paxillin and focal adhesion kinase (FAK ), but the mechanism is not understood. Activation of integrins during adhesion of cells to extracellular matrix, or stimulation of quiescen t cell monolayers with G protein-coupled receptor ligands including br adykinin, bombesin, endothelin, vasopressin, and lysophosphatidic acid , also induces tyrosine phosphorylation of paxillin and FAK and format ion of focal adhesions, These effects are generally independent of pro tein kinase C but are inhibited by agents that prevent cytoskeletal as sembly or block activation of the small molecular weight G protein Rho . This report demonstrates that tyrosine phosphorylation of paxillin a nd FAK elicited by stimulation of muscarinic m3 receptors with the ace tylcholine analog carbachol is inhibited by soluble peptides containin g the arginine-glycine-aspartate motif (the recognition site for integ rins found in adhesion proteins such as fibronectin) but is unaffected by peptides containing the inactive sequence arginine-glycine-glutama te. Tyrosine phosphorylation elicited by carbachol, but not by cell ad hesion to fibronectin, is reduced by the protein kinase C inhibitor GF 109203X, The response to carbachol is dependent on the presence of fi bronectin, Moreover, immunofluorescence studies show that carbachol tr eatment induces formation of stress fibers and focal adhesions. These results suggest that muscarinic receptor stimulation activates integri ns via a protein kinase C-dependent mechanism, The activated integrins transmit a signal into the cell's interior leading to tyrosine phosph orylation of paxillin and FAK, This represents a novel mechanism for r egulation of tyrosine phosphorylation by muscarinic receptors.