Slack B E
Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston MA 02118, USA.
Proc Natl Acad Sci U S A. 1998 Jun 23;95(13):7281-6. doi: 10.1073/pnas.95.13.7281.
The G protein-coupled m1 and m3 muscarinic acetylcholine receptors increase tyrosine phosphorylation of several proteins, including the focal 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 quiescent cell monolayers with G protein-coupled receptor ligands including bradykinin, bombesin, endothelin, vasopressin, and lysophosphatidic acid, also induces tyrosine phosphorylation of paxillin and FAK and formation of focal adhesions. These effects are generally independent of protein kinase C but are inhibited by agents that prevent cytoskeletal assembly or block activation of the small molecular weight G protein Rho. This report demonstrates that tyrosine phosphorylation of paxillin and FAK elicited by stimulation of muscarinic m3 receptors with the acetylcholine analog carbachol is inhibited by soluble peptides containing the arginine-glycine-aspartate motif (the recognition site for integrins found in adhesion proteins such as fibronectin) but is unaffected by peptides containing the inactive sequence arginine-glycine-glutamate. Tyrosine phosphorylation elicited by carbachol, but not by cell adhesion to fibronectin, is reduced by the protein kinase C inhibitor GF 109203X. The response to carbachol is dependent on the presence of fibronectin. Moreover, immunofluorescence studies show that carbachol treatment induces formation of stress fibers and focal adhesions. These results suggest that muscarinic receptor stimulation activates integrins via a protein kinase C-dependent mechanism. The activated integrins transmit a signal into the cell's interior leading to tyrosine phosphorylation of paxillin and FAK. This represents a novel mechanism for regulation of tyrosine phosphorylation by muscarinic receptors.
G蛋白偶联的毒蕈碱型m1和m3乙酰胆碱受体可增加多种蛋白质的酪氨酸磷酸化，包括粘着斑相关蛋白桩蛋白和粘着斑激酶（FAK），但其机制尚不清楚。细胞与细胞外基质粘附过程中整合素的激活，或用包括缓激肽、蛙皮素、内皮素、血管加压素和溶血磷脂酸在内的G蛋白偶联受体配体刺激静止的细胞单层，也可诱导桩蛋白和FAK的酪氨酸磷酸化以及粘着斑的形成。这些效应通常与蛋白激酶C无关，但可被阻止细胞骨架组装或阻断小分子量G蛋白Rho激活的试剂所抑制。本报告表明，用乙酰胆碱类似物卡巴胆碱刺激毒蕈碱型m3受体所引发的桩蛋白和FAK的酪氨酸磷酸化，可被含有精氨酸-甘氨酸-天冬氨酸基序（在诸如纤连蛋白等粘附蛋白中发现的整合素识别位点）的可溶性肽所抑制，但不受含有无活性序列精氨酸-甘氨酸-谷氨酸的肽的影响。卡巴胆碱引发的酪氨酸磷酸化，而非细胞与纤连蛋白的粘附所引发的酪氨酸磷酸化，可被蛋白激酶C抑制剂GF 109203X所降低。对卡巴胆碱的反应依赖于纤连蛋白的存在。此外，免疫荧光研究表明，卡巴胆碱处理可诱导应力纤维和粘着斑的形成。这些结果提示，毒蕈碱受体刺激通过蛋白激酶C依赖性机制激活整合素。激活的整合素将信号传递到细胞内部，导致桩蛋白和FAK的酪氨酸磷酸化。这代表了毒蕈碱受体调节酪氨酸磷酸化的一种新机制。
