Pleiotropic Coupling of G Protein-coupled Receptors to the Mitogen-activated Protein Kinase Cascade
ROLE OF FOCAL ADHESIONS AND RECEPTOR TYROSINE KINASES*
- From The Howard Hughes Medical Institute and the Departments of Medicine, Surgery, and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
Abstract
G protein-coupled receptors (GPCRs) initiate Ras-dependent activation of the Erk 1/2 mitogen-activated protein kinase cascade by stimulating recruitment of Ras guanine nucleotide exchange factors to the plasma membrane. Both integrin-based focal adhesion complexes and receptor tyrosine kinases have been proposed as scaffolds upon which the GPCR-induced Ras activation complex may assemble. Using specific inhibitors of focal adhesion complex assembly and receptor tyrosine kinase activation, we have determined the relative contribution of each to activation of the Erk 1/2 cascade following stimulation of endogenous GPCRs in three different cell types. The tetrapeptide RGDS, which inhibits integrin dimerization, and cytochalasin D, which depolymerizes the actin cytoskeleton, disrupt the assembly of focal adhesions. In PC12 rat pheochromocytoma cells, both agents block lysophosphatidic acid (LPA)- and bradykinin-stimulated Erk 1/2 phosphorylation, suggesting that intact focal adhesion complexes are required for GPCR-induced mitogen-activated protein kinase activation in these cells. In Rat 1 fibroblasts, Erk 1/2 activation via LPA and thrombin receptors is completely insensitive to both agents. Conversely, the epidermal growth factor receptor-specific tyrphostin AG1478 inhibits GPCR-mediated Erk 1/2 activation in Rat 1 cells but has no effect in PC12 cells. In HEK-293 human embryonic kidney cells, LPA and thrombin receptor-mediated Erk 1/2 activation is partially sensitive to both the RGDS peptide and tyrphostin AG1478, suggesting that both focal adhesion and receptor tyrosine kinase scaffolds are employed in these cells. The dependence of GPCR-mediated Erk 1/2 activation on intact focal adhesions correlates with expression of the calcium-regulated focal adhesion kinase, Pyk2. In all three cell types, GPCR-stimulated Erk 1/2 activation is significantly inhibited by the Src kinase inhibitors, herbimycin A and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine (PP1), suggesting that Src family nonreceptor tyrosine kinases represent a point of convergence for signals originating from either scaffold.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grants HL16037 (to R. J. L.) and DK02352 and DK55524 (to L. M. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Supported by National Institutes of Health Medical Scientist Training Program Grant T32GM-07171.
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↵§ An Investigator with the Howard Hughes Medical Institute. To whom correspondence should be addressed: The Howard Hughes Medical Institute, Box 3821, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-2974; Fax: 919-684-8875.
- Abbreviations:
- GPCR
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G protein-coupled receptor
- MAP
-
mitogen-activated protein
- FAK
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focal adhesion kinase
- RTK
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receptor tyrosine kinase
- EGF
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epidermal growth factor
- LPA
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lysophosphatidic acid
- PP1
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4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine
- PAGE
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polyacrylamide gel electrophoresis
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- Received October 22, 1998.
- Revision received February 9, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
