Key points are not available for this paper at this time.
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. 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. Many GPCRs 1The abbreviations used are: GPCR, G protein-coupled receptor; MAP, mitogen-activated protein; FAK, focal adhesion kinase; RTK, receptor tyrosine kinase; EGF, epidermal growth factor; LPA, lysophosphatidic acid; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine; PAGE, polyacrylamide gel electrophoresis initiate Ras-dependent activation of the Erk 1/2 MAP kinase cascade by inducing the tyrosine phosphorylation of proteins that serve as scaffolds for the plasma membrane recruitment of Ras guanine nucleotide exchange factors. Receptor stimulation results in a rapid increase in the tyrosine phosphorylation of docking proteins, such as Shc (1Ptasznik A. Traynor-Kaplan A. Bokoch G.M. J. Biol. Chem. 1995; 270: 19969-19973Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 2Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar) and Gab1 (3Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (588) Google Scholar), followed by the Grb2-mediated recruitment of the Ras guanine nucleotide exchange factor, mSos, to the plasma membrane (4van Biesen T. Hawes B.E. Luttrell D.K. Krueger K.M. Touhara K. Porfiri E Sakaue M. Luttrell L.M. Lefkowitz R.J. Nature. 1995; 376: 781-784Crossref PubMed Scopus (526) Google Scholar). These tyrosine phosphorylation events are sensitive to the inhibition of Src family nonreceptor tyrosine kinases in many cell types (2Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, 3Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (588) Google Scholar,5Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (880) Google Scholar). Although the requirement for tyrosine kinases in GPCR-mediated Erk 1/2 activation has been well documented, the proximal signaling events whereby these receptors initiate tyrosine phosphorylation remain poorly understood. Recent data have implicated FAK family kinases and RTKs, both of which regulate the activity of Src kinases, as proximal mediators of GPCR-induced tyrosine phosphorylation. FAKs are nonreceptor tyrosine kinases that compose part of the focal adhesion complex. These complexes assemble on αβ integrin heterodimers following integrin engagement of extracellular matrix proteins. Following recruitment, FAKs autophosphorylate and provide docking sites for several signaling proteins, including c-Src and Grb2 (6Clark E.A. Brugge J.S Science. 1995; 268: 233-239Crossref PubMed Scopus (2822) Google Scholar). In many cell types, stimulation of Gi- or Gq-coupled receptors causes FAK activation (7Hordijk P.L. Verlaan I. van Corven E.J. Moolenaar W.H. J. Biol. Chem. 1994; 269: 645-651Abstract Full Text PDF PubMed Google Scholar, 8Rodriguez-Fernandez J.L. Rozengurt E J. Biol. Chem. 1996; 271: 27895-27901Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9Luttrell L.M. Daaka Y. Della Rocca G.J. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31648-31656Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). This activation is cell adhesion-dependent, because disruption of focal adhesions prevents the response (10Slack B.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7281-7286Crossref PubMed Scopus (56) Google Scholar). In neuronal cells, stimulation of either LPA or bradykinin receptors activates the calcium-regulated FAK family kinase, Pyk2 (11Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1254) Google Scholar), and overexpression of Pyk2 mutants that are either catalytically inactive or unable to bind to c-Src prevents GPCR-induced Erk 1/2 activation (5Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (880) Google Scholar, 11Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1254) Google Scholar). In other systems, however, GPCR-mediated Erk 1/2 activation is apparently dissociable from FAK phosphorylation (7Hordijk P.L. Verlaan I. van Corven E.J. Moolenaar W.H. J. Biol. Chem. 1994; 269: 645-651Abstract Full Text PDF PubMed Google Scholar, 8Rodriguez-Fernandez J.L. Rozengurt E J. Biol. Chem. 1996; 271: 27895-27901Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9Luttrell L.M. Daaka Y. Della Rocca G.J. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31648-31656Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Classical RTKs, such as the receptor for epidermal growth factor (EGF), are single transmembrane domain proteins that dimerize and transphosphorylate upon ligand binding. Tyrosine phosphorylation of RTKs promotes their association with SH2 or PTB domain-containing signaling proteins, which assemble on the receptor to form a Ras activation complex (12Medema R.H. Bos J.L. Crit. Rev. Oncog. 1993; 4: 615-661PubMed Google Scholar). “Transactivation” of RTKs following GPCR stimulation has been implicated in GCPR-mediated activation of Erk 1/2 (13Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 14Rao G.N. Delafontaine P. Runge M.S. J. Biol. Chem. 1995; 270: 27871-27875Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 15Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1329) Google Scholar). In Rat 1 fibroblasts and COS-7 cells, inhibition of EGF receptor function inhibits LPA-, endothelin-1-, and thrombin receptor-mediated tyrosine phosphorylation of Shc and Gab1 and activation of Erk 1/2 (3Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (588) Google Scholar, 15Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1329) Google Scholar). In this model, the transactivated RTK forms the structural core of a GPCR-induced mitogenic signaling complex, as receptor phosphorylation creates docking sites for the components of the Ras activation complex. We compared the role of focal adhesions and EGF receptors in mediating Erk 1/2 activation via endogenously expressed LPA, thrombin, and bradykinin receptors in three different cell types: PC12 rat pheochromocytoma cells, Rat 1 fibroblasts, and HEK-293 embryonic kidney cells. Surprisingly, we found that the preferred scaffold was independent of the specific Gi/Gq-coupled GPCR being stimulated. Rather, the utilization of scaffolds varied between cell types, with PC-12 cells and Rat 1 fibroblasts apparently representing opposite ends of a continuum. In PC-12 cells GPCR-mediated Erk 1/2 activation was almost exclusively focal adhesion-dependent, whereas in Rat 1 fibroblasts it was almost exclusively RTK-dependent. In HEK-293 cells, both scaffolds contributed to the GPCR signal. Utilization of the focal adhesion scaffold correlated with signaling via pertussis toxin-insensitive G proteins and with cellular expression of the calcium-regulated FAK family kinase, Pyk2. In each case, GPCR-stimulated Erk 1/2 activation was sensitive to Src kinase inhibitors, suggesting that a critical role of both scaffolds is to support the GPCR-induced activation of Src family nonreceptor tyrosine kinases. Cytochalasin D, EGF, herbimycin A, tyrphostin AG1478, and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine (PP1) were from Calbiochem. Bordetella pertussis toxin was from List Biologicals. LPA and bradykinin were from Sigma. The tetrapeptides H3N+-arginine-glycine-aspartate-serine-COO−(RGDS) and H3N+-arginine-glycine-glutamate-serine-COO−(RGES) and the thrombin agonist hexapeptide H3N+-serine-phenylalanine-leucine-leucine-arginine-asparagine-CONH2(SFLLRN) were synthesized at the Howard Hughes Medical Institute peptide facility (Duke University Medical Center). Rat pheochromocytoma PC12 cells, Rat-1 fibroblasts, and HEK-293 cells were from the American Type Culture Collection. PC12 cells were maintained in with and at in a Rat-1 fibroblasts were maintained in with and HEK-293 cells were maintained in with with and the of and Pyk2 tyrosine phosphorylation, cells to in were in for stimulation was at in following with inhibitors, as in the were with and in 1 1 1 were by and to a protein of 1 a of the cell was to for gel electrophoresis and of Erk 1/2 phosphorylation of was of a of protein G with at complexes were with and with in and by Tyrosine phosphorylation or the of proteins was by protein was a of FAK was a of and Pyk2 was a of each with as proteins on were by and by the of Erk 1/2 phosphorylation, of cell were by and Erk 1/2 phosphorylation was by protein a of MAP kinase with as of Erk 1/2 phosphorylation was of to and on a of Erk 1/2 phosphorylation, were of and to of endogenous GPCRs of the Erk 1/2 cascade in a of cell types, we Erk 1/2 phosphorylation following stimulation of Rat and HEK-293 cells with for LPA, thrombin, or bradykinin of these receptors has been to both pertussis and signals from to and G proteins Corven E.J. A. K. T. Moolenaar W.H. Full Text PDF PubMed Scopus Google Scholar, van E. van E. J. J. 1993; PubMed Scopus Google Scholar, J.M. PubMed Scopus Google Scholar). Erk 1/2 activation via endogenous EGF receptors was determined as a for cellular and 1 GPCR-induced Erk 1/2 phosphorylation in each of the three cell In PC12 cells, both and bradykinin-stimulated Erk 1/2 phosphorylation was pertussis toxin-insensitive 1 In these cells, the thrombin agonist a stimulation of Erk 1/2 phosphorylation In and Erk 1/2 phosphorylation in Rat 1 fibroblasts was completely pertussis 1 In HEK-293 cells, pertussis toxin partially the LPA and thrombin receptor 1 Erk 1/2 phosphorylation was pertussis whereas the response to was pertussis This to pertussis toxin that Gi/Gq-coupled GPCRs the Erk 1/2 cascade via G protein in different cell types. Erk 1/2 phosphorylation was insensitive to pertussis toxin in all three cell in the tyrosine kinase inhibitors herbimycin A and significantly inhibited LPA-, and Erk 1/2 phosphorylation in Rat and HEK-293 cells. Although EGF receptor at the employed both herbimycin A and Erk 1/2 phosphorylation. These data that Src kinase activation to both the and Erk 1/2 Src family kinases with both integrin-based focal adhesion complexes T. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) and receptor tyrosine kinases (12Medema R.H. Bos J.L. Crit. Rev. Oncog. 1993; 4: 615-661PubMed Google Scholar), it is that signals originating from either sensitive to Src Gi/Gq-coupled receptors in different cell types focal adhesions or RTKs as signaling the of these scaffolds for of the in GPCR-mediated Erk 1/2 this we determined the relative dependence of GPCR signals on the of focal adhesions and receptor tyrosine kinases in Rat and HEK-293 cells. FAK family kinases, and Pyk2 as and have been to autophosphorylate in response to GPCR stimulation (7Hordijk P.L. Verlaan I. van Corven E.J. Moolenaar W.H. J. Biol. Chem. 1994; 269: 645-651Abstract Full Text PDF PubMed Google Scholar, 8Rodriguez-Fernandez J.L. Rozengurt E J. Biol. Chem. 1996; 271: 27895-27901Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9Luttrell L.M. Daaka Y. Della Rocca G.J. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31648-31656Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 11Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1254) Google Scholar). in A, and Rat 1 cells of and Pyk2 expression as by protein Although all three the neuronal PC12 cells Pyk2. In HEK-293 cells and Rat 1 cells Pyk2 In PC12 cells, LPA, and EGF stimulation tyrosine phosphorylation of both Pyk2 and that both kinases, are following GPCR stimulation focal adhesions are required for the activation of FAK family kinases and of complexes T. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, T. Biol. 1997; PubMed Scopus Google Scholar, J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). assembly of focal adhesions both M. K. J. Biol. 1996; PubMed Scopus Google Scholar) and to the extracellular matrix Nature. 1996; 383: PubMed Scopus Google Scholar). the which the integrin ligand found in extracellular matrix proteins such as have been to block integrin Full Text PDF PubMed Scopus Google Scholar, K.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and disrupt the of focal adhesions. are by of actin following to cytochalasin In cells, integrin the inhibits and tyrosine phosphorylation of and (10Slack B.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7281-7286Crossref PubMed Scopus (56) Google Scholar). the to which intact focal adhesions required for GPCR-stimulated Erk 1/2 activation, we determined the of and cytochalasin on Erk 1/2 phosphorylation in Rat and HEK-293 cells. In each tyrosine phosphorylation of was as a for the of focal adhesion in stimulation of LPA, thrombin, or EGF receptors the tyrosine phosphorylation of that each of these receptors focal adhesion complex of cells with the RGDS but the inhibited phosphorylation in each of the three cell In PC12 cells, and bradykinin-stimulated Erk 1/2 phosphorylation, phosphorylation, was inhibited by the RGDS peptide A, In LPA and Erk 1/2 phosphorylation in Rat 1 fibroblasts was completely insensitive to the RGDS peptide the inhibition of phosphorylation In HEK-293 cells, LPA and Erk 1/2 phosphorylation was partially inhibited EGF receptor-mediated Erk 1/2 activation was insensitive to the RGDS peptide in all three cell that intact focal adhesions are required for stimulation of Erk 1/2 by results were cytochalasin to focal adhesion in cytochalasin and EGF phosphorylation in and HEK-293 cells with the RGDS cytochalasin completely and Erk 1/2 phosphorylation in PC12 cells A, In Rat 1 fibroblasts, and Erk 1/2 phosphorylation was cytochalasin whereas a inhibition of and Erk 1/2 phosphorylation was in HEK-293 cells RTKs including the EGF (3Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (588) Google Scholar, 15Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1329) Google Scholar, L.M. Della Rocca G.J. van Biesen T. Luttrell D.K. Lefkowitz R.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), growth factor (13Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar), and growth G.N. Delafontaine P. Runge M.S. J. Biol. Chem. 1995; 270: 27871-27875Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) receptors in response to GPCR In Rat 1 and COS-7 cells, inhibition of EGF receptor GPCR-mediated MAP kinase activation (3Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (588) Google Scholar, 15Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1329) Google Scholar). both RTKs and focal adhesions represent docking sites for proteins in the of either or both function as a scaffold for GPCR-mediated activation of Erk in the EGF receptor-specific tyrphostin AG1478 has different on GPCR-stimulated Erk 1/2 phosphorylation in Rat and HEK-293 cells. to tyrphostin AG1478 no effect on LPA-, or FAK phosphorylation in of the three cell whereas inhibition the EGF effect to Erk 1/2 activation, and bradykinin-stimulated Erk 1/2 phosphorylation in PC12 cells was insensitive to tyrphostin AG1478 A, In tyrphostin AG1478 inhibited both and Erk 1/2 activation in Rat 1 fibroblasts In HEK-293 cells, the tyrphostin inhibition of and Erk 1/2 phosphorylation the of GPCR-stimulated Erk 1/2 activation to tyrphostin AG1478 was opposite the of inhibitors of focal adhesion these results that focal adhesions and RTKs function as scaffolds for GPCR-mediated Erk 1/2 data that both focal adhesions and RTKs function to support activation of the Erk 1/2 MAP kinase cascade following activation of endogenous Gi/Gq-coupled a with these In PC12 cells, Gi/Gq-coupled receptors such as for LPA and bradykinin Erk 1/2 activation via pertussis toxin-insensitive G proteins and a focal scaffold. The of pertussis toxin is with the that the receptor Erk 1/2 activation in PC12 cells H. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the of EGF receptor to GPCR-stimulated Erk 1/2 activation in these cells. Rat 1 fibroblasts apparently represent the opposite of a continuum. In these cells, LPA and thrombin receptors Erk 1/2 activation via pertussis G proteins and of the EGF PC12 cells, GPCR-stimulated Erk 1/2 activation in these cells is by the disruption of focal adhesion HEK-293 cells apparently both as these cells and Erk 1/2 activation that is partially pertussis and partially sensitive to inhibitors of focal adhesion complex assembly and of EGF receptor from either scaffold apparently on Src family nonreceptor tyrosine kinases, as Src inhibitors Erk 1/2 activation in each cell Gi/Gq-coupled receptors are to the Erk via both tyrosine and B.E. van Biesen T. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). data that a of the Erk 1/2 phosphorylation by LPA receptors in Rat 1 fibroblasts and by LPA and thrombin receptors in HEK-293 cells is insensitive to both and EGF kinase This tyrosine may protein kinase Erk 1/2 activation, which we have is and herbimycin in HEK-293 cells B.E. van Biesen T. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). Although it is that GPCR-mediated Erk 1/2 activation from focal and scaffolds are the factors that scaffold are poorly understood. data that cell expression of calcium-regulated FAK kinases such as Pyk2 in neuronal (11Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1254) Google Scholar) or cells I. I. Schlessinger J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) may GPCRs the focal adhesion complex as a signaling scaffold. Pyk2 and their domain and their and (11Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1254) Google Scholar) but to significantly in their by extracellular activation of the Pyk2 apparently by a upon both cellular adhesion and a or protein kinase (11Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1254) Google Scholar, J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). phosphorylation of both endogenous Pyk2 and following cell adhesion in rat cells, but Pyk2 phosphorylation is by with or C. C. A. J.L. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). This of Pyk2 and activity a for their in the of MAP kinase with GPCR-induced is from Erk 1/2 activation in Rat 1 cells J.L. Rozengurt E J. Biol. Chem. 1996; 271: 27895-27901Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9Luttrell L.M. Daaka Y. Della Rocca G.J. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31648-31656Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), which Pyk2. Conversely, overexpression of Pyk2 in cells is to Erk 1/2 activation that is and Src (5Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (880) Google Scholar). RTKs, including for growth factor, EGF, and growth (13Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 14Rao G.N. Delafontaine P. Runge M.S. J. Biol. Chem. 1995; 270: 27871-27875Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 15Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1329) Google Scholar). In a cell GPCR-stimulated Erk 1/2 activation may of in cells, which endogenous EGF LPA stimulation results in Erk 1/2 activation that is upon of growth factor EGF receptors are expressed in these cells, signaling in EGF A. H. A. Herrlich P. Ullrich A. G. T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). Although such data that for the of RTKs may the RTK are poorly understood. In COS-7 cells, EGF receptor is pertussis and inhibited by of G protein L.M. Della Rocca G.J. van Biesen T. Luttrell D.K. Lefkowitz R.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, A. H. A. Herrlich P. Ullrich A. G. T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). Conversely, protein kinase EGF receptor has been in HEK-293 cells receptors EMBO J. 1997; 16: PubMed Scopus Google Scholar). the role of Src family kinases in GPCR stimulation of Erk of Src kinases by the J. Courtneidge S.A. E. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), LPA (2Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar), M. B. T. 1995; PubMed Scopus (167) Google Scholar), peptide (1Ptasznik A. Traynor-Kaplan A. Bokoch G.M. J. Biol. Chem. 1995; 270: 19969-19973Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar), (2Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, J. Courtneidge S.A. E. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), and J. Courtneidge S.A. E. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar) receptors has been of c-Src to Pyk2 is required for because a point of Pyk2 that complex with c-Src as a of GPCR-stimulated Erk 1/2 activation (5Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (880) Google Scholar). inhibition of Src kinase activity either c-Src mutants (2Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar) or agents (3Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (588) Google Scholar) LPA receptor-mediated tyrosine phosphorylation of Shc and Gab1 and Erk 1/2 activation in COS-7 cells. Although these data support a role for Src kinases of both FAK family kinases and transactivated RTKs, that Src kinase activity may role in GPCR-induced RTK of Src kinase LPA and receptor-mediated EGF receptor phosphorylation in COS-7 cells L.M. Della Rocca G.J. van Biesen T. Luttrell D.K. Lefkowitz R.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). In stimulation has been to association of c-Src with the EGF receptor independent of EGF receptor suggesting that c-Src activation may EGF receptor S. K. H. T. T. H. H. K.M. Y. T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). has in the whereby GPCRs activation of the Erk 1/2 MAP kinase In GPCR-stimulated Erk 1/2 activation the assembly of a Ras activation complex on the plasma which is upon tyrosine phosphorylation of proteins such as Shc and Gab1 and recruitment of data that both focal adhesions and RTKs function as scaffolds for the assembly of this complex and that the preferred scaffold is determined by the cellular in which the receptor is is in expression to the employed by GPCRs which expression in of the of these different scaffolds of the of and signals originating from We and M. for and for peptide and and
Rocca et al. (Sat,) studied this question.