A β-Arrestin-dependent Scaffold Is Associated with Prolonged MAPK Activation in Pseudopodia during Protease-activated Receptor-2-induced Chemotaxis

Cell motility during wound healing and inflammation is often dependent on the ability of the cell to sense a gradient of agonist. The first step in this process is the extension of a pseudopod in the direction of the agonist, and a diverse set of signals mediate pseudopod extension by different receptors. We have reported previously that protease-activated receptor-2 (PAR-2), a proinflammatory receptor that is highly expressed in motile cells such as neutrophils, macrophages, and tumor cells, is one of a growing family of receptors that utilizes a β-arrestin-dependent mechanism for activation of the 42–44-kDa members of the MAPK family (extracellular signal-regulated kinases 1 and 2; ERK1/2). β-Arrestin-bound PAR-2 serves as a scaffold to sequester a pool of activated ERK1/2 in the cytosol; however, a specific role for the sequestered kinase activity has not been established. We now show that PAR-2 activation promotes ERK1/2- and β-arrestin-dependent reorganization of the actin cytoskeleton, polarized pseudopodia extension, and chemotaxis. Using subcellular fractionation, confocal microscopy, and physical isolation of pseudopodial proteins, we demonstrate that the previously identified PAR-2/β-arrestin/ERK1/2 scaffolding complex is enriched in the pseudopodia, where it appears to prolong ERK1/2 activation. These studies suggest that the formation of a β-arrestin/ERK1/2 signaling complex at the leading edge may be involved in localized actin assembly and chemotaxis and provide the first example of a distinct cellular consequence of β-arrestin-sequestered ERK1/2 activity. Cell motility during wound healing and inflammation is often dependent on the ability of the cell to sense a gradient of agonist. The first step in this process is the extension of a pseudopod in the direction of the agonist, and a diverse set of signals mediate pseudopod extension by different receptors. We have reported previously that protease-activated receptor-2 (PAR-2), a proinflammatory receptor that is highly expressed in motile cells such as neutrophils, macrophages, and tumor cells, is one of a growing family of receptors that utilizes a β-arrestin-dependent mechanism for activation of the 42–44-kDa members of the MAPK family (extracellular signal-regulated kinases 1 and 2; ERK1/2). β-Arrestin-bound PAR-2 serves as a scaffold to sequester a pool of activated ERK1/2 in the cytosol; however, a specific role for the sequestered kinase activity has not been established. We now show that PAR-2 activation promotes ERK1/2- and β-arrestin-dependent reorganization of the actin cytoskeleton, polarized pseudopodia extension, and chemotaxis. Using subcellular fractionation, confocal microscopy, and physical isolation of pseudopodial proteins, we demonstrate that the previously identified PAR-2/β-arrestin/ERK1/2 scaffolding complex is enriched in the pseudopodia, where it appears to prolong ERK1/2 activation. These studies suggest that the formation of a β-arrestin/ERK1/2 signaling complex at the leading edge may be involved in localized actin assembly and chemotaxis and provide the first example of a distinct cellular consequence of β-arrestin-sequestered ERK1/2 activity. A number of stimuli are able to promote cytoskeletal reorganization and random migration, but directed migration (or chemotaxis) is dependent upon the ability of the cell to sense and respond to a gradient of agonist (1Parent C.A. Devreotes P.N. Science. 1999; 284: 765-770Crossref PubMed Scopus (735) Google Scholar). The first step in chemotaxis is the formation of a leading edge, where a pseudopod is extended in the direction of the gradient. Receptors that promote chemotaxis must then be able to communicate with actin machinery to direct the formation of a leading edge. A number of common signaling proteins are implicated in pseudopod extension and chemotaxis, including members of the MAPK 1The abbreviations used are: MAPK, mitogen-activated protein kinase; GFP, green fluorescent protein; ARR-GFP, GFP-tagged β-arrestin-1; ARR319–418-GFP, dominant negative β-arrestin-1; ERK, extracellular signal-regulated kinase; ERK-KR, dominant negative ERK2; IGF-1R, insulin-like growth factor-I receptor; MI, migration index; PAR, protease-activated receptor; P2AP, PAR-2 activating peptide; p-ERK, phosphorylated ERK1/2; P1AP, PAR-1 activating peptide; MEK, MAPK/ERK kinase; HLB, Hypotonic lysis buffer; MES, 4-morpholineethane-sulfonic acid; CT, Celltracker; DAPI, 4,6-diamidino-2-phenylindoledihydrochloride hydrate. family: extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38 MAPK. ERK1/2 phosphorylation of one member of the Wiscott-Aldrich syndrome family of proteins (Wave-1/Scar-1) and myosin light chain kinase have been implicated in platelet-derived growth factor, integrin, and lysophosphatidic acid-induced cell motility (2Miki H. Fukuda M. Nishida E. Takenawa T. J. Biol. Chem. 1999; 274: 27605-27609Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 3Klemke R.L. Cai S. Giannini A.L. Gallagher P.J. de Lanerolle P. Cheresh D.A. J. Cell Biol. 1997; 137: 481-492Crossref PubMed Scopus (1107) Google Scholar, 4Brahmbhatt A.A. Klemke R.L. J. Biol. Chem. 2003; 278: 13016-13025Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), whereas p38 MAPK activity was found to be important for CXCR-4 receptorinduced chemotaxis (5Sun Y. Cheng Z. Ma L. Pei G. J. Biol. Chem. 2002; 277: 49212-49219Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). More recently, localization of ERK1/2 to the leading edge of migrating fibroblasts was shown to be involved in lysophosphatidic acid-induced pseudopod extension, although the mechanism by which it is localized is unclear (4Brahmbhatt A.A. Klemke R.L. J. Biol. Chem. 2003; 278: 13016-13025Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Although the nuclear effects of ERK1/2 on gene regulation are more widely studied, an understanding of how ERK1/2 activity is controlled in the cytosol by association with different scaffolding complexes is emerging. Recent studies suggest that the cytosolic effects of ERK1/2 may not occur in response to any signal that converges on this pathway and are not a default result of ERK1/2 that is leftover in the cytosol (6Hughes P.E. Oertli B. Hansen M. Chou F.L. Willumsen B.M. Ginsberg M.H. Mol. Biol. Cell. 2002; 13: 2256-2265Crossref PubMed Scopus (41) Google Scholar). This model would require that an additional level of control over MAPK activity be exerted. Studies on a number of cell surface receptors have demonstrated a role for components of the endocytotic machinery, such as the clathrin adaptor protein, β-arrestin, in both the activation and subcellular localization of MAPKs (7Miller W. Lefkowitz R.J. Curr. Opin. Cell Biol. 2001; 13: 139-145Crossref PubMed Scopus (283) Google Scholar). There is a growing body of evidence to suggest that these “endosomal scaffolds” can both link activation of MAPKs to specific receptors and determine the ultimate subcellular localization of the active kinases (7Miller W. Lefkowitz R.J. Curr. Opin. Cell Biol. 2001; 13: 139-145Crossref PubMed Scopus (283) Google Scholar). Furthermore, recent genetic studies (5Sun Y. Cheng Z. Ma L. Pei G. J. Biol. Chem. 2002; 277: 49212-49219Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 8Fong A.M. Premont R.T. Richardson R.M. Yu Y.R. Lefkowitz R.J. Patel D.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7478-7483Crossref PubMed Scopus (269) Google Scholar) have suggested that β-arrestins are required for immune cell chemotaxis, and an enticing hypothesis is that endosomal scaffolds can restrict kinase activity to the leading edge to promote localized actin assembly and filament organization. One of the first examples of β-arrestin-dependent localization of MAPKs came from work on protease-activated receptor-2 (PAR-2), a member of the recently identified family of G protein-coupled receptors that are self-activated by tethered ligands exposed upon proteolytic cleavage of their extracellular N termini. PAR-2 is cleaved and activated by pancreatic trypsin, mast cell tryptase, coagulation factors (factor VII/Xa) and a membrane-bound serine protease (MBSPII) to mediate proinflammatory responses (9Camerer E. Huang W. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5255-5260Crossref PubMed Scopus (618) Google Scholar, 10Dery O. Bunnett N.W. Biochem. Soc. Trans. 1999; 27: 246-254Crossref PubMed Scopus (52) Google Scholar, 11Takeuchi T. Harris J.L. Huang W. Yan K.W. Coughlin S.R. Craik C.S. J. Biol. Chem. 2000; 275: 26333-26342Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). A synthetic peptide (PAR-2 activating peptide or P2AP), corresponding to the tethered ligand sequence, will specifically activate PAR-2 in the absence of enzymatic cleavage (12Dery O. Corvera C.U. Steinhoff M. Bunnett N.W. Am. J. Physiol. 1998; 274: C1429-C1452Crossref PubMed Google Scholar). Our early studies showed that ERK1/2 activation by PAR-2 is facilitated by β-arrestin and involves the formation of a large scaffolding complex (Stokes radius ∼6–7 nm) that sequesters the activated kinases in the cytosol, thus preventing their nuclear translocation and presumably leading to specific phosphorylation of cytosolic or membrane-associated proteins. PAR-2 also uses a ras-independent pathway to activate ERK1/2, in contrast to the classic pathway used by receptor tyrosine kinases and many G protein-coupled receptors (13DeFea K.A. Zalevsky J. O. Bunnett N.W. J. Cell Biol. 2000; PubMed Scopus Google Scholar). have been for ERK1/2 and kinase activation F.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, R.J. Lefkowitz R.J. Science. 2000; PubMed Google Scholar, A. Lefkowitz R.J. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) and for receptor activation of ERK1/2 A. S. Lefkowitz R.J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). β-arrestin-dependent of ERK1/2 such as demonstrated for and insulin-like growth factor-I receptor the nuclear Y. S. H. Lefkowitz R.J. Science. 1999; PubMed Scopus Google Scholar, K.A. O. Bunnett N.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar, Y. Lefkowitz R.J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). such as the PAR-1 to not require β-arrestin for activation of ERK1/2 P.J. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). one the specific effects of MAPK activation by a receptor as on cell mechanism of and scaffolding complexes with which the kinases PAR-2 is highly expressed in neutrophils, mast cells, and tumor cells, where it has been suggested to promote cytoskeletal but the mechanism by which it unclear L. A. A. 2001; Google Scholar). We that PAR-2 chemotaxis the formation of a scaffolding ERK1/2 and kinases the where can activate actin machinery and to polarized pseudopod we on the regulation of ERK1/2 pathway and show the that PAR-2 activation in actin pseudopod and chemotaxis whereas receptors that activate ERK1/2 by different and that and dominant negative of β-arrestin or chemotaxis but not random and that phosphorylated ERK1/2 is enriched with PAR-2 in the pseudopodia, where activity is from or and P2AP, and and by PAR-1 activating peptide was and by the of and was by and PAR-2 and GFP-tagged and GFP-tagged have been previously (13DeFea K.A. Zalevsky J. O. Bunnett N.W. J. Cell Biol. 2000; PubMed Scopus Google Scholar). and from Lefkowitz PAR-2 GFP-tagged and GFP-tagged from Bunnett of negative was a from of GFP-tagged was by of and of and by The was from Cell PAR-2 and actin and protein A from from was from from and was from Cell and of Cell cells a from Bunnett of and cells from cells in with cells in with and cell at with cells with PAR-2 or with by by cell for by and as previously (13DeFea K.A. Zalevsky J. O. Bunnett N.W. J. Cell Biol. 2000; PubMed Scopus Google Scholar), and with to PAR-2 and of cell for and with β-arrestin and as previously (13DeFea K.A. Zalevsky J. O. Bunnett N.W. J. Cell Biol. 2000; PubMed Scopus Google Scholar), and for localization of phosphorylated was used to the and cells for for and with P2AP, P1AP, or for at and by in of Hypotonic lysis 1 and 1 2 and at for nuclear one in and for at in and at for to and cytosolic in of protein from by by with to actin and A of actin was in and nuclear and was in that of cytosolic and to and in and with 1 of to of protein at and cells with 1 extracellular or to for and cells, with or for or for in in and with for 1 at by for 1 at the 1 was to cells with for in cytoskeletal MES, in in cytoskeletal for at in and with for 1 at by confocal on a with of cells also a and Cell and pseudopod extension with or on both with for in or with cells the and to and for in or in or to the to a gradient or to both and to a for or with in cells or cell on the surface with a and cells and pseudopodia on an with and in of and number was was by of pseudopodia and by from the and at on a the of or or in cells with GFP, and as that cells and of agonist for cells from both with Cell for in and by set on of cells, with and of 1 cells that both and and 2 cells that cells that not from the cell from and of of and cells in for for in cell number and and are as over for and and This not for in cell from migration as the of cells that by the of cells cell as of cells that by the number of cells that for migration and a to one of on cell was as Klemke R.L. J. Cell Biol. 2002; PubMed Scopus Google Scholar). with ARR-GFP, the of as and with for to pseudopodia in and in pseudopodial proteins, cell on the surface with and pseudopodia on the lysis 1 1 protease Cell body proteins in a of proteins from cell and pseudopod by by with to p-ERK, PAR-2 and of and used to determine and PAR-2 in studies have suggested that PAR-2 ERK1/2, in the of the kinases a scaffolding complex that sequesters their activity in the cytosol, by a mechanism distinct from that by receptor tyrosine kinases such as the (13DeFea K.A. Zalevsky J. O. Bunnett N.W. J. Cell Biol. 2000; PubMed Scopus Google Scholar, T. B. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the that PAR-2 actin reorganization by an cells, with PAR-2 on extracellular and with or for with or with the which and was with and an in actin but in formation of pseudopodia of both pseudopodia and was by with PAR-2 the that PAR-2 chemotaxis whereas receptors that activate ERK1/2 by we the effects of PAR-2 activation on cell migration with that of a model of the classic receptor tyrosine kinase and PAR-1 receptor for that ERK1/2 by a PAR-2 cell used in these cells and a cell growth factor-I receptor is highly expressed in cells, and PAR-1 is expressed in both cell as by not was a and random migration was from chemotaxis by the response of cells to a of agonist to and of with a gradient of agonist to the This is used to a cells that are able to sense a gradient of agonist will by a and to in in a number of cells that random migration will in response to a gradient and a of agonist Klemke R.L. J. Cell Biol. 2002; PubMed Scopus Google Scholar, D.A. J. PubMed Scopus Google Scholar). A gradient of a in cell migration over and in cell migration response to a of P2AP, migration was by as a or a gradient not migration in cell to random migration, as both a and a gradient of cell migration A and cell migration in response to was in cells, which we to of receptor or PAR-2 chemotaxis, random migration, and PAR-1 not motility in the cell the that are to activate the role of MAPK activity in chemotaxis, we migration in the of and and a p38 and chemotaxis by and in cells, whereas cells, and chemotaxis by and whereas migration that ERK1/2 but not p38 MAPK activation is involved in chemotaxis MAPK and ERK1/2 can the of p38 MAPK may be of ERK1/2 random migration was by and was not by or random and directed migration have for MAPK activity. that the to and a for ERK1/2 effects of these we motility in the of cells with or and on of or cells from both of the with Cell and by and as A of migration in cells is shown in chemotaxis was by and by random migration, however, was and by of These with that ERK1/2 is required for chemotaxis. Although ERK1/2 may be a to for random migration, it is not an and these suggest the receptors different for directed and random PAR-2 in a of demonstrated previously that PAR-2 ERK1/2 by a β-arrestin-dependent in of a pool of activated kinase in the and PAR-1 have been shown to nuclear translocation of ERK1/2 (13DeFea K.A. Zalevsky J. O. Bunnett N.W. J. Cell Biol. 2000; PubMed Scopus Google Scholar, T. B. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Coughlin S.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We that upon PAR-2 the endosomal ERK1/2 is in a where it actin assembly at the leading edge of a PAR-2 specifically sequester a pool of activated ERK1/2 to a membrane-associated we subcellular on cells with P2AP, or for and of activated ERK1/2 in and both and cells, PAR-2 activation cytosolic and but nuclear ERK1/2 was a in cytosolic and nuclear in cells, and a in both cytosolic and nuclear in cells and a in cytosolic in cells in was in in response to or and with and demonstrated the of not We the localization of activated ERK1/2 to a in response to PAR-2 by and by and to Although a nuclear translocation of ERK1/2, activated ERK1/2 was in the cytosol PAR-2 activation in of activated ERK1/2 in the cytosol, where it may be with the with proteins and ERK1/2 with in the ERK1/2 in the with a a for protein during pseudopodia formation was Klemke R.L. J. Cell Biol. 2002; PubMed Scopus Google Scholar). cells to this have large cell such as cells pseudopodia and thus the pseudopodial protein is for This is to the migration that with are to cell body are physical of cell body from demonstrate pseudopodia with are shown in to was in migration a in pseudopodia pseudopodial proteins, both the cell from the or the pseudopodia from the are lysis and of proteins are then by of protein from control cells not been to pseudopodia are as a with to proteins shown previously (13DeFea K.A. Zalevsky J. O. Bunnett N.W. J. Cell Biol. 2000; PubMed Scopus Google Scholar) to be in PAR-2 endosomal we able to determine that β-arrestin, and phosphorylated ERK1/2 are found in the and are highly and β-arrestin is enriched in the pseudopodia with the cell body that ERK1/2 activity is sequestered the growing pseudopodia of polarized cells where it be actin machinery or proteins with cell was found in the cell that was cell body of pseudopodial ERK1/2 not enriched in the whereas in cell or ERK1/2 activation appears to to by of PAR-2 ERK1/2 activation is in the pseudopodia that a pool of the ERK1/2 is sequestered by PAR-2 at the leading edge, where activity is that this pool of activated ERK1/2 was with β-arrestin complex was of PAR-2 we of ERK1/2 and of PAR-2 β-arrestin and ERK1/2 can be and studies demonstrated that of a GFP-tagged dominant negative of corresponding to the clathrin ERK1/2 activation and cytosolic (13DeFea K.A. Zalevsky J. O. Bunnett N.W. J. Cell Biol. 2000; PubMed Scopus Google Scholar). that ERK1/2 activity with or a dominant negative PAR-2 chemotaxis and that components of the previously identified β-arrestin-dependent scaffolding complex are localized to the pseudopodia, we the of on pseudopodia formation and chemotaxis. cells with or with or Cell migration as in and pseudopodia formation as in pseudopodia formation by pseudopodia extension was by of ARR319–418-GFP, that of and of random β-arrestin was required for chemotaxis was the migration in cells with migration was by of MI, of whereas migration was that formation of pseudopodia, as with a cell PAR-2 is one of a growing family of receptors that the scaffolding of β-arrestin for activation and localization of MAPKs (7Miller W. Lefkowitz R.J. Curr. Opin. Cell Biol. 2001; 13: 139-145Crossref PubMed Scopus (283) Google Scholar). There is evidence that these endosomal scaffolds a role in the subcellular of MAPKs and their and responses (13DeFea K.A. Zalevsky J. O. Bunnett N.W. J. Cell Biol. 2000; PubMed Scopus Google Scholar, R.J. Lefkowitz R.J. Science. 2000; PubMed Google Scholar, A. S. Lefkowitz R.J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, K.A. O. Bunnett N.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar, S. Y. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar). Although one that of β-arrestin can gene cytosolic of A. Lefkowitz R.J. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar), a role for β-arrestin-dependent endosomal scaffolds in a cellular response has been Our work demonstrated that complexes activated ERK1/2 in the cytosol (13DeFea K.A. Zalevsky J. O. Bunnett N.W. J. Cell Biol. 2000; PubMed Scopus Google Scholar), but the localization and role of the sequestered kinase activity in PAR-2 responses we demonstrate that chemotaxis is dependent on both ERK1/2 and Using a of and we also show that phosphorylated ERK1/2 is at the in response to activation of but not in response to or PAR-1 and that it is highly enriched in the pseudopodia of migrating Furthermore, the active ERK1/2 is found in pseudopodia, with components and of a previously identified endosomal that the complex to ERK1/2 and kinases at the leading edge of motile and ERK1/2 for β-arrestin in cell motility is a to has been demonstrated in immune studies have demonstrated that β-arrestins are for and cytoskeletal reorganization and cell motility A.M. Premont R.T. Richardson R.M. Yu Y.R. Lefkowitz R.J. Patel D.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7478-7483Crossref PubMed Scopus (269) Google Scholar, M. T. J.L. R.J. Cell Biol. 2002; PubMed Scopus Google Scholar), but the studies provide the first evidence for β-arrestin-dependent endosomal scaffolds cell and pseudopodia a receptor for that not require β-arrestin for activation of ERK1/2, not direct chemotaxis in the cell and but not PAR-1 has been shown to direct chemotaxis in immune cells, and tumor cells Coughlin S.R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, P.J. M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, S. E. M. T. M. S. J. 2002; PubMed Scopus Google Scholar), migration in L. A. A. 2001; Google the to promote chemotaxis in these although it can activate ERK1/2, a role for the that random migration was not dependent on ERK1/2 or β-arrestin this the ability of PAR-2 to promote localization of ERK1/2 at the where it is with β-arrestin for to PAR-1 activation in localization of the is with a model in which the PAR-2 endosomal scaffold sequesters a pool of active ERK1/2 at the leading edge to actin This result not that receptors that localization of ERK1/2 will promote chemotaxis, it that receptors activate is widely that are for pseudopod the mechanism for PAR-2 is and may to be by receptors. We to that whereas ERK1/2 activation to to of PAR-2 activation in cell or in activated ERK1/2 was enriched in pseudopodial of PAR-2 activation The pseudopodia a of protein, and of protein from cell in a pool of active ERK1/2 is we would be able to it although we not be able to it in ERK1/2 was not enriched in the pseudopodia, we that activity is at the leading edge of the ERK1/2 may be from by association with the scaffolding ERK1/2 for phosphorylated ERK1/2 during pseudopodial extension, or it be by in the A for on these and studies (13DeFea K.A. Zalevsky J. O. Bunnett N.W. J. Cell Biol. 2000; PubMed Scopus Google Scholar), we a model for cell migration that is in cells sense a gradient of PAR-2 agonist such as trypsin, mast cell tryptase, and coagulation factors in an activation of PAR-2 in and protein kinase activation. protein kinase phosphorylation of the PAR-2 promotes β-arrestin association with and formation of an endosomal scaffold the ERK1/2, and proteins. studies suggest clathrin is in the complex and enriched in the and ERK1/2 is then and of ERK1/2 are in this and in the one of may the first ERK1/2 activity is by of association with a complex the where it can proteins involved in actin such as members of the (2Miki H. Fukuda M. Nishida E. Takenawa T. J. Biol. Chem. 1999; 274: 27605-27609Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), myosin light chain kinases R.L. Cai S. Giannini A.L. Gallagher P.J. de Lanerolle P. Cheresh D.A. J. Cell Biol. 1997; 137: 481-492Crossref PubMed Scopus (1107) Google Scholar), extracellular proteins (6Hughes P.E. Oertli B. Hansen M. Chou F.L. Willumsen B.M. Ginsberg M.H. Mol. Biol. Cell. 2002; 13: 2256-2265Crossref PubMed Scopus (41) Google Scholar, P.E. M. J. Ginsberg M.H. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), or These promote localized actin in pseudopod extension and cell the association of ERK1/2 with a β-arrestin scaffold on the may to ERK1/2 and kinases such as the leading edge, but the complex may as the pseudopodia ERK1/2 to proteins involved in cell ERK1/2 activity may result from of ERK1/2 for or signaling at the leading edge may Cell migration a of signal activation and and of this pathway is to by of the for β-arrestin in ERK1/2 activation. in the the complex is and PAR-2 is and in of PAR-2 to the for of signaling O. H. Bunnett N.W. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). the mechanism of PAR-2 chemotaxis is important from a as as a cell PAR-2 is highly expressed in a number of both and cells, the including and tumor cells and the including and both and a is evidence PAR-2 to wound healing and tumor L. A. A. 2001; Google Scholar, B. P.J. 1999; PubMed Scopus Google Scholar, R.J. P. Am. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, S.R. T. 2001; Google Scholar) that chemotaxis has both and a more cell these studies provide with the of of may that the PAR-2 endosomal scaffold is with a number of cytoskeletal proteins at the leading edge of the We Klemke and for in the pseudopodia Bunnett for PAR-2 and GFP-tagged Lefkowitz for for dominant negative for Zalevsky for of the Zalevsky for and Zalevsky for