Constitutive Protease-activated Receptor-2-mediated Migration of MDA MB-231 Breast Cancer Cells Requires Both β-Arrestin-1 and -2

Protease-activated receptor-2 (PAR-2) is activated by trypsin-like serine proteases and can promote cell migration through an ERK1/2-dependent pathway, involving formation of a scaffolding complex at the leading edge of the cell. Previous studies also showed that expression of a dominant negative fragment of β-arrestin-1 reduces PAR-2-stimulated internalization, ERK1/2 activation, and cell migration; however, this reagent may block association of many proteins, including β-arrestin-2 with clathrin-coated pits. Here we investigate the role of PAR-2 in the constitutive migration of a metastatic breast cancer cell line, MDA MB-231, and use small interfering RNA to determine the contribution of each β-arrestin to this process. We demonstrate that a trypsin-like protease secreted from MDA MB-231 cells can promote cell migration through autocrine activation of PAR-2 and this correlates with constitutive localization of PAR-2, β-arrestin-2, and activated ERK1/2 to pseudopodia. Addition of MEK-1 inhibitors, trypsin inhibitors, a scrambled PAR-2 peptide, and silencing of β-arrestins with small interfering RNA also reduce base-line migration of MDA MB-231 cells. In contrast, a less metastatic PAR-2 expressing breast cancer cell line does not exhibit constitutive migration, pseudopodia formation, or trypsin secretion; in these cells PAR-2 is more uniformly distributed around the cell periphery. These data demonstrate a requirement for both β-arrestins in PAR-2-mediated motility and suggest that autocrine activation of PAR-2 by secreted proteases may contribute to the migration of metastatic tumor cells through β-arrestin-dependent ERK1/2 activation. Protease-activated receptor-2 (PAR-2) is activated by trypsin-like serine proteases and can promote cell migration through an ERK1/2-dependent pathway, involving formation of a scaffolding complex at the leading edge of the cell. Previous studies also showed that expression of a dominant negative fragment of β-arrestin-1 reduces PAR-2-stimulated internalization, ERK1/2 activation, and cell migration; however, this reagent may block association of many proteins, including β-arrestin-2 with clathrin-coated pits. Here we investigate the role of PAR-2 in the constitutive migration of a metastatic breast cancer cell line, MDA MB-231, and use small interfering RNA to determine the contribution of each β-arrestin to this process. We demonstrate that a trypsin-like protease secreted from MDA MB-231 cells can promote cell migration through autocrine activation of PAR-2 and this correlates with constitutive localization of PAR-2, β-arrestin-2, and activated ERK1/2 to pseudopodia. Addition of MEK-1 inhibitors, trypsin inhibitors, a scrambled PAR-2 peptide, and silencing of β-arrestins with small interfering RNA also reduce base-line migration of MDA MB-231 cells. In contrast, a less metastatic PAR-2 expressing breast cancer cell line does not exhibit constitutive migration, pseudopodia formation, or trypsin secretion; in these cells PAR-2 is more uniformly distributed around the cell periphery. These data demonstrate a requirement for both β-arrestins in PAR-2-mediated motility and suggest that autocrine activation of PAR-2 by secreted proteases may contribute to the migration of metastatic tumor cells through β-arrestin-dependent ERK1/2 activation. One of the earliest steps in tumor cell metastasis is the reorganization of the actin cytoskeleton and initiation of cell migration (1Condeelis J.S. Wyckoff J.B. Bailly M. Pestell R. Lawrence D. Backer J. Segall J.E. Semin. Cancer Biol. 2001; 11: 119-128Crossref PubMed Scopus (117) Google Scholar). A variety of extracellular signaling molecules including growth factors and chemokines can promote these events and recent evidence suggests trypsin-like proteases secreted from tumor cells might generate migratory signals through the activation of cell surface protease-activated receptor-2 (PAR-2) 1The abbreviations used are: PAR-2, protease-activated receptor-2; ERK1/2, extracellular signal-regulated kinase-2; AP, activating peptide; hAP, human AP (SLIGKV-NH2); 2f-AP, 2-furoyl-LIGRL-ornithine-NH2; scr-AP, scrambled PAR-2 peptide; SBTI, soybean trypsin inhibitor; PIC, protease inhibitor mixture; CM, conditioned medium; ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA.1The abbreviations used are: PAR-2, protease-activated receptor-2; ERK1/2, extracellular signal-regulated kinase-2; AP, activating peptide; hAP, human AP (SLIGKV-NH2); 2f-AP, 2-furoyl-LIGRL-ornithine-NH2; scr-AP, scrambled PAR-2 peptide; SBTI, soybean trypsin inhibitor; PIC, protease inhibitor mixture; CM, conditioned medium; ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA. (2Ducroc R. Bontemps C. Marazova K. Devaud H. Darmoul D. Laburthe M. Life Sci. 2002; 70: 1359-1367Crossref PubMed Scopus (43) Google Scholar, 3Kamath L. Meydani A. Foss F. Kuliopulos A. Cancer Res. 2001; 61: 5933-5940PubMed Google Scholar, 4Li Y. Sarkar F.H. Cancer Lett. 2002; 186: 157-164Crossref PubMed Scopus (110) Google Scholar, 5D'Andrea M.R. Derian C.K. Santulli R.J. Andrade-Gordon P. Am. J. Pathol. 2001; 158: 2031-2041Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 6Hjortoe G.M. Petersen L.C. Albrektsen T. Sorensen B.B. Norby P.L. Mandal S.K. Pendurthi U.R. Rao L.V.M. Blood. 2004; 103: 3029-3037Crossref PubMed Scopus (222) Google Scholar, 7Greenberg D.L. Mize G.J. Takayama T.K. Biochemistry. 2003; 42: 702-709Crossref PubMed Scopus (66) Google Scholar). PAR-2 is highly expressed in a variety of normal and tumor cells and is activated by nanomolar concentrations of several serine proteases, including trypsin, tryptase, Factors VIIa and Xa, and membrane-type serine protease-1 (MT-SP1I) (8Camerer E. Huang W. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5255-5260Crossref PubMed Scopus (604) Google Scholar, 9Dery O. Corvera C.U. Steinhoff M. Bunnett N.W. Am. J. Physiol. 1998; 274: C1429-C1452Crossref PubMed Google Scholar, 10Takeuchi 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 (389) Google Scholar). Proteolytic cleavage of the PAR-2 extracellular N terminus reveals a tethered ligand that binds to and activates the receptor. Activating peptides (APs) corresponding to the tethered ligand sequence SLIGKV/RL (human/mouse) and a chemically modified version of AP (2-furoyl-LIGRL-ornithine-NH2 (2f-AP)) also activate PAR-2, in the absence of proteolytic cleavage (9Dery O. Corvera C.U. Steinhoff M. Bunnett N.W. Am. J. Physiol. 1998; 274: C1429-C1452Crossref PubMed Google Scholar, 11McGuire J.J. Saifeddine M. Triggle C.R. Sun K. Hollenberg M.D. J. Pharmacol. Exp. Ther. 2004; 309: 1124-1131Crossref PubMed Scopus (113) Google Scholar). A number of PAR-2-mediated events, including activation of ERK1/2 and cell migration are blocked by expression of a dominant negative fragment of β-arrestin-1 that encodes the clathrin binding domain (12DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (683) Google Scholar). However, the specificity of this reagent is unclear as it acts by binding β-arrestin contact sites on clathrin and may affect other clathrin-regulated processes as well. In recent studies, we demonstrated that PAR-2 promotes chemotaxis by a mechanism involving localization of activated ERK1/2 and its upstream regulatory kinases to the pseudopodia. This pseudopodial localization appears to occur through the formation of a scaffolding complex containing β-arrestin(s) that we refer to as an endosomal scaffold (12DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (683) Google Scholar, 13Ge L. Ly Y. Hollenberg M. DeFea K. J. Biol. Chem. 2003; 278: 34418-34426Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). While β-arrestins were originally thought to mediate signal termination, there has been a growing body of evidence that they can also serve as signaling scaffolds for a number of receptors (14Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (699) Google Scholar, 15McDonald P.H. Chow C.W. Miller W.E. Laporte S.A. Field M.E. Lin F.T. Davis R.J. Lefkowitz R.J. Science. 2000; 290: 1574-1577Crossref PubMed Google Scholar, 16Miller W. Lefkowitz R.J. Curr. Opin. Cell Biol. 2001; 13: 139-145Crossref PubMed Scopus (279) Google Scholar) and that further functional differences exist between the two β-arrestin family members. Recent studies using siRNA to specifically knockdown either β-arrestin-1 or 2 suggest that β2AR primarily utilizes β-arrestin-2 for internalization, while the angiotensin II type 1a receptor (AT1aR) can use either for internalization but requires β-arrestin-2 for signaling to ERK1/2 (17Ahn S. Nelson C.D. Garrison T.R. Miller W.E. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1740-1744Crossref PubMed Scopus (188) Google Scholar). Additional differences may exist between other cell types and receptors. The role of β-arrestins in cell migration is supported by genetic studies demonstrating impaired CXCR-mediated motility in lymphocytes from β-arrestin-2 knock-out mice (18Fong 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 (261) Google Scholar). However, the specific β-arrestin family member(s) that mediate(s) PAR-2-promoted cell motility has not been investigated nor has the role of β-arrestins in tumor cell migration. The purpose of these studies was 2-fold. First, using siRNA to specifically knockdown each β-arrestin, we wished to determine which β-arrestins mediate ERK1/2 activation and chemotaxis downstream of PAR-2. Second, using the metastatic breast cancer cells (MDA MB-231) that migrate constitutively and cells with low metastatic potential (MDA MB-468) (19Zhang R.D. Fidler I.J. Price J.E. Invasion Metastasis. 1991; 11: 204-215PubMed Google Scholar, 20Gordon L.A. Mulligan K.T. Maxwell-Jones H. Adams M. Walker R.A. Jones J.L. Int. J. Cancer. 2003; 106: 8-16Crossref PubMed Scopus (108) Google Scholar) that do not migrate without stimulation, we wished to investigate how PAR-2 and β-arrestins might modulate cell migration in a metastatic tumor cell. Materials—All chemicals were from Sigma or Fisher Scientific unless otherwise stated. MDA MB-468 and MDA MB-231 cells were from American Type Tissue Culture Collection. Human PAR-2 peptides SLIGKV-NH2 (hAP) and 2f-AP and scrambled PAR-2 peptide (scr-AP) were synthesized by Genemed Inc. Trypsin was from Worthington Chemicals. hAP was used at 50 μm, 2f-AP was used at 50 nm, and trypsin was used at 1 nm unless otherwise stated. The following antibodies were used: mouse anti-pRaf, Raf, and β-arrestin-1 (Pharmingen); mouse anti-PAR-2 Sam11 (Zymed Laboratories Inc.); rabbit anti-PAR-2 B5 (Dr. Morley Hollenberg, University of Calgary); rabbit β-arrestin-1 and -2 were generated in the Lefkowitz laboratory; rabbit anti-ERK1 antibody (Santa Cruz Biotechnology). Anti-phospho-p42/44 MAPK (pERK) and MEK1 inhibitor PD98059 were from Cell Signaling Inc. Antibody dilutions for Western blotting were as follows: pERK (1:2000), ERK1/2 (1:2000), PAR-2 (SAM11, 1:250), β-arrestin-1/2 (1: 100), phospho-Raf (1:1000), total Raf (1:1000), total ERK1 (1:1000), and histone (1:250). Cell Culture—MDA MB-231 and MDA MB-468 cells were grown in Leibowitz's L15 medium, supplemented with 14 mm NaHCO3 and 10% fetal calf serum and maintained at 37 °C, were for and inhibitor 2 and mm or hAP were for as and cells were in normal by in and with and μm, and were on a in were with a MDA cells were by in on a and were 2 using A with of and is in the siRNA synthesized siRNA β-arrestin-2, β-arrestin-1 and with RNA and were from in and were with of siRNA, using pERK studies, cells were siRNA and to chemotaxis cells were for and siRNA and migration and pseudopodia cells were in serum with for migration and for pseudopodia AP, or were to the and cells or pseudopodia and cell were as L. Ly Y. Hollenberg M. DeFea K. J. Biol. Chem. 2003; 278: 34418-34426Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). inhibitor a of serine protease 1 soybean trypsin inhibitor 2 or 2 or PD98059 was to the cells for 2 and migration as were for with 50 AP for at 37 °C, by in of and as L. Ly Y. Hollenberg M. DeFea K. J. Biol. Chem. 2003; 278: 34418-34426Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). of was using and pERK were to total for each Trypsin the of the of was to cells and were for was by and by by with of 1 of trypsin was and were with trypsin using and were using or and were a of of and were used to determine and differences of PAR-2 in MDA MB-231 investigate the contribution of PAR-2 in tumor cell we the base-line and PAR-2-stimulated migration through in MDA MB-231 and the less metastatic MDA MB-468 cells. We that PAR-2 migration in a variety of tumor cells but these two for MDA MB-231 cells the while MDA MB-468 cells the base-line migration and trypsin of the cell with metastatic the total number of cells was in MDA MB-231 cells in the MDA MB-468 cells Addition of PAR-2 activating peptide (hAP) or trypsin to the in a and in MDA MB-231 cell migration and a and in MDA MB-468 cell migration, Previous studies demonstrated that PAR-2 are as a is less a to the at a migratory L. Ly Y. Hollenberg M. DeFea K. J. Biol. Chem. 2003; 278: 34418-34426Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). investigate the of MDA MB-231 cell migration was by PAR-2, we in the of a scrambled PAR-2 peptide; peptides containing a scrambled tethered ligand sequence been demonstrated to PAR-2 activation J.J. Saifeddine M. Triggle C.R. Sun K. Hollenberg M.D. J. Pharmacol. Exp. Ther. 2004; 309: 1124-1131Crossref PubMed Scopus (113) Google Scholar, Saifeddine M. Hollenberg M.D. J. Pharmacol. Exp. Ther. 2002; PubMed Scopus Google Scholar). MDA MB-231 cell migration by 10% that constitutive PAR-2 activation to migratory the that constitutive PAR-2 activation might to the of serine protease in the medium, we investigated the of a protease inhibitor for or protease on MDA MB-231 cell migration. the role of we the and MDA MB-231 cell migration by and 50 while and a in MDA MB-231 migration, with in this cell line that of PAR-2 cell migration L. Meydani A. Foss F. Kuliopulos A. Cancer Res. 2001; 61: 5933-5940PubMed Google Scholar). demonstrate that PAR-2 was for the motility and that the protease were not we hAP or 2f-AP in the of 2f-AP is a PAR-2 that the as at nanomolar concentrations J.J. Saifeddine M. Triggle C.R. Sun K. Hollenberg M.D. J. Pharmacol. Exp. Ther. 2004; 309: 1124-1131Crossref PubMed Scopus (113) Google Scholar). hAP and 2f-AP cell migration to and 10% of in cells that constitutive proteolytic cleavage of PAR-2 to the migratory of MDA MB-231 cells. not block PAR-2-stimulated migration in MDA MB-468 cells not further that the were not with the of on MDA MB-231 cell migration, of to MDA MB-231 cells in of cell processes and of hAP in and and Previous studies showed that MEK1 and expression of a dominant negative PAR-2-stimulated migration in and MDA MB-468 cells (12DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (683) Google Scholar, 13Ge L. Ly Y. Hollenberg M. DeFea K. J. Biol. Chem. 2003; 278: 34418-34426Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Addition of PD98059 base-line MDA MB-231 cell migration by PAR-2 was through ERK1/2 to promote migration. with this base-line ERK1/2 activation was in from MDA MB-231 with MDA MB-468 and of hAP ERK1/2 in both MDA MB-231 and MDA MB-468 cells MDA MB-231 of trypsin to block MDA MB-231 cell migration suggests they might trypsin, leading to autocrine activation of PAR-2. We the trypsin secreted from MDA MB-231 and MDA MB-468 cells by Western of conditioned Trypsin in the of MDA MB-231 cells and to while trypsin in the of MDA MB-468 cells The of trypsin of conditioned was by the of to a trypsin and was to in the low nanomolar We the of from MDA MB-231 cells to or MDA MB-468 cells to promote chemotaxis of MDA MB-468 cells. While MDA MB-231 in a in cell migration, MDA MB-468 of with cell migration demonstrating that the in the is a serine trypsin a in cell migration PAR-2 to in MDA MB-231 of total cell suggests that both MDA MB-231 and MDA MB-468 cells of PAR-2 but the of We that PAR-2 is by the in and these the by an of cells. by an is both and as it of cell and K. The may These of PAR-2 are more in cell and we are the role of PAR-2 in its signaling reveals that PAR-2 localization in MDA MB-231 cells appears to at the leading while in the MDA MB-468 cells it is distributed more uniformly around the cell surface of MDA MB-231 cells with trypsin in of PAR-2 around the cell while of MDA MB-468 cells with hAP in of surface PAR-2 at In MDA a of PAR-2 is to both the as has been by several other S.K. L.M. Bunnett N.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and the to constitutive receptor activation. to from the of cell migration is that to the leading determine PAR-2 is constitutively in pseudopodia with other of the endosomal scaffold (12DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (683) Google Scholar, 13Ge L. Ly Y. Hollenberg M. DeFea K. J. Biol. Chem. 2003; 278: 34418-34426Abstract Full Text Full Text PDF PubMed Scopus (177) Google we pseudopodia and cell from MDA MB-231 cells as L. Ly Y. Hollenberg M. DeFea K. J. Biol. Chem. 2003; 278: 34418-34426Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, J. Cell Biol. 2002; PubMed Scopus Google Scholar). MDA MB-231 cells were pseudopodia in to in MDA MB-468 cells are and in for In MDA MB-231 β-arrestin-2, and are to the pseudopodia ERK1/2 and are distributed between pseudopodia and cell the are specifically at the leading A by and J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar) that was specifically with but not with the that of signaling molecules to the leading edge might promote migration. the modified of PAR-2 are While of the PAR-2 is in the of the and of the are to the cell body studies are to determine and a role in localization of PAR-2 to the pseudopodia and cell migration. These data are with the that trypsin secreted from MDA MB-231 cells activates PAR-2 in its a signaling complex at the leading edge of the cell. MDA MB-231 Cell on and data showed that receptor internalization with a dominant negative fragment of β-arrestin-1 PAR-2-stimulated ERK1/2 activation and motility (12DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (683) Google Scholar, 13Ge L. Ly Y. Hollenberg M. DeFea K. J. Biol. Chem. 2003; 278: 34418-34426Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Recent evidence suggests that β-arrestins are not family and while receptors to on β-arrestin for both signaling and signal use for internalization and for is unclear which β-arrestin PAR-2 and the dominant negative β-arrestin-1 used can both and may a more on We used siRNA to knockdown β-arrestin-1 and -2 expression either or and that expression of either of the β-arrestin-1 or -2 base-line MDA MB-231 cell migration 10% and 50 while knockdown of both β-arrestins in a investigate the of PAR-2 and β-arrestins on cell migration was through the pathway, we the of siRNA knockdown on PAR-2-stimulated cell in the of in the to of base-line cell migration, and of hAP or 2f-AP migration. In the of PIC, siRNA knockdown of β-arrestin-1 expression cell migration, and knockdown of β-arrestin-2 reduces migration by that PAR-2-stimulated migration requires both β-arrestin-1 and -2 and the of they each in PAR-2 determine a of in the role of β-arrestins in cell migration was ERK1/2 activation, we the β-arrestin-1 and -2 siRNA on ERK1/2 activation of cells with to base-line PAR-2 of either β-arrestin-1 or -2 expression PAR-2 ERK1/2 by both β-arrestins contribute to PAR-2-stimulated ERK1/2 activation and that the for β-arrestin-1 and -2 in cell migration might downstream of In the studies we evidence the that secreted proteases the metastatic potential of tumor cells by migration (2Ducroc R. Bontemps C. Marazova K. Devaud H. Darmoul D. Laburthe M. Life Sci. 2002; 70: 1359-1367Crossref PubMed Scopus (43) Google Scholar, 4Li Y. Sarkar F.H. Cancer Lett. 2002; 186: 157-164Crossref PubMed Scopus (110) Google Scholar, 5D'Andrea M.R. Derian C.K. Santulli R.J. Andrade-Gordon P. Am. J. Pathol. 2001; 158: 2031-2041Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 10Takeuchi 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 (389) Google Scholar) and a mechanism for these We that a highly metastatic breast cancer cell line, MDA MB-231, trypsin and constitutive migration, while a less metastatic tumor cell line, MDA does migration of MDA MB-231 cells is by the of serine protease or a scrambled PAR-2 activation of PAR-2 by specific activating peptides cell migration in the of protease to or These data suggest that the contribution of serine proteases to MDA MB-231 cell migration is by PAR-2. While other serine proteases of activating PAR-2 may as trypsin is a of the and its in MDA MB-231 cells suggests it may an role in constitutive PAR-2 activation and cell migration in these cells. this we that from MDA MB-231 cells can promote migration of the less metastatic MDA and this is by with serine protease While autocrine activation of PAR-2 is to for of the constitutive migration of these and other tumor the data a trypsin-like proteases contribute to the metastatic potential of a cell by activating migratory through receptors and a role for β-arrestin scaffolding of signaling A specific requirement for β-arrestin-2 in motility has been demonstrated using β-arrestin-1 and -2 knock-out mice (18Fong 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 (261) Google Scholar). of the studies is the evidence that both β-arrestins are for PAR-2-mediated they are not for receptors that to a is with studies by demonstrating that the two β-arrestin in specific (17Ahn S. Nelson C.D. Garrison T.R. Miller W.E. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1740-1744Crossref PubMed Scopus (188) Google Scholar, 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 (261) Google Scholar, E. J. R.J. G.M. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Lin Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: Google Scholar, S. S.K. H. Lefkowitz R.J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google and in the of actin are in studies from in and K. in Previous studies in demonstrated that migration is β-arrestins and ERK1/2 and appears to of the activated kinases to the pseudopodia on endosomal (12DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (683) Google Scholar, 13Ge L. Ly Y. Hollenberg M. DeFea K. J. Biol. Chem. 2003; 278: 34418-34426Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Here we that the of this endosomal scaffold are constitutively to pseudopodia in MDA MB-231 cells. both β-arrestin-1 and -2 are for cell migration, β-arrestin-2 is to a in the with the that the two β-arrestins may in cell a variety of breast cancer cell demonstrated that MDA MB-468 cells exhibit low of cell with MDA MB-231 and are not metastatic in a mouse (19Zhang R.D. Fidler I.J. Price J.E. Invasion Metastasis. 1991; 11: 204-215PubMed Google Scholar, 20Gordon L.A. Mulligan K.T. Maxwell-Jones H. Adams M. Walker R.A. Jones J.L. Int. J. Cancer. 2003; 106: 8-16Crossref PubMed Scopus (108) Google Scholar, Y. A. A. A. Int. J. Cancer. 2002; PubMed Scopus Google Scholar). The that less metastatic tumor cells both PAR-2 and migrate in to its activation is a number of proteases can activate PAR-2 (8Camerer E. Huang W. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5255-5260Crossref PubMed Scopus (604) Google Scholar, O. Bunnett N.W. PubMed Scopus Google the that in a might the metastatic potential of a cell metastasis of tumor cells they the the tumor cells are thought to metastasis as well. However, tumor cells may for cell migration, which they can of cell studies are to determine trypsin and PAR-2 endosomal scaffold formation are in a of tumor PAR-2 is for metastasis of in the of serine protease and PAR-2 in and the role of β-arrestins in this process. The use of serine protease as is not a and trypsin and been used in the of as and E. J. R.J. G.M. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, J.E. J. PubMed Scopus Google Scholar, M.D. J. PubMed Scopus Google Scholar, M. S. A. PubMed Scopus Google Scholar). the however, the of serine proteases as a of tumor metastasis has been an of While the has been on the role of proteases in the tumor and the studies suggest that activation of cell migration through cell surface receptors may also contribute to the of serine We are to Morley Hollenberg of for B5 to Ly for and to Zalevsky for of the with

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