Constitutive protease-activated receptor-2-mediated migration of MDA MB-231 breast cancer cells requires both beta-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 beta-arrestin-1 reduces PAR-2-stimulated internalization, ERK1/2 activation, and cell migration; however, this reagent may block association of many proteins, including beta-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 beta-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, beta-arrestin-2, and activated ERK1/2 to pseudopodia. Addition of MEK-1 inhibitors, trypsin inhibitors, a scrambled PAR-2 peptide, and silencing of beta-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 beta-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 beta-arrestin-dependent ERK1/2 activation.
Division of Biomedical Sciences, Biochemical and Molecular Biology Program and Cellular, Molecular and Developmental Biology Program, University of California, Riverside, CA 92521, USA.
Lan
L
Ge
Sudha K
SK
Shenoy
Robert J
RJ
Lefkowitz
Kathryn
K
DeFea
D019390:Arrestins / Q000378:metabolism / Q000502:physiology*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000201:enzymology / Q000473:pathology*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D002633:Chemotaxis; D002966:Clathrin / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009362:Neoplasm Metastasis; D011554:Pseudopodia / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D044464:Receptor, PAR-2 / Q000502:physiology*; D013347:Subcellular Fractions; D013997:Time Factors; D014162:Transfection; D014357:Trypsin / Q000378:metabolism / Q000494:pharmacology; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605253:ARRB1 protein, human; C000605254:ARRB2 protein, human; D019390:Arrestins; D002966:Clathrin; D034741:RNA, Small Interfering; D044464:Receptor, PAR-2; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D014357:Trypsin