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Plasmin is a major extracellular protease that elicits intracellular signals to mediate platelet aggregation, chemotaxis of peripheral blood monocytes, and release of arachidonate and leukotriene from several cell types in a G protein-dependent manner. Angiostatin, a fragment of plasmin(ogen), is a ligand and an antagonist for integrin α9β1. Here we report that plasmin specifically interacts with α9β1 and that plasmin induces migration of cells expressing recombinant α9β1 (α9-Chinese hamster ovary (CHO) cells). Migration was dependent on an interaction of the kringle domains of plasmin with α9β1 as well as the catalytic activity of plasmin. Angiostatin, representing the kringle domains of plasmin, alone did not induce the migration of α9-CHO cells, but simultaneous activation of the G protein-coupled protease-activated receptor (PAR)-1 with an agonist peptide induced the migration on angiostatin, whereas PAR-2 or PAR-4 agonist peptides were without effect. Furthermore, a small chemical inhibitor of PAR-1 (RWJ 58259) and a palmitoylated PAR-1-blocking peptide inhibited plasmin-induced migration of α9-CHO cells. These results suggest that plasmin induces migration by kringle-mediated binding to α9β1 and simultaneous proteolytic activation of PAR-1. [ABSTRACT FROM AUTHOR]

Urokinase-type plasminogen activator (uPA) induces cell adhesion and chemotactic movement, uPA signaling requires its binding to uPA receptor (uPAR/CD87), but how glycosylphosphatidylinositol-anchored uPAR mediates signaling is unclear, uPAR is a ligand for several integrins (e.g. α[sub 5]β[sub 1]) and supports cell-cell interaction by binding to integrins on apposing cells (in trans). We studied whether binding of uPAR to α[sub 5]β[sub 1] in cis is invalved in adhesion and migration of Chinese hamster ovary cells in response to immobilized uPA. This process was temperature-sensitive and required mitogen-activated protein kinase activation. Anti-uPAR antibody or depletion of uPAR blocked, whereas overexpression of uPAR enhanced, cell adhesion to uPA. Adhesion to uPA was also blocked by deletion of the growth factor domain (GFD) of uPA and by anti-GFD antibody, whereas neither the isolated uPA kringle nor serine protease domain supported adhesion directly. Interestingly, anti-α[sub 5] antibody, RGD peptide, and function-blocking mutations in α[sub 5]β[sub 1] blocked adhesion to uPA. uPA-induced cell migration also required GFD, uPAR, and α[sub 5]β[sub 1], but α[sub 5]β[sub 1] alone did not support uPA-induced adhesion and migration. Thus, binding of uPA causes uPAR to act as a ligand for α[sub 5]β[sub 1] to induce cell adhesion, intracellular signaling, and cell migration. We demonstrated that uPA induced RGD-dependent binding of uPAR to α[sub 5]β[sub 1] in solution. These results suggest that uPA-induced adhesion and migration of Chinese hamster ovary cells occurs as a consequence of (a) uPA binding to uPAR through GFD, (b) the subsequent binding of a uPA-uPAR complex to α[sub 5]β[sub 1] via uPAR, and (c) signal transduction through α[sub 5]β[sub 1]. [ABSTRACT FROM AUTHOR]

ADAMs (a disintegrin and metalloproteases) are members of the metzincin superfamily of metalloproteases. Among integrins binding to disintegrin domains of ADAMs are alpha(9)beta(1) and alpha(v)beta(3), and they bind in an RGD-independent and an RGD-dependent manner, respectively. Human ADAM15 is the only ADAM with the RGD motif in the disintegrin domain. Thus, both integrin alpha(9)beta(1) and alpha(v)beta(3) recognize the ADAM15 disintegrin domain. We determined how these integrins recognize the ADAM15 disintegrin domain by mutational analysis. We found that the Arg(481) and the Asp-Leu-Pro-Glu-Phe residues (residues 488-492) were critical for alpha(9)beta(1) binding, but the RGD motif (residues 484-486) was not. In contrast, the RGD motif was critical for alpha(v)beta(3) binding, but the other residues flanking the RGD motif were not. As the RX(6)DLPEF alpha(9)beta(1) recognition motif (residues 481-492) is conserved among ADAMs, except for ADAM10 and 17, we hypothesized that alpha(9)beta(1) may recognize disintegrin domains in all ADAMs except ADAM10 and 17. Indeed we found that alpha(9)beta(1) bound avidly to the disintegrin domains of ADAM1, 2, 3, and 9 but not to the disintegrin domains of ADAM10 and 17. As several ADAMs have been implicated in sperm-oocyte interaction, we tested whether the functional classification of ADAMs, based on specificity for integrin alpha(9)beta(1), applies to sperm-egg binding. We found that the ADAM2 and 15 disintegrin domains bound to oocytes, but the ADAM17 disintegrin domain did not. Furthermore, the ADAM2 and 15 disintegrin domains effectively blocked binding of sperm to oocytes, but the ADAM17 disintegrin domain did not. These results suggest that oocytes and alpha(9)beta(1) have similar binding specificities for ADAMs and that alpha(9)beta(1), or a receptor with similar specificity, may be involved in sperm-egg interaction during fertilization. As alpha(9)beta(1) is a receptor for many ADAM disintegrins and alpha(9)beta(1) and ADAMs are widely expressed, alpha(9)beta(1)-ADAM interaction may be of a broad biological importance.