Although plasminogen activator inhibitor-1 (PAI-1) is known to stimulate cell migration, little is known about underlying mechanisms. We show that both active and inactive (e.g. cleaved) PAI-1 can activate the Jak/Stat signaling system and stimulate cell migration in chemotaxis, haptotaxis, chemokinesis, and wound healing assays. Moreover, antibodies to the LDL receptor-related protein (LRP) and an LRP antagonist (RAP) blocked these motogenic effects of PAI-1, while a PAI-1 mutant that did not bind to LRP failed to activate the Jak/Stat signaling pathway or to stimulate cell migration. PAI-1 had no chemotactic effect on LRP-deficient cells. These results indicate that LRP is a signaling molecule, that it mediates the migration-promoting activity of PAI-1, and that this activity does not require intact, biologically active PAI-1. Activation of this LRP-dependent signaling pathway by PAI-1 may begin to explain how the inhibitor stimulates cell migration in a variety of normal and pathological processes.

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Although the urokinase receptor (uPAR) binds to vitronectin (VN) and promotes the adhesion of cells to this matrix protein, the biochemical details of this interaction remain unclear. VN variants were employed in BIAcore experiments to examine the uPAR-VN interaction in detail and to compare it to the interaction of VN with other ligands. Heparin and plasminogen bound to VN fragments containing the heparin-binding domain, indicating that this domain was functionally active in the recombinant peptides. However, no significant binding was detected when uPAR was incubated with this domain, and neither heparin nor plasminogen competed with it for binding to VN. In fact, uPAR only bound to fragments containing the somatomedin B (SMB) domain, and monoclonal antibodies (mAbs) that bind to this domain competed with uPAR for binding to VN. Monoclonal antibody 8E6 also inhibited uPAR binding to VN, and this mAb was shown to recognize sulfated tyrosine residues 56 and 59 in the region adjacent to the SMB domain. Destruction of this site by acid treatment eliminated mAb 8E6 binding but had no effect on uPAR binding. Thus, there appears to be a single binding site for uPAR in VN, and it is located in the SMB domain and is distinct from the epitope recognized by mAb 8E6. Inhibition of uPAR binding to VN by mAb 8E6 probably results from steric hindrance. Vitronectin (VN)1 is a 75-kDa adhesive glycoprotein. It circulates in blood in a monomeric ("closed," "native") form, but is converted into a multimeric ("extended," "opened," "denatured") form when incorporated into the extracellular matrix or treated with urea (1, 2). The extended form of VN binds to specific receptors on cells (3, 4) and to various other molecules such as the C5b-9 complement complex (5), the thrombin-antithrombin III complex (6, 7), plasminogen activator inhibitor 1 (PAI-1) (8 -10), uPAR (11), heparin (1, 2, 12, 13), collagen (14 -16), plasminogen (13, 17), and ␤-endorphin (18). These interactions not only promote the attachment, spreading, and growth of cells (19 -21) but also influence the coagulation, fibrinolytic, and complement systems (22,23).Although a number of investigators have attempted to identify the binding site(s) in VN for these molecules, the literature remains somewhat controversial. For example, three different regions of the VN molecule have been proposed to contain the binding sites for uPAR and PAI-1. The first region, the somatomedin B (SMB) domain (residues 1-44) was identified from direct binding studies (9, 24 -27) and from studies showing that soluble SMB competes with uPAR and PAI-1 for binding to denatured VN (9). The second region in VN that has been implicated in uPAR and PAI-1 binding is the heparin binding (HB) domain (residues 348 -370). Thus, synthetic peptides from this domain interfere with both uPAR (21) and PAI-1 (28) binding to VN. Moreover, mAb 8E6 (which has been mapped to the HB domain (Ref. 13)) also inhibits the binding of PAI-1 to VN. Although a third region in VN (residues 115-121) was shown to have weak PAI-...

The N-terminal cysteine-rich somatomedin B (SMB) domain (residues 1-44) of the human glycoprotein vitronectin contains the high-affinity binding sites for plasminogen activator inhibitor-1 (PAI-1) and the urokinase receptor (uPAR). We previously showed that the eight cysteine residues of recombinant SMB (rSMB) are organized into four disulfide bonds in a linear uncrossed pattern (Cys(5)-Cys(9), Cys(19)-Cys(21), Cys(25)-Cys(31), and Cys(32)-Cys(39)). In the present study, we use an alternative method to show that this disulfide bond arrangement remains a major preferred one in solution, and we determine the solution structure of the domain using NMR analysis. The solution structure shows that the four disulfide bonds are tightly packed in the center of the domain, replacing the traditional hydrophobic core expected for a globular protein. The few noncysteine hydrophobic side chains form a cluster on the outside of the domain, providing a distinctive binding surface for the physiological partners PAI-1 and uPAR. The hydrophobic surface consists mainly of side chains from the loop formed by the Cys(25)-Cys(31) disulfide bond, and is surrounded by conserved acidic and basic side chains, which are likely to contribute to the specificity of the intermolecular interactions of this domain. Interestingly, the overall fold of the molecule is compatible with several arrangements of the disulfide bonds. A number of different disulfide bond arrangements were able to satisfy the NMR restraints, and an extensive series of conformational energy calculations performed in explicit solvent confirmed that several disulfide bond arrangements have comparable stabilization energies. An experimental demonstration of the presence of alternative disulfide conformations in active rSMB is provided by the behavior of a mutant in which Asn(14) is replaced by Met. This mutant has the same PAI-1 binding activity as rVN1-51, but its fragmentation pattern following cyanogen bromide treatment is incompatible with the linear uncrossed disulfide arrangement. These results suggest that active forms of the SMB domain may have a number of allowed disulfide bond arrangements as long as the Cys(25)-Cys(31) disulfide bond is preserved.