Rac mediates cytoskeletal rearrangements and increased cell motility induced by urokinase-type plasminogen activator receptor binding to vitronectin.

The urokinase-type plasminogen activator receptor (uPAR) is involved in the regulation of cell motility in a variety of cell types. We show here that expression of human uPAR in growing murine fibroblasts leads to a dramatic reorganization of the actin cytoskeleton. uPAR expression induces multiple rapidly advancing protrusions that resemble the leading edge of migrating cells. The cytoskeletal changes are independent of uPA and activation of the RGD-binding activity of integrins but require uPAR binding to vitronectin (VN). The actin reorganization is blocked by coexpression of dominant negative versions of either Rac (N17Rac) or p130Cas, but not by inhibitors of Cdc42 or Rho, and is accompanied by a Rac-dependent increase in cell motility. In addition, a fourfold increase in the level of activated Rac is induced by uPAR expression. We conclude that uPAR interacts with VN both to initiate a p130Cas/Rac-dependent signaling pathway leading to actin reorganization and increased cell motility and to act as an adhesion receptor required for these responses. This mechanism may play a role in uPAR-mediated regulation of cell motility at sites where VN and uPAR are co-expressed, such as malignant tumors.

Signaling to Rho GTPases.

Rho GTPases regulate many important processes in all eukaryotic cells, including the organization of the actin cytoskeleton, gene transcription, cell cycle progression, and membrane trafficking. Their activity is regulated by signals originating from different classes of surface receptors including G-protein-coupled receptors, tyrosine kinase receptors, cytokine receptors, and adhesion receptors. Recent work has identified multiple mechanisms by which receptors can signal to Rho GTPases and this will be the major focus of this review. In addition, there is growing evidence for cross-talk within the Rho GTPase family as well as between the Rho and Ras GTPase families. These signaling networks are thought to provide the cooperative and coordinated interactions that are crucial for regulating complex biological processes such as cell migration.

Kinetic analysis of integrin-dependent cell adhesion on vitronectin--the inhibitory potential of plasminogen activator inhibitor-1 and RGD peptides.

Distinct binding interactions between cell-surface receptors and extracellular matrix components are characteristic of multifunctional adhesion proteins such as vitronectin. The close proximity of binding sites for alpha(v)-integrins and plasminogen activator inhibitor-1 (PAI-1) on vitronectin may have consequences for cell adhesion and migration, or for the localized inhibition of plasminogen activators. In this study, the kinetics and reversibility of vitronectin-dependent cell adhesion via alpha(v)-integrins was investigated using RGD peptides and PAI-1 as competitors. Active, but not latent or cleaved PAI-1, and RGD peptides were effective in preventing cell adhesion to vitronectin provided the inhibitor was present at the time of cell seeding. In a concentration-dependent manner urokinase or thrombin abrogated the inhibitory effect of PAI-1. Following cell seeding onto a vitronectin substratum, delayed addition of RGD peptides or active PAI-1 (10-20 min post-seeding) resulted in the loss of their inhibitory potential. These data were supported by experiments in a purified system where delayed addition of active PAI-1 could no longer prevent vitronectin binding to immobilized alpha(v)beta3, while a cyclic RGD peptide gave some moderate inhibition. The apparent stabilization of vitronectin-integrin contacts was observed with immobilized native or multimeric vitronectin but not with the more rigid form of denatured, aggregated multimers. These results demonstrate that the cell adhesive properties of vitronectin depend on its conformational flexibility and can be tightly regulated in a spatio-temporal manner through direct competition of cellular integrins by soluble or matrix-bound factors such as PAI-1.

Binding of urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex to the endocytosis receptors alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein and very-low-density lipoprotein receptor involves basic residues in the inhibitor.

The complex of the type-1 plasminogen activator inhibitor (PAI-1) and its target proteinases, the urokinase and tissue-type plasminogen activators (uPA and tPA), but not the free components, bind with high affinity to the endocytosis receptors alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha2MR/LRP) and very-low-density lipoprotein receptor (VLDLR). To characterize the molecular interaction between the complexes and the receptors, alanine codons were introduced into the human PAI-1 cDNA to replace the four basic residues, Arg-78, Lys-82, Arg-120 and Lys-124, as double mutations. The purified recombinant mutant proteins, rPAI-1/R78A-K124A and rPAI-1/K82A-R120A, produced by the yeast Pichia pastoris, were indistinghuisable from wild-type recombinant and natural human PAI-1 with respect to inhibitory activity against uPA, stability of SDS-resistant complexes with uPA, and vitronectin binding. Radiolabelled mutant uPA.PAI-1 complexes bound with a 10- to 20-fold, and 3- to 7-fold reduced affinity to purified alpha2MR/LRP and VLDLR respectively. alpha2MR/LRP-mediated endocytosis of the mutant complexes by COS-1 cells was reduced to 48 and 38% of the level of endocytosis of wild-type PAI-1. Binding of the mutant complexes to the uPA receptor was not affected. These findings suggest that the binding mode of the uPA.PAI-1 complex to both alpha2MR/LRP and VLDLR is similar. The four residues are surface exposed in the region defined by alpha-helix D and beta-strand 1A in the serine protease inhibitor (serpin) structure. Our study represents the first identification of residues in a surface region implicated in molecular recognition of protease.serpin complexes by endocytosis receptors of the low-density lipoprotein receptor family.

The urokinase-type plasminogen activator system in cancer metastasis: a review.

The urokinase-type plasminogen activator (u-PA) system consists of the serine proteinases plasmin and u-PA; the serpin inhibitors alpha2-anti-plasmin, PAI-1 and PAI-2; and the u-PA receptor (u-PAR). Two lines of evidence have strongly suggested an important and apparently causal role for the u-PA system in cancer metastasis: results from experimental model systems with animal tumor metastasis and the finding that high levels of u-PA, PAI-1 and u-PAR in many tumor types predict poor patient prognosis. We discuss here recent observations related to the molecular and cellular mechanisms underlying this role of the u-PA system. Many findings suggest that the system does not support tumor metastasis by the unrestricted enzyme activity of u-PA and plasmin. Rather, pericellular molecular and functional interactions between u-PA, u-PAR, PAI-1, extracellular matrix proteins, integrins, endocytosis receptors and growth factors appear to allow temporal and spatial re-organizations of the system during cell migration and a selective degradation of extracellular matrix proteins during invasion. Differential expression of components of the system by cancer and non-cancer cells, regulated by paracrine mechanisms, appear to determine the involvement of the system in cancer cell-directed tissue remodeling. A detailed knowledge of these processes is necessary for utilization of the therapeutic potential of interfering with the action of the system in cancers.

Plasminogen activator inhibitor-1 represses integrin- and vitronectin-mediated cell migration independently of its function as an inhibitor of plasminogen activation.

Cell migration involves the integrins, their extracellular matrix ligands, and pericellular proteolytic enzyme systems. We have studied the role of plasminogen activator inhibitor-1 (PAI-1) in cell migration, using human amnion WISH cells and human epidermoid carcinoma HEp-2 cells in an assay measuring migration from microcarrier beads and a modified Boyden-chamber assay. Active, but not latent or reactive center-cleaved, PAI-1 inhibited migration. A PAI-1 mutant without ability to inhibit plasminogen activation was as active as wild-type PAI-1 as a migration inhibitor, showing that inhibition of plasminogen activation was not involved. PAI-1 specifically interfered with intergrin- and vitronectin-mediated migration: Migration onto vitronectin-coated but not onto fibronectin-coated surfaces was inhibited by PAI-1, a cyclic RGD peptide inhibited migration, and both cell lines expressed vitronectin-binding alpha v-integrins. In addition, active PAI-1, but not latent or reactive center-cleaved PAI-1, inhibited vitronectin binding to integrins in an in vitro binding assay, without affecting binding of fibronectin. Monoclonal antibodies against the urokinase receptor, another vitronectin binding protein, did not affect cell migration in the beads assay, while some inhibitory effect was observed in the Boyden-chamber assay. We conclude that PAI-1, independently of its role as a proteinase inhibitor, inhibits cell migration by competing for vitronectin binding to integrins, while the interference of PAI-1 with binding of vitronectin to the urokinase receptor may play a secondary role. These data define a novel function for the serpin PAI-1, enabling it to regulate cell migration over vitronectin-rich extracellular matrix in the body.

Conformational changes of the reactive-centre loop and beta-strand 5A accompany temperature-dependent inhibitor-substrate transition of plasminogen-activator inhibitor 1.

We have studied conformational changes of type-1 plasminogen-activator inhibitor (PAI-1) during a temperature-dependent inhibitor-substrate transition by measuring susceptibility of the molecule to non-target proteinases. When incubated at 0 degree C instead of the normally used 37 degrees C, a tenfold decrease in the specific inhibitory activity of active PAI-1 was observed. Accordingly, PAI-1 was recovered in a reactive-centre-cleaved form from incubations with urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) at 0 degree C, but not at 37 degrees C. It thus behaved as a substrate for the target proteinases at the lower temperature. Active PAI-1 was exposed to a variety of non-target proteinases, including elastase, papain, thermolysin, trypsin, and V8 proteinase. It was found that specific peptide bonds in the reactive centre loop (RCL) and strand 5 in beta-sheet A (s5A) had a temperature-dependent proteolytic susceptibility, while the P17-P16 (E332-S333) bond, forming the hinge between s5A and the RCL, showed indistinguishable susceptibility to proteolysis by V8 proteinase at 0 degree and 37 degrees C. In latent and reactive-centre-cleaved PAI-1, all the bonds were resistant to proteolysis at the higher as well as the lower temperature. An anti-PAI-1 monoclonal antibody maintained the inhibitory activity of PAI-1 and prevented reactive centre cleavage at 0 degree C, and thus prevented substrate behaviour. Concomitantly, it caused specific changes in proteolytic susceptibility of s5A and the RCL, but it did not affect cleavage of the P17-P16 bond by V8 proteinase. Our observations suggest that temperature-dependent conformational changes of beta-sheet A and the RCL determine whether the serpin act as an inhibitor or a substrate. Furthermore they suggest that the RCL of PAI-1 is fully extracted from beta-sheet A in the inhibitory as well as in the substrate form, favoring a so-called induced conformational state model to explain why inhibitory activity requires partial insertion of the RCL into beta-sheet A.

Very low density lipoprotein receptor binds and mediates endocytosis of urokinase-type plasminogen activator-type-1 plasminogen activator inhibitor complex.

Very low density lipoprotein receptor (VLDL-R) was found to be expressed in bovine mammary gland and the human breast carcinoma cell line MCF-7 as an M(r) 105,000 variant, and in Chinese hamster ovary (CHO) cells transfected with human VLDL-R cDNA as an M(r) 130,000 variant. The receptor was purified by ligand affinity chromatography with immobilized M(r) 40,000 receptor-associated protein (RAP). The purified receptor was found to bind urokinase-type plasminogen activator-type-1 plasminogen activator inhibitor complex (u-PA.PAI-1), while there was no or very weak binding of active site blocked u-PA (DFP-u-PA), PAI-1 or u-PA-type-2 plasminogen activator inhibitor complex. The binding of u-PA.PAI-1 was blocked by RAP. The transfected CHO cells had an efficient, RAP-sensitive endocytosis of u-PA.PAI-1, severalfold higher than non-transfected parental CHO cells. u-PA.PAI-1 endocytosis was partially inhibited by DFP-u-PA, which blocks binding of the complex to the u-PA receptor. RAP and DFP-u-PA sensitive u-PA.PAI-1 endocytosis was also observed in MCF-7 cells, which were without detectable levels of other RAP-binding endocytosis receptors. These results show that VLDL-R represents a novel endocytosis mechanism for u-PA receptor-bound u-PA.PAI-1.

Differential regulation of urokinase-type-1 inhibitor complex endocytosis by phorbol esters in different cell lines is associated with differential regulation of alpha 2-macroglobulin receptor and urokinase receptor expression.

Complex between urokinase and its type-1 inhibitor (uPA-PAI-1) may, when bound to the urokinase receptor (uPAR), be endocytosed by an ensuing binding of the complex to the multiligand receptors alpha (2)-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) and glycoprotein 330 (gp330). We have found that phorbol esters regulate endocytosis of uPA-PAI-1 differently in different cell lines. In COS-1 cells, expressing uPAR and high levels of alpha 2MR/LRP under basal conditions, phorbol esters cause a time-dependent decrease in endocytosis concomitantly with a parallel down-regulation of alpha 2MR/LRP expression. An up-regulation of uPAR expression was also observed. General endocytosis via the clathrin-coated pit pathway was not affected by PMA treatment, as judged from measurements of transferrin endocytosis. In LLC-PK1 cells, expressing alpha 2MR/LRP but not uPAR under basal conditions, phorbol esters transiently increase endocytosis in parallel with a transient induction of uPAR expression, while there was virtually no change in alpha 2MR/LRP expression. Differential regulation of endocytosis therefore seems to be caused by differential regulation of the receptors, with either the alpha 2MR/LRP-level (in COS)-1 cells) or the uPAR-level (in LLC-PK1 cells) being rate-limiting.

Very low density lipoprotein receptor from mammary gland and mammary epithelial cell lines binds and mediates endocytosis of M(r) 40,000 receptor associated protein.

We here report that the M(r) 40,000 receptor associated protein (RAP), previously found to bind to alpha 2-macroglobulin receptor/low density lipoprotein receptor related protein (alpha 2MR/LRP) and glycoprotein 330 (gp330), binds to an M(r) 105,000 membrane protein from bovine mammary gland, human mamma tumors and mammary epithelial cell lines. We have purified this protein from bovine and human sources. N-terminal amino acid sequencing and immunoblotting analyses showed that the protein was identical or closely related to very low density lipoprotein receptor (VLDL-R). Experiments with the human mamma carcinoma cell line MCF-7 showed that this receptor was able to mediate an efficient endocytosis of RAP. These novel findings strongly suggest that RAP functions as a modulator of ligand binding to VLDL-R, similarly to alpha 2MR/LRP and gp330.

Regions involved in binding of urokinase-type-1 inhibitor complex and pro-urokinase to the endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Evidence that the urokinase receptor protects pro-urokinase against binding to the endocytic receptor.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) binds several ligands, including complex between the two chain urokinase-type plasminogen activator (uPA) and type-1 plasminogen activator inhibitor (PAI-1), and the single chain zymogen pro-urokinase (pro-uPA). We have used truncated variants of uPA and PAI-1 as well as Fab fragments of monoclonal antibodies with known epitopes to identify regions in the uPA.PAI-1 complex and in pro-uPA involved in binding to alpha 2MR/LRP.uPA.PAI-1 complex bound with high affinity (EC50 about 0.4 nM) via contacts in the PAI-1 moiety as well as the uPA serine proteinase domain and the uPAA chain. Pro-uPA bound with lower affinity (EC50 about 10 nM), and efficient binding to alpha 2MR/LRP was dependent on contact with both the A chain and the serine proteinase domain. We analyzed the effect of complex formation with the urokinase receptor since this is the primary target for binding of uPA.PAI-1 and pro-uPA at the cell surface, and since it has been demonstrated that urokinase receptor-bound uPA.PAI-1 complex is internalized following interaction with alpha 2 MR/LRP (Nykjaer, A., Petersen, C. M., Møller, B., Jensen, P.H., Moestrup, S.K., Holtet, T.L., Etzerodt M., Thøgersen, H.C., Munch, M., Andreasen, P.A., and Gliemann, J. (1992) J. Biol. Chem. 267, 14543-14546). Soluble recombinant urokinase receptor blocked the binding of pro-uPA to alpha 2MR/LRP but caused only a slight reduction in the affinity for binding of uPA.PAI-1. Moreover, pro-uPA bound to the urokinase receptor at the cell surface was not internalized and degraded unless activated to uPA and complexed with PAI-1. We conclude that pro-uPA is protected against degradation via alpha 2MR/LRP when bound to uPAR due to shielding of a binding contact in the A chain, whereas the affinity of uPAR-bound uPA.PAI-1 complex for binding to alpha 2MR/LRP remains sufficient to allow rapid internalization and degradation.

Receptor-mediated endocytosis of plasminogen activators and activator/inhibitor complexes.

Recent findings have elucidated the mechanism for clearance from the extracellular space of the two types of plasminogen activators, urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA), and their type-1 inhibitor (PAI-1). Activator/PAI-1 complexes and uncomplexed t-PA bind to the multi-ligand receptors alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR) and epithelial glycoprotein 330 (gp330). These receptors mediate endocytosis and degradation of u-PA/PAI-1 complex bound to the glycosyl phosphatidyl inositol-anchored urokinase receptor (u-PAR) on cell surfaces, and participate, in cooperation with other receptors, in hepatic clearance of activator/PAI-1 complexes and uncomplexed t-PA from blood plasma. The alpha 2MR- and gp330-mediated endocytosis of a ligand (u-PA/PAI-1 complex) initially bound to another receptor (u-PAR) is a novel kind of interaction between membrane receptors. Binding to alpha 2MR and gp330 is a novel kind of molecular recognition of serine proteinases and serpins.