The vitronectin binding area of plasminogen activator inhibitor‐1, mapped by mutagenesis and protection against an inactivating organochemical ligand

Tumor overexpression of urokinase-type plasminogen activator (uPA) and its specific inhibitor SerpinE1 (plasminogen activator inhibitor type-1) correlates with poor prognosis and increased metastatic potential. Conversely, tumor expression of uPA and another specific inhibitor, SerpinB2 (plasminogen activator inhibitor type-2), are associated with favorable outcome and relapse-free survival. It is not known how overexpression of these uPA inhibitors results in such disparate outcomes. A possible explanation may be related to the presence of a proposed low density lipoprotein receptor (LDLR)-binding motif in SerpinE1 responsible for mitogenic signaling via ERK that is absent in SerpinB2. We now show that complementation of such a LDLR-binding motif in SerpinB2 by mutagenesis of two key residues enabled high affinity binding to very LDLR (VLDLR). Furthermore, the VLDLR-binding SerpinB2 form behaved in a manner indistinguishable from SerpinE1 in terms of enhanced uPA-SerpinB2 complex endocytosis and subsequent ERK phosphorylation and cell proliferation; that is, the introduction of the LDLR-binding motif to SerpinB2 was necessary and sufficient to allow it to acquire characteristics of SerpinE1 associated with malignancy. In conclusion, this study defines the structural elements underlying the distinct interactions of SerpinE1 versus SerpinB2 with endocytic receptors and how differential VLDLR binding impacts on downstream cellular behavior. This has clear relevance to understanding the paradoxical disease outcomes associated with overexpression of these serpins in cancer.SerpinE1 (plasminogen activator inhibitor type-1, PAI-1) 4 and SerpinB2 (plasminogen activator inhibitor type-2, PAI-2) are both efficient inhibitors of the urokinase-type plasminogen activator (uPA), a key enzyme in the tissue remodeling process. Combined tumor overexpression of uPA, its receptor (uPAR), and SerpinE1 (serine protease inhibitor E1) is strongly correlated with poor patient prognosis (1-3) and metastatic potential (4 -8). Consequently, uPA and SerpinE1 expression is recommended as an independent prognostic indicator in node-negative breast cancer (9). On the contrary, tumor expression of SerpinB2 is correlated with increased relapse-free survival in breast cancer (and possibly other cancer types) (10). Further, the relationship between outcome and high SerpinB2 levels in node-negative breast cancer is only relevant if uPA levels are also elevated (1, 11), suggesting that the inhibitory relationship between uPA and SerpinB2 mediates this functionality.Binding of uPA to uPAR has been reported to induce the activation of numerous cell type-specific signaling pathways, such as MAPK/ERK, JAK/STAT, Src, focal adhesion kinase, and Rac, thereby affecting cell adhesion, migration, differentiation, and apoptosis (12). Because uPAR is not a transmembrane protein, being linked to the cell surface by a glycosylphosphatidylinositol anchor, uPA/uPAR signaling is mediated by integrins and several other adaptor molecules, including members of the lo...

Section: Discussionmentioning

confidence: 83%

Dependence on Endocytic Receptor Binding via a Minimal Binding Motif Underlies the Differential Prognostic Profiles of SerpinE1 and SerpinB2 in Cancer

Cochran

Croucher

Lobov

et al. 2011

“…A recombinant form of vitronectin lacking the somatomedin B domain (r⌬sBVN) was expressed in the baculovirus FastBac system and purified as described (32). PAI-1 was produced according to the method outlined by Jensen et al (21). The numbering used to denote the amino acid positions in PAI-1 throughout the present work is Ser-1-Ala-2-Val-3-His-4 -His-5, according to Andreasen et al (35).…”

Section: Methodsmentioning

confidence: 99%

“…The binding site for the SMB domain on PAI-1 has been well defined by x-ray crystallography (15, 31) and site-directed mutagenesis (19,21). Because the mutants identified in this study (K82E, R117E/ R120E, R103A/M112A/Q125A, K71A/R78A, Y81A/K82A, and W177F) interact in the latent form at a site that is separate from the recognition sequences for the SMB domain, it was important to complete our analysis and test for any effects of these mutagenized forms of PAI-1 in their active conformation on binding to full-length vitronectin, which contains the SMB domain.…”

Section: Comparison Of the Binding Of Active And Latent Forms Of Pai-mentioning

confidence: 99%

Characterization of a Site on PAI-1 That Binds to Vitronectin Outside of the Somatomedin B Domain

Schar

Jensen

Christensen

et al. 2008

Vitronectin and plasminogen activator inhibitor-1 (PAI-1) are proteins that interact in the circulatory system and pericellular region to regulate fibrinolysis, cell adhesion, and migration. The interactions between the two proteins have been attributed primarily to binding of the somatomedin B (SMB) domain, which comprises the N-terminal 44 residues of vitronectin, to the flexible joint region of PAI-1, including residues Arg-103, Met-112, and Gln-125 of PAI-1. A strategy for deletion mutagenesis that removes the SMB domain demonstrates that this mutant form of vitronectin retains PAI-1 binding (Schar, C. R., Blouse, G. E., Minor, K. M., and Peterson, C. B. (2008) J. Biol. Chem. 283, 10297-10309). In the current study, the complementary binding site on PAI-1 was mapped by testing for the ability of a battery of PAI-1 mutants to bind to the engineered vitronectin lacking the SMB domain. This approach identified a second, separate site for interaction between vitronectin and PAI-1. The binding of PAI-1 to this site was defined by a set of mutations in PAI-1 distinct from the mutations that disrupt binding to the SMB domain. Using the mutations in PAI-1 to map the second site suggested interactions between ␣-helices D and E in PAI-1 and a site in vitronectin outside of the SMB domain. The affinity of this second interaction exhibited a K D value ϳ100-fold higher than that of the PAI-1-somatomedin B interaction. In contrast to the PAI-1-somatomedin B binding, the second interaction had almost the same affinity for active and latent PAI-1. We hypothesize that, together, the two sites form an extended binding area that may promote assembly of higher order vitronectin-PAI-1 complexes.Vitronectin is a circulatory protein that interacts with several other plasma components that regulate coagulation and fibrinolysis, cell lysis via the complement cascade, and cell binding and migration in an integrin-dependent and urokinase-type plasminogen activator receptor-dependent fashion (1, 2). An interaction with vitronectin that has been widely studied occurs with the serine protease inhibitor (or serpin), PAI-1, 2 the primary inhibitor of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Most circulating PAI-1 is found in a complex with vitronectin (3). The formation of the complex between vitronectin and PAI-1 is presumed to be physiologically significant in several respects. Binding of vitronectin to PAI-1 stabilizes the active conformation of the serpin, increasing the half-life for its conversion to the inactive, so-called latent state, and maintains its protease inhibitory properties for a prolonged period (4, 5). PAI-1 binds to the N-terminal ϳ50-amino acid somatomedin B (SMB) domain of vitronectin. The binding of PAI-1 competes with the binding of the urokinase-type plasminogen activator receptor to this domain and with the binding of integrins to the adjacent RGD sequence (for a review, see Ref. 6). Moreover, PAI-1 has been reported to induce oligomerization of vitronectin (7-10).Th...

“…Multimeric vitronectin was prepared by denaturation of the protein in 8 M urea for 2 h at room temperature followed by dialysis into PBS (140 mM NaCl, 3 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4). Recombinant human wild-type PAI-1 and recombinant stable PAI-1 mutant, 14-1B (38), used in the ELISA assays were both purchased from Molecular Innovations, Inc. PAI-1 used in the Biacore experiments and latency transition assays was produced according to the method outlined in Jensen et al (28). The PAI-1 protein preparations were tested for activity using a urokinase inhibition assay; all behaved similarly and fully inhibited urokinase.…”

Section: Methodsmentioning

confidence: 99%

“…Conversely, the complementary vitronectin binding site within PAI-1 has been thoroughly investigated. A region between helices D, E, and F in PAI-1 termed the flexible joint region was proposed to comprise the primary binding site for vitronectin by Lawrence et al (27) in 1994 and was finally fully mapped to this region by Jensen et al (28) in 2002. The epitope was later confirmed by x-ray crystallography studies of the PAI-1-somatomedin B complex (9).…”

mentioning

confidence: 99%

A Deletion Mutant of Vitronectin Lacking the Somatomedin B Domain Exhibits Residual Plasminogen Activator Inhibitor-1-binding Activity

Schar

Blouse

Minor

et al. 2008

Vitronectin and plasminogen activator inhibitor-1 (PAI-1) are important physiological binding partners that work in concert to regulate cellular adhesion, migration, and fibrinolysis. The high affinity binding site for PAI-1 is located within the N-terminal somatomedin B domain of vitronectin; however, several studies have suggested a second PAI-1-binding site within vitronectin. To investigate this secondary site, a vitronectin mutant lacking the somatomedin B domain (r⌬sBVN) was engineered. The short deletion had no effect on heparin-binding, integrin-binding, or cellular adhesion. Binding to the urokinase receptor was completely abolished while PAI-1 binding was still observed, albeit with a lower affinity. Analytical ultracentrifugation on the PAI-1-vitronectin complex demonstrated that increasing NaCl concentration favors 1:1 versus 2:1 PAI-1-vitronectin complexes and hampers formation of higher order complexes, pointing to the contribution of charge-charge interactions for PAI-1 binding to the second site. Furthermore, fluorescence resonance energy transfer between differentially labeled PAI-1 molecules confirmed that two independent molecules of PAI-1 are capable of binding to vitronectin. These results support a model for the assembly of higher order PAI-1-vitronectin complexes via two distinct binding sites in both proteins.