Kinetic Analysis of the Interaction between Vitronectin and the Urokinase Receptor

Yuushi Okumura,Yuichi Kamikubo,Scott A Curriden,Jieyi Wang,Tatsuto Kiwada,Shiroh Futaki,Kouki Kitagawa,David J Loskutoff

doi:10.1074/jbc.m111225200

Abstract

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.

Highlights

The urokinase receptor binds to vitronectin (VN) and promotes the adhesion of cells to this matrix protein, the biochemical details of this interaction remain unclear
Our results indicate that the somatomedin B (SMB) domain is the only high affinity binding site for uPAR and PAI-1 in VN, and that the binding site for monoclonal antibodies (mAbs) 8E6 is distinct from the binding sites for uPAR and PAI-1
We directly compared the ability of uPAR, PAI-1, plasminogen, and heparin to bind to VN fragments lacking either the SMB domain or the heparin binding (HB) domain

Summary

Introduction

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. Monoclonal antibody 8E6 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. A third region in VN (residues 115–121) was shown to have weak PAI-1 binding activity [29], the relative importance of this region for PAI-1 and uPAR binding remains to be established Another potential problem for the interpretation of VN binding studies concerns the molecular forms of VN employed (i.e. native closed form versus the more opened denatured form). We constructed and purified several recombinant VN variants, and developed a series of mAbs against VN We employed these molecules in real-time biomolecular interaction analysis experiments (BIAcore) to more precisely identify the uPAR and PAI-1 binding sites in denatured VN, and to determine the kinetic constants for their interactions.

Methods

Results

Conclusion