Urokinase Regulates Vitronectin Binding by Controlling Urokinase Receptor Oligomerization

Abstract

Adhesion of monocytes to the extracellular matrix is mediated by a direct high affinity interaction between cell-surface urokinase-type plasminogen activator (uPA) receptor (uPAR) and the extracellular matrix protein vitronectin. We demonstrate a tight connection between uPA-regulated uPAR oligomerization and high affinity binding to immobilized vitronectin. We find that binding of soluble uPAR (suPAR) to immobilized vitronectin is strictly ligand-dependent with a linear relationship between the observed binding and the concentration of ligand added. Nevertheless, a comparison of experimentally obtained binding curves to those generated using a simple equilibrium model suggests that the high affinity vitronectin-binding pro-uPA⅐suPAR complex contains two molecules of suPAR. In co-immunoprecipitation experiments, using different epitopetagged suPAR molecules, suPAR/suPAR co-immunoprecipitation displayed a similar uPA dose dependence as that observed for vitronectin binding, demonstrating that the high affinity vitronectin-binding complex indeed contains oligomeric suPAR. Structurally, the kringle domain of uPA was found to be critical for the formation of the vitronectin-binding competent complex because the amino-terminal fragment, but not the growth factor-like domain, behaved as a full-length uPA. Our data represent the first demonstration of functional, ligand-induced uPAR oligomerization having extensive implications for glycosylphosphatidylinositolanchored receptors in general, and for the biology of the uPA/uPAR system in particular.Cell migration and invasion are important processes in many patho/physiological conditions such as tumor invasion, angiogenesis, and inflammation. Plasminogen activators, their inhibitors, and their cell-surface receptor(s) play central roles in these processes by regulating extracellular proteolysis, cell adhesion, and signal transduction. In tissues, extracellular proteolysis is controlled by the production of plasmin that is generated by plasminogen activators, mainly urokinase (uPA) 1 (1), which binds to a specific membrane receptor, uPAR.Fully processed human uPAR is a 45-55-kDa glycoprotein linked to the outer membrane leaflet by a glycosylphosphatidylinositol lipid anchor (2). The protein is composed of three homologous domains with a disulfide bonding pattern characteristic of the uPAR/Ly-6 superfamily (3).Besides providing the cells with the means to perform directed extracellular matrix degradation, binding of uPA to uPAR has profound effects on cell adhesion, migration, and proliferation (4 -6). Although binding to uPAR is always required, these latter processes are often independent of the proteolytic activity of uPA, strongly suggesting that other protein interactions are involved.Indeed, several data indicate that a conformational change in uPAR is capable of profoundly modifying its biological properties. First, it has been shown that uPA binding to uPAR causes the appearance of novel binding sites for vitronectin (Vn) (7-10), thrombospondin (8), uPAR-associated ...

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

“…uPAR Binding Assay-The binding of uPAR to recombinant vitronectin was measured as described by Sidenius et al (44) with slight modification. The wells of a microtiter plate were coated with multimeric vitronectin and recombinant vitronectin at a concentration of 15 nM in PBS.…”

Conformational Regulation of Urokinase Receptor Function

“…With a view to this proposition, it is noteworthy that ample evidence exists in the literature from different laboratories to suggest that uPA binding modulates the interaction between uPAR and vitronectin both at the biochemical (8,18,19) and the cellular level (9, 10, 20 -25). The molecular basis underlying this dependence is, however, still controversial, and several models, including uPAR dimerization (9,19,26), direct interactions with integrins (27)(28)(29), or other adaptor proteins (30,31) have been advocated.…”

“…The molecular basis underlying this dependence is, however, still controversial, and several models, including uPAR dimerization (9,19,26), direct interactions with integrins (27)(28)(29), or other adaptor proteins (30,31) have been advocated. In the present study, we have revisited this molecular interplay guided by the structural data obtained recently on this ternary complex (17,18), and we now present independent functional data pointing to a crucial role of the molecular flexibility in uPAR.…”

Soluble urokinase plasminogen activator receptor—A valuable biomarker in systemic lupus erythematosus?

“…This fragment has been demonstrated to take part in stem cell mobilization [52] and leukocyte trafficking [26,53,54]. Full-length suPAR seems to retain both uPA as well as vitronectin-binding capacity [55,56], but in contrast to cell-attached uPAR, fulllength suPAR cannot be cleaved by uPA [57]. Due to this discrepancy, is has been speculated that suPAR could possibly retain uPA and vitronectin from cell bound uPAR and thereby inhibit uPA-uPARvitronectin mediated proteolysis, cell adhesion and migration [2].…”