Interaction of single-chain urokinase with its receptor induces the appearance and disappearance of binding epitopes within the resultant complex for other cell surface proteins

Abstract

Binding of urokinase-type plasminogen activator (uPA) to its glycosylphosphatidylinositol-anchored receptor (uPAR) initiates signal transduction, adhesion, and migration in certain cell types. To determine whether some of these activities may be mediated by associations between the uPA/uPAR complex and other cell surface proteins, we studied the binding of complexes composed of recombinant, soluble uPA receptor (suPAR) and single chain uPA (scuPA) to a cell line (LM-TK- fibroblasts) that does not express glycosylphosphatidylinositol (GPI)-anchored proteins to eliminate potential competition by endogenous uPA receptors. scuPA induced the binding of suPAR to LM-TK- cells. Binding of labeled suPAR/scuPA was inhibited by unlabeled complex, but not by scuPA or suPAR added separately, indicating cellular binding sites had been formed that are not present in either component. Binding of the complex was inhibited by low molecular weight uPA (LMW-uPA) indicating exposure of an epitope found normally in the isolated B chain of two chain uPA (tcuPA), but hidden in soluble scuPA. Binding of LMW-uPA was independent of its catalytic site and was associated with retention of its enzymatic activity. Additional cell binding epitopes were generated within suPAR itself by the aminoterminal fragment of scuPA, which itself does not bind to LM-TK- cells. When scuPA bound to suPAR, a binding site for alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2 MR/LRP) was lost, while binding sites for cell-associated vitronectin and thrombospondin were induced. In accord with this, the internalization and degradation of cell-associated tcuPA and tcuPA-PAI- 1 complexes proceeded less efficiently in the presence of suPAR. Further, little degradation of suPAR was detected, suggesting that cell- bound complex dissociated during the initial stages of endocytosis. Thus, the interaction of scuPA with its receptor causes multiple functional changes within the complex including the dis-appearance of an epitope in scuPA involved in its clearance from the cell surface and the generation of novel epitopes that promote its binding to proteins involved in cell adhesion and signal transduction.

Vitronectin Concentrates Proteolytic Activity on the Cell Surface and Extracellular Matrix by Trapping Soluble Urokinase Receptor-Urokinase Complexes

“…The interaction between uPA/suPAR and VN was further investigated in LM-TK Ϫ cells, a mouse fibroblastic cell line that has been shown in earlier studies to be negative for uPAR surface expression. 36 125 I-suPAR did not bind to LM-TK Ϫ cells, and also 125 I-Gly158scuPA alone showed very little specific binding. In contrast, the uPA/suPAR-complex bound specifically to these cells, as the binding of 125 I-suPAR was substantially induced after coaddition of Gly158scuPA (Fig 4A).…”

“…20 Hence, we propose that the binding of the complex to LM-TK Ϫ cells is mediated predominantly by VN, whereas in a previous report, partial interaction was also attributed to thrombospondin-1. 36 The plasminogen activator potential on the cell surface or the matrix of cells was drastically enhanced by the uPA/suPAR-complex and could be inhibited by MoAb-13H1 or active PAI-1. These observations underline the central role of VN also as adaptor component for the control of pericellular proteolysis, which is relevant for tissue remodelling.…”

Urokinase-derived peptides regulate vascular smooth muscle contraction in vitro and in vivo

“…Signal transduction through the urokinase receptor has been implicated in cell differentiation, adhesion, and migration (31,59). Urokinase has been proposed to induce a conformational change in uPAR that promotes its interaction with vitronectin (52,60,61), complements receptor 3 (62), and modulates ␤ 2 -integrin function through intracellular signaling (63,64). However, the determinants in urokinase and within the uPA receptor that promote signal transduction remain incompletely defined.…”

“…The effect of Å6 on the plasminogen activator activity of scuPA/suPAR complexes was performed as described (52). Briefly, equimolar complexes between scuPA and suPAR were generated at 50-fold final concentration.…”

Proteolysis is the most fundamental property of malignancy and its inhibition may be used therapeutically (Review)

“…HMW-uPA can be cleaved at the A-chain into a short chain A (A1), forming low molecular weight uPA (LMW-uPA) and an amino-terminal fragment. LMW-uPA is able to activate plasminogen, however, it does not bind to the uPA receptor (uPAR) ( 61 , 62 ). A C/T single-nucleotide polymorphism (SNP) in codon 141 of urokinase (known as P141L) may be associated with late onset Alzheimer’s disease, decreased affinity for fibrin-binding ( 63 ) and reduced risk of Helicobacter pylori infection ( 64 ).…”