title: 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.
authors.affiliation: Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia 19104, USA.
authors.forename: A A
authors.initials: AA
authors.lastname: Higazi
authors.forename: R H
authors.initials: RH
authors.lastname: Upson
authors.forename: R L
authors.initials: RL
authors.lastname: Cohen
authors.forename: J
authors.initials: J
authors.lastname: Manuppello
authors.forename: J
authors.initials: J
authors.lastname: Bognacki
authors.forename: J
authors.initials: J
authors.lastname: Henkin
authors.forename: K R
authors.initials: KR
authors.lastname: McCrae
authors.forename: M Z
authors.initials: MZ
authors.lastname: Kounnas
authors.forename: D K
authors.initials: DK
authors.lastname: Strickland
authors.forename: K T
authors.initials: KT
authors.lastname: Preissner
authors.forename: J
authors.initials: J
authors.lastname: Lawler
authors.forename: D B
authors.initials: DB
authors.lastname: Cines
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D017261:Glycosylphosphatidylinositols / Q000172:deficiency / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology*; D011485:Protein Binding; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D012995:Solubility; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000378:metabolism
chemical_list: D000939:Epitopes; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D000511:alpha-Macroglobulins; D014568:Urokinase-Type Plasminogen Activator