Urokinase regulates vitronectin binding by controlling urokinase receptor oligomerization.
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-uPA.suPAR complex contains two molecules of suPAR. In co-immunoprecipitation experiments, using different epitope-tagged 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 glycosylphosphatidylinositol-anchored receptors in general, and for the biology of the uPA/uPAR system in particular.
Sidenius
Nicolai
N
Molecular Genetics Unit, DIBIT, Università Vita-Salute San Raffaele, 20132 Milan, Italy. sidenius.nicolai@hsr.it
Andolfo
Annapaola
A
Fesce
Riccardo
R
Blasi
Francesco
F
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D021122:Protein Subunits; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000302:isolation & purification; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D020298:U937 Cells; D014568:Urokinase-Type Plasminogen Activator / Q000302:isolation & purification / Q000378:metabolism*; D019096:Vitronectin / Q000302:isolation & purification / Q000378:metabolism*
D004792:Enzyme Precursors; C524898:PLAUR protein, human; D021122:Protein Subunits; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D019096:Vitronectin; D014568:Urokinase-Type Plasminogen Activator