Abstract
Human urokinase-type plasminogen activator (uPA) binds rapidly and with high affinity to a number of human cell types; this localizes plasmin generation to the close environment of the cell surface. uPA binding to HeLa and U937 cells is mediated by a single class of sites with an affinity of 3.4 +/- 1.3 x 10(-10) M. Binding is abolished by treatment of the cells with trypsin. Chemical cross-linking of Mr 55,000 125I-uPA to the surface of HeLa and U937 cells with disuccinimidyl suberate or with formaldehyde results in the formation of a labeled complex of Mr 100,000, suggesting a Mr of 45,000 +/- 5,000 for the receptor or a subunit thereof. When cells solubilized in Triton X-114 are subjected to heat-induced phase separation, unoccupied receptor, receptor-bound 125I-uPA, and cross-linked 125I-uPA-receptor complex all partition in the detergent phase, whereas the unbound ligand remains in the aqueous phase; similar phase partitioning is observed with endogenous uPA-receptor complexes from cultured human and murine cells. Thus, uPA bound at the cell surface is tightly associated with an amphiphilic membrane protein. Interaction of uPA with this plasma membrane receptor is species-specific, since human uPA fails to bind to murine cells, and murine uPA does not bind to human cells. Finally, incubation of HeLa cells in the presence of epidermal growth factor or phorbol 12-myristate 13-acetate results, over a period of 24 h, in a progressive change in uPA binding: an approximately 10-fold increase in the number of sites is accompanied by a 10-fold decrease in their affinity. Cross-linking and phase partitioning of 125I-uPA bound to epidermal growth factor- or phorbol 12-myristate 13-acetate-treated cells indicate that, as in control conditions, it is associated with a Mr 45,000 cell surface amphiphilic polypeptide.
Highlights
Sites with an affinity of 3.4 f 1.3 X 10”’ M
HeLa cells were incubated in the presenceof epidermal growth factor (EGF) (Fig. 9) or phorbol 12-myristate 13-acetate (PMA) for various periods of time, and specific binding of 12sI-uPA wasthen determined.A Scatchard analysis of these data revealed that after 24 h in the with synthetic peptides (Appella et al, 1987), the sequences of which correspond to thegrowth factor-like domain of the presence of EGF, the number of uPA-binding sites had increased about 10-fold; mouse (Belin et al, 1985) or human (Gunzler et al, 1982) their affinity was decreased 10-fold
Microscopic analysis revealed that proteolytic plaques formed around most of the cells incubated with uPA; the size of the plaques was much larger around EGF- and PMA-treated cells than around control cells, while the number of plaques was essentially the same in all cultures
Summary
U937 and HeLa cells were incubated with M , 55,000 lZ5I- ity, did not bind to HeLa cells, nor did it prevent binding of uPA and increasing concentrations of unlabeled uPA, and the the M , 55,000 enzyme (not shown); the catalytic B chain of cell-associated radioactivity was counted (Fig. 1). The eluted fraction was analyzed by SDS-PAGE cells(Fig. 2, A and B ) revealed the presence of a labeled and autoradiography Under both reducing (Fig. 3) and nonspeciesmigrating at an apparentM , of 100,000(panel A, lanes reducing (not shown) conditions, a single band was observed 4-7;panel B, lane 6 ) , which corresponds presumably to a with an apparent M , of 45,000. 100,000speciesobtained upon DSS-mediated cross-linking of 6 cell-bound lZ5I-uPAwas exclusively found in the detergent phase afterTriton X-114 phase separation (not shown). PMA, the amounotf receptor-bound uPAwas essentially the resultsarethemean (+ S.D.) of threedeterminations.Forboth complete sample and detergent phase, the difference between total and nonspecific binding was highly significant ( p < 0.001)
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