FIGURE 2.

Cr-u-PAR supports u-PA-dependent Pg activation that cannot be down-regulated by receptor cleavage. A, specific binding of tcu-PA to u-PAR leads to Pn generation that can be blocked by preincubation with excess CMK·u-PA. Cells were first incubated with or without 100 nm of CMK·u-PA for 30 min at 37 °C followed by 10 nm of tcu-PA. Cell-surface u-PA activity was measured via a Pg activation assay as described under “Experimental Procedures.” B, proteolysis of u-PAR results in a decrease in cell-surface Pn generation. Cells expressing wt u-PAR (white bars) or cr-u-PAR (black bars) were subjected to limited proteolysis with 10 nm chymotrypsin or 100 nm tcu-PA, and residual intact u-PAR was detected by incubation with 10 nm of tcu-PA followed by a Pg activation assay. Data represent the mean and S.D. of three individual replicates.