Figure 4.

uPAR binding ability of phosphorylated and “phosphorylation-like” prouPA variants. (A) 30 × 10⁶ subconfluent A431 cells were metabolically labeled with [³²P]orthophosphate for 24 h, the conditioned medium was then removed, and receptor-bound pro-uPA was extracted by treating the cells with a total of 10 ml of acidic buffer for 5 min. Half of the neutralized acid wash was incubated with 5B4-agarose (lane 2) or glycine-blocked agarose (lane 1), and the resulting matrix-bound proteins were analyzed by 12.5% SDS-PAGE under reducing conditions. (B) Serine phosphorylated pro-uPA was purified from A431 cell line by the Fe³⁺ chromatography procedure, whereas the histidine-tagged proteins were purified by Ni-NTA chromatography from the conditioned medium of HeLa stable transfectants. The result of a competition between ¹²⁵I-ATF (10⁵ cpm/sample) and the indicated nanomolar concentrations of unlabeled urinary uPA (•), Pser-uPA (▴), His-pro-uPA^wt (□ ), His-pro-uPA^138E(▵), His-pro-uPA^138E/303E (○) to U937 cell uPARs is shown. Cell-bound radioactivity is reported as a percentage of the maximal binding in the absence of competitor (¹²⁵I-ATF specific binding to control cells in the absence of competitor, 3,000 dpm). Data are shown as the mean of three independent experiments performed in duplicate; standard deviations are indicated by error bars.