Integrin α_Mβ₂ binds HMW-tc-uPA and uPAR enhances the interaction.

(A) Adhesion to uPA. HEK293 cells (2 × 10⁵cells/well) expressing uPAR or β₂-integrin receptors or both were seeded onto wells of microtiter plates coated with HMW-sc-uPA, HMW-tc-uPA, DIP–HMW-tc-uPA (HMW-tc-uPA was treated with 5 mM DFP for 2 hours at room temperature and extensively dialyzed against PBS), or BSA. Cells were allowed to adhere for 30 minutes at 37°C. After several washings, the number of adherent cells was quantified using the Cyquant Cell Proliferation Kit as described in “Materials and methods.” The data are means ± SEM of quadruple measurements from 3 independent experiments. (B) Direct binding of tc-uPA. HEK α_Mβ₂ and mock cells were incubated in DMEM/F-12 medium/1 mM Mg²⁺/0.1% BSA with 200 nM Alexa 488–HMW-tc-uPA for 0 to 4 hours at 37°C. Cell-bound uPA was detected by FACS. (C) Migration to uPA. HEK293 cells were added (2 × 10⁵ cells/well) to the upper chamber of transwells in a serum-free DMEM/F-12 medium. HMW-sc-uPA or HMW-tc-uPA was added to the lower chamber (100 nM). The cells were allowed to migrate for 6 hours in a humidified incubator at 37°C and 5% CO₂. The number of migrated cells was determined as described in “Materials and methods.” The data are expressed as means ± SEM of duplicate wells from 3 independent experiments.