uPA:PAI-1-independent uPAR uptake does not require small GTPases activity.

<p>Panel A: constitutive uPAR uptake assessed by immunofluorescence of HEK293-uPAR cells transiently transfected with DNAs encoding GFP tagged mutants of RhoA, Rac1 and Cdc42. A monoclonal anti-uPAR antibody (R3) was bound to the cell surface, cells incubated for 30 minutes at 37°C. Cells were washed, fixed and processed for immunofluorescence. Images (a–b) show uPAR internalization in the presence of dominant negative RhoA, (c–d) the same in the presence of dominant negative Rac1-GFP. The bottom panel (e–f) represents uPAR internalization with dominant negative Cdc42-GFP. None of the mutants inhibited the constitutive endocytic route responsible for uPAR uptake. Data are representative of 2–4 different experiments. Scale bars 10 µm. Panel B: biotinylation assay of ligand-independent uPAR uptake in HEK293-uPAR cells transiently transfected with GFP-tagged dominant-negative mutants of RhoA, Rac-1 and Cdc42. After biotinylation, cells were incubated at 37°C for 15 minutes, lysed, immunoprecipitated with anti-rabbit uPAR antibodies and blotted with mouse monoclonal uPAR antibody or with HRP streptavidin. Western Blot analysis indicates that none of the small GTPase mutants used had a major inhibitory effect on uPAR constitutive uptake. Panel C shows the quantification of uPAR endocytosis by flow cytometry in cells transfected with GFP or small-GTPases-GFP constructs as indicated. Cells were incubated with a polyclonal anti uPAR antibody bound to PE-Neutravidin at 4°C, incubated at 37°C for 15 minutes and then analysed for internalized receptor in GFP expressing cells gating upon GFP-positive cells. These results indicate that small GTPases do not play a role in constitutive uPAR endocytosis.