Dimerization controls the lipid raft partitioning of uPAR/CD87 and regulates its biological functions

Abstract

The urokinase-type plasminogen activator receptor (uPAR/CD87) is a glycosylphosphatidylinositol-anchored membrane protein with multiple functions in extracellular proteolysis, cell adhesion, cell migration and proliferation. We now report that cell surface uPAR dimerizes and that dimeric uPAR partitions preferentially to detergent-resistant lipid rafts. Dimerization of uPAR did not require raft partitioning as the lowering of membrane cholesterol failed to reduce dimerization and as a transmembrane uPAR chimera, which does not partition to lipid rafts, also dimerized efficiently. While uPA bound to uPAR independently of its membrane localization and dimerization status, uPA-induced uPAR cleavage was strongly accelerated in lipid rafts. In contrast to uPA, the binding of Vn occurred preferentially to raft- associated dimeric uPAR and was completely blocked by cholesterol depletion.

Fig. 1. uPAR partitions to two biochemically distinct membrane domains. (A) Western blot analysis of uPAR membrane localization. 293/uPAR cells were lysed in buffer containing 1% Triton X-100 and subjected to sucrose density gradient ultracentrifugation (see Materials and methods). Equal volumes of the resulting fractions (10 µl) were probed for uPAR using a polyclonal anti-uPAR antibody in immunoblotting. The same fractions were also probed with polyclonal antibodies to β1-integrin and the transferrin receptor, respectively. 293/uPAR cells were first treated with CD (10 mM, 1 h, 37°C) and then subjected to flotation analysis as described above. (B) 293/uPAR-TM-expressing cells were subjected to the same analysis. (C) 293/uPAR cells were detergent lysed in buffer containing 1% Brij-58 at 37°C and subjected to flotation analysis (see Materials and methods). The data presented are representative of several independent experiments.

Fig. 2. Dimeric cell surface uPAR revealed by chemical cross linking and co-immunoprecipitation. (A) Western blot analysis of uPAR dimerization by chemical cross linking. 293/uPAR cells were treated with the chemical cross linker BS³ as indicated below the panel, washed and lysed. Equal amounts of total protein were separated by SDS–PAGE and analyzed by immunoblotting using a polyclonal anti-uPAR antibody. Similar data were observed in several experiments. (B) Affinity purification of cross-linked uPAR. Lysates obtained from large-scale cross linking experiments carried out on 293/uPAR cells were loaded onto an anti-uPAR antibody affinity column. After washing, bound protein was eluted, concentrated and separated by SDS–PAGE. The slab gel was silver stained and the band corresponding to the uPAR adduct (marked by an asterisk) excised, subjected to trypsin digestion and analyzed by mass spectrometry (see Supplementary data). (C) Lysates from 293 cells expressing either wild-type uPAR (WT), FLAG-tagged uPAR (FLAG) or both (WT+FLAG) were immunoprecipitated with an anti-uPAR antibody (R4) which recognizes both wild-type and FLAG-tagged uPAR, or with an anti-FLAG antibody (M2) which recognizes only the FLAG-tagged receptor. The immunoprecipitated material was fractionated by SDS–PAGE and analyzed by immunblotting using an anti-uPAR antibody (R2) which recognizes only wild-type uPAR (upper panel). To ensure that appropriate immunoprecipitation had been achieved, the blots were also probed with a polyclonal uPAR antibody which recognizes all forms of the receptor (α-uPAR-PC, lower panel). Similar results were obtained in at least three independent experiments.

Fig. 3. Dimerization regulates uPAR partitioning to lipid rafts. (A) Differential raft partitioning of monomeric and dimeric uPAR. 293/uPAR cells were incubated in the presence (lower panel) or absence (upper panel) of CD (10 mM, 1 h, 37°C), treated with BS³ (0.5 mM, 30 min, 4°C), and analyzed by flotation and immunoblotting. (B) Flotation analysis of BS³-treated 293/uPAR cells lysed at 37°C in buffer containing 1% Brij-58. (C) Differential raft-partitioning of dimeric uPAR analyzed by co-immunoprecipitation. Flotation analysis was carried out on lysates of 293 cells expressing both wild-type and FLAG-tagged uPAR. Raft fractions (2, 3 and 4) and non-raft fractions (6, 7 and 8) were pooled and subjected to co-immunoprecipitation analysis as described in the legend to Figure 2C. The total uPAR present in each membrane compartment was visualized by immunoblotting with a polyclonal anti-uPAR antibody. Prior to immunoprecipitation, all fractions were equalized for detergent and sucrose concentration. The results presented are representative of two (C) or more (A and B) independent experiments.

Fig. 4. Dimerization of uPAR does not require its association with lipid rafts. (A) The effect of CD treatment on chemical cross linking. 293/uPAR cells were cholesterol depleted using CD and treated with BS³ as indicated below the panel, and the cell lysates analyzed by western blotting. (B) The effect of CD treatment on uPAR co-immunoprecipitation. 293 cells transfected with a combination of uPAR-WT and uPAR-FLAG were treated with CD as indicated and analyzed by co-immunoprecipitation as described in the legend to Figure 2C. (C) Chemical cross linking of uPAR-TM. 293/uPAR-TM cells were treated with chemical cross linker as indicated and cell lysates analyzed by western blotting as above.

Fig. 5. Lipid raft- and dimerization-independent binding of uPA to uPAR. (A) 293/uPAR cells were incubated with ¹²⁵I-labeled uPA (1 nM, filled bars) or RAP (3 nM, open bars). After removal of unbound reagents the cells were lysed in Triton X-100 buffer and subjected to flotation analysis. Fractions were collected and the radioactivity was determined by γ-counting. The level of radioactivity in each fraction is shown as percentage of the sum of radioactivity in all fractions. The data represents the mean ±SD of two independent binding/flotation experiments. The percentage of total radioactivity associated with the raft and non-raft fractions is indicated below each graph. (B) To determine the amount of ¹²⁵I-labeled uPA specifically associated with uPAR, raft and non-raft fractions were pooled and subjected to immunoprecipitation using a polyclonal antibody against uPAR (α-uPAR) or an irrelevant antibody (control). After washing, precipitated radioactivity was determined by γ-counting. (C) 293/uPAR cells were treated with BS³ (upper panel) or CD and BS³ (lower panel), as described previously, and subsequently to flotation analysis. Fractions were immunoprecipitated with biotinylated uPA and analysed by SDS–PAGE and immunoblotting using an anti-uPAR antibody.

Fig. 6. uPA-mediated uPAR cleavage is accelerated in lipid rafts. (A) 293/uPAR cells were treated for various times (0, 1, 5, 15, 60 and 120 min) at 37°C with 3 nM uPA in DMEM supplemented with 0.1% BSA. After the incubation, cells were moved to ice, washed in ice-cold PBS, lysed in Triton X-100 containing buffer, and subjected to flotation analysis as described above. uPAR and cleaved uPAR (D2D3) were identified by immunoblotting analysis using a polyclonal anti-uPAR antibody. (B) Flotation fractions corresponding to the raft and non-raft material were pooled, aliquots were reduced and deglycosylated, fractionated by SDS–PAGE and immunoblotted with a polyclonal anti-uPAR antibody. (C) The intensity of the bands corresponding to full-length uPAR in (B) was quantified by densitometry and plotted as a percentage of the intensity observed in cells not exposed to uPA (0 min). The data represents the mean value of two independent experiments. (D) Lysates of 293/uPAR cells exposed to uPA (3 nM) for different lengths of time were immunoprecipitated with M2 and blotted with R2 to reveal the co-immunoprecipitated uPAR, or with a polyclonal anti-uPAR antibody to reveal total uPAR.

Fig. 7. Dimeric uPAR directs the selective binding of Vn to lipid rafts. (A) 293/uPAR cells incubated with ¹²⁵I-labeled Vn were lysed and subjected to flotation analysis. The resulting fractions were assayed for radioactivity. The level of radioactivity in each fraction is shown as a percentage of the sum of radioactivity in all fractions. The data represents the mean ±SD of two independent binding/flotation experiments. The percentage of total radioactivity associated with the raft and non-raft fractions is indicated. (B) To determine the amount of ¹²⁵I-labeled Vn specifically associated with uPAR, the raft and non-raft fractions were pooled and subjected to immunoprecipitation using a polyclonal antibody against uPAR (α-uPAR) or an irrelevant antibody (ct. Ab). After washing, the radioactivity bound to the beads was determined by γ-counting. (C) Cells treated with BS³ (upper panel) or CD followed by BS³ (lower panel) were lysed, subjected to flotation analysis and aliquots of the resulting fractions were immunoprecipitated with biotinylated Vn(1–97). uPAR was visualized using an anti-uPAR polyclonal antibody. (D) Adhesion of 293/uPAR cells to Vn- or Fn-coated wells was allowed to proceed for 30 min at 37°C, with or without pre-treatment of cells with 10 mM CD for 1 h at 37°C (see Materials and methods). Adherent cells were quantified after fixing by staining with crystal violet. The data represent the mean ±SD of three independent experiments, each carried out in quadruplicate.