We have shown previously that the urokinase-type plasminogen activator receptor (uPAR) physically associates with β2 integrins on human leukocyte membranes. We now report that uPAR associates with certain members of the β1 and β3 integrin families expressed by a nonhematopoietic fibrosarcoma cell line (HT1080) when adherent to certain extracellular matrix molecules. Flow cytometry studies indicated that HT1080 cells expressed uPAR and β1 and β3 integrins. Double staining immunofluorescence was used to label uPAR and β1 and β3 integrins. The staining patterns of uPAR and β1 integrins were strikingly similar when attached to fibronectin, laminin, or vitronectin but not polylysine-coated substrates. Resonance energy transfer (RET) between uPAR and β1 integrins was observed, especially at focal adhesion plaques; this indicates that these molecules are within about 7 nm of each other on these cell membranes. uPAR and β3 integrin coclustering and RET were also observed on tumor cells adherent to vitronectin but not to fibronectin, laminin, or polylysine-coated surfaces. Because N-acetyl-d-glucosamine was found previously to inhibit β2 integrin-uPAR association, we tested the effect of saccharides on the β1-uPAR and β3-uPAR colocalization and RET. Colocalization and RET between uPAR and β1 or β3 integrins were effectively inhibited by N-acetyl-d-glucosamine on extracellular matrix-coated surfaces. To better define which members of β1 and β3 integrin families associate with uPAR, we studied the association of several α subunits with uPAR on tumor cells. We found that: (a) α5 colocalizes with uPAR on cells attached to fibronectin-coated surfaces; (b) α5 and αv colocalize with uPAR on cells adherent to vitronectin; and (c) α3 and α6 associate with uPAR on cells attached to laminin. In further support of physical associations between integrins and uPAR on tumor cells, uPAR was found to coimmunoprecipitate with β1 integrins in Brij-58 lysates of HT1080 cells (as detected by anti-uPAR Western blotting of material isolated from an anti-β1 integrin immunoaffinity column). Thus, uPAR may laterally associate with integrins of tumor cells when attached to specific extracellular matrix elements to enable directional proteolysis for tumor cell migration and invasion.

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This work was supported by NIH Grant AI/CA27409 and the Research Stimulation Fund of Wayne State University (to H. R. P.) and NIH Grants CA39064 and CA42246 (to R. F. T.).

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