UROKINASE-TYPE PLASMINOGEN-ACTIVATOR RECEPTORS ASSOCIATE WITH BETA(1)AND BETA(3) INTEGRINS OF FIBROSARCOMA CELLS - DEPENDENCE ON EXTRACELLULAR-MATRIX COMPONENTS

We have shown previously that the urokinase-type plasminogen activator receptor (uPAR) physically associates with beta(2) integrins on human leukocyte membranes. We now report that uPAR associates with certain members of the beta(1) and beta(3) integrin families expressed by a no nhematopoietic fibrosarcoma cell line (HT1080) when adherent to certai n extracellular matrix molecules. Flow cytometry studies indicated tha t HT1080 cells expressed uPAR and beta(1) and beta(3) integrins. Doubl e staining immunofluorescence was used to label uPAR and beta(1) and b eta(3) integrins. The staining patterns of uPAR and beta(1) integrins were strikingly similar when attached to fibronectin, laminin, or vitr onectin but not polylysine-coated substrates. Resonance energy transfe r (RET) between uPAR and beta(1) integrins was observed, especially at focal adhesion plaques; this indicates that these molecules are withi n about 7 nm of each other on these cell membranes. uPAR and beta(3) i ntegrin coclustering and RET were also observed on tumor cells adheren t to vitronectin but not to fibronectin, laminin, or polylysine-coated surfaces. Because N-acetyl-D-glucosamine was found previously to inhi bit beta(2) integrin-uPAR association, we tested the effect of sacchar ides on the beta(1)-uPAR and beta(3)-uPAR colocalization and RET. Colo calization and RET between uPAR and beta(1) or beta(3) integrins were effectively inhibited by N-acetyl-D-glucosamine on extracellular matri x-coated surfaces. To better define which members of beta(1) and beta( 3) integrin families associate with uPAR, we studied the association o f several or subunits with uPAR on tumor cells. We found that: (a) alp ha(5) colocalizes with uPAR on cells attached to fibronectin-coated su rfaces; (b) alpha(5) and alpha(v) colocalize with uPAR on cells adhere nt to vitronectin; and (c) alpha(3) and alpha(6) associate with uPAR o n cells attached to laminin. In further support of physical associatio ns between integrins and uPAR on tumor cells, uPAR was found to coimmu noprecipitate with beta(1) integrins in Brij-58 lysates of HT1080 cell s (as detected by anti-uPAR Western blotting of material isolated from an anti-beta(1) integrin immunoaffinity column). Thus, uPAR may later ally associate with integrins of tumor cells when attached to specific extracellular matrix elements to enable directional proteolysis for t umor cell migration and invasion.