Analysis of integrin receptors for laminin and type IV collagen on metastatic B16 melanoma cells
Ramos, D.M.; Berston, E.D.; Kramer, R.H.
Cancer Research 50(3): 728-734
1990
ISSN/ISBN: 0008-5472 PMID: 2153445 Document Number: 356569
As tumor cells invade surrounding tissue, they adhere to various extracellular matrix components. Previously we reported that B16-BL6 melanoma cell adhesion to both basement membrane and purified protein substrates was blocked by antibody to .beta.1-integrin adhesion receptors (R. H. Kramer et al., Cancer Res., 49:393-402, 1989). In the present study we found using immunofluorescent staining, that .beta.1-integrin complexes were colocalized with vinculin in focal adhesion plaques on laminin, type IV collagen, and fibronectin substrates. To identify potential adhesion receptors on B16 cells, the cells were surface-labeled with 125I, solubilized with detergent, and chromatographed on laminin-, type IV collagen-, and fibronectin-Sepharose columns. On laminin-Sepharose, an integrin heterodimer complex was elucted with EDTA that contained a .beta.1 chain at Mr 120,000 and and .alpha. subunit at Mr 140,000 (nonreduced). This complex was specific for laminin and failed to bind to collagen- or fibronectin-Sepharose columns. Immunoprecipitation with specific monoclonal antibody identified this complex as .alpha.6.beta.1 (VLA-6). Furthermore, monoclonal antibody to the .alpha.6.beta.1 complex effectively blocked the attachment of B16-BL6 cells to laminin but did not affect adhesion to fibronectin or type IV collagen. We recovered a different integrin complex from type IV collagen-Sepharose columns that was composed of a .beta.1 chain and an .alpha. chain of Mr 180,000 (nonreduced). This same complex also exhibited a weak affinity for laminin-affinity chromatography. The laminin-binding complex and the type IV collagen-binding complex were clearly distinct from the fibronectin-binding receptor and were not eluted by arginyl-glycylaspartate-containing peptides. The results suggest that the B16 melanoma cells express multiple integrin-related receptors that appear to mediate cell adhesion to basement membrane matrices.