Analysis of integrin receptors for laminin and type IV collagen on metastatic B16 melanoma cells.
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 eluted with EDTA that contained a beta 1 chain at Mr 120,000 and an 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-glycyl-aspartate-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.
Ramos
D M
DM
Department of Stomatology, University of California, San Francisco 94143.
Berston
E D
ED
Kramer
R H
RH
D000818:Animals; D002448:Cell Adhesion; D002846:Chromatography, Affinity; D003094:Collagen / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D016023:Integrins / Q000378:metabolism*; D007526:Isoelectric Point; D007797:Laminin / Q000378:metabolism*; D008546:Melanoma, Experimental / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008970:Molecular Weight; D009362:Neoplasm Metastasis; D011233:Precipitin Tests; D011956:Receptors, Cell Surface / Q000378:metabolism; D038981:Receptors, Collagen; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic / Q000378:metabolism; D017474:Receptors, Laminin
D016023:Integrins; D007797:Laminin; D011956:Receptors, Cell Surface; D038981:Receptors, Collagen; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic; D017474:Receptors, Laminin; D003094:Collagen