Detergent extracts of primary rat hepatocytes and neonatal cardiac fibroblasts -were applied to collagen type I-Sepharose in the presence of 1 mM MnCl2. Elution of bound proteins by 10 mM EDTA yielded one β1-integrin heterodimer from hepatocytes with an Mr of 180,000/115,000 under nonreducing conditions. Two β1-integrins with r's (nonreduced) of 180,000/ 115,000 and 145,000/115,000 could be isolated from surface-iodinated fibroblasts. A monoclonal antibody, 3A3, directed against the rat homolog of the human integrin VLA-1, precipitated the affinity-purified Mr 180,000/115,000 heterodimer, establishing the relatedness of the Mr 180,000 subunit to the α1-chain of the β1-integrin subfamily. Both the α1β1-integrin and the 145,000/β1-integrin heterodimers bound specifically to Sepharose beads derivatized with the collagen fragment α1(I) CB3, -which lacks RGD sequences. Immunofluorescence staining using the 3A3 monoclonal antibody revealed that the rat α1β1-integrin was present at focal adhesion sites of fibroblasts grown on native collagen type I- but not on fibronectin-coated substrates, although both types of substrates supported the formation of β1-integrin containing focal adhesions. Similarly, hepatocytes cultured on substrata coated with collagen type I (but not fibronectin) were stained in a patchy pattern localized to the cell periphery by 3A3 IgG. Furthermore, 3A3 IgG completely inhibited the attachment of hepatocytes to collagen type I, whereas under identical conditions the attachment of fibroblasts to these substrates was inhibited only by approximately 40%. The attachment of both hepatocytes and cardiac fibroblasts to fibronectin was unaffected by the presence of the 3A3 antibody. Collectively these data show that a rat homolog of the human VLA-1 heterodimer both biochemically and functionally fulfills the criteria of a single collagen receptor on rat hepatocytes. In contrast, rat cardiac fibroblasts utilize two different collagen-binding integrins to adhere to collagen, one of which is the rat homolog of the human VLA-1 heterodimer. Furthermore α1(I) CB3 contains cell binding sites for β1-integrins.
ASJC Scopus subject areas
- Cell Biology