Cell adhesion to tenascin-X - Mapping of cell adhesion sites and identification of integrin receptors

Adhesive properties of tenascin-X (TN-X) were investigated using TN-X purified from bovine skin and recombinant proteins encompassing the RGD sequence located within the tenth fibronectin type-III domain, and the fibrinogen-like domain. Osteosarcoma (MG63) and bladder carcinoma cells (ECV304) cells were shown to adhere to purified TN-X, but did not spread and did not assemble actin stress fibers. Both cell types adhered to recombinant proteins harboring the contiguous fibronectin type-III domains 9 and 10 (FNX 9-10) but not to the FNX 10 domain alone. This adhesion to FNX 9-10 was shown to be mediated by alpha v beta 3 integrin, was inhibited by RGD peptides and was strongly reduced in proteins mutated within the RGD site. As antibodies against alpha v beta 3 integrin had no effects on cell adhesion to purified TN-X, we suggest that the RGD sequence is masked in intact TN-X. Cell attachment to the recombinant TN-X fibrinogen domain (FbgX) and to purified TN-X was greater for MG63 than for ECV304 cells. A beta 1-containing integrin was shown to be involved in MG63 cell attachment to FbgX and to purified TN-X. Although the existence of other cell interaction sites is likely in this huge molecule, these similar patterns of adhesion and inhibition suggest that the fibrinogen domain might be a dominant site in the whole molecule.