NON-RGD DOMAINS OF OSTEOPONTIN PROMOTE CELL-ADHESION WITHOUT INVOLVING ALPHA-V INTEGRINS

Osteopontin (OPN) is an integrin-binding secreted protein that contain s an Arg-Gly-Asp (RGD) amino acid sequence and binds to various cell t ypes via RGD-mediated interaction with the alpha v beta 3 integrin. We have identified a cell line whose binding to OPN does not require RGD or alpha v interactions. We compared the ability of two murine cell l ines, L929 fibroblastic cells and B16-BL6 melanoma cells, to interact with OPN (from human milk, and recombinant human and mouse OPN) as wel l as recombinant OPN prepared to include either the N-terminal or C-te rminal halves but lacking the RGD sequence. Both cell lines adhered to GRGDS peptides coupled to BSA, and these interactions were inhibited by addition of GRGDS (but not GRGES) peptides or a monoclonal antibody specific to the alpha v integrin subunit. Adhesion of L929 cells to O PN was also dependent on the RGD sequence and the alpha v integrin sub unit. However, the binding of B16-BL6 cells was not inhibited by eithe r GRGDS peptides or the anti-alpha v antibody. B16-BL6 (but not L929) cells were also able to adhere to and spread on both N-terminal and C- terminal OPN proteins that lack the RGD sequence, and these interactio ns were not inhibited by either GRGDS peptides or anti-alpha v antibod y. Together these results indicate that B16-BL6 cells can adhere to OP N by interactions that are independent of either the RGD sequence or t he alpha v integrin subunit, and suggest that some cells can interact with additional, non-RGD binding sites in OPN. (C) 1996 Wiley-Liss, In c.