SITE-DIRECTED MUTAGENESIS OF THE ARGININE-GLYCINE-ASPARTIC ACID SEQUENCE IN OSTEOPONTIN DESTROYS CELL-ADHESION AND MIGRATION FUNCTIONS

Osteopontin (OPN) is a secreted calcium-binding phosphoprotein produced in a variety of normal and pathological contexts, including tissue mineralization and cancer. OPN contains a conserved RGD (arg-gly-asp) amino acid sequence that has been implicated in binding of OPN to cell surface integrins. To determine whether the RGD sequence in OPN is required for adhesive and chemotactic functions, we have introduced two site-directed mutations in the RGD site of the mouse OPN cDNA, in which the RGD sequence was either deleted or mutated to RGE (arg-gly-glu). in order to test the effect of these mutations an OPN function, we expressed control and mutated mouse OPN in E. coli as recombinant glutathione-S-transferase (GST)-OPN fusion proteins. Control mouse GST-OPN was functional in cell adhesion assays, supporting attachment and spreading of mouse (malignant PAP2 ras-transformed NIH 373, and, to a lesser extent, normal NIH 373 fibroblasts) and human (MDA-MB-435 breast cancer, and normal gingival fibroblast) cells. In contrast, neither of the RGD-mutated OPN proteins (''delRGD'' or ''RGE'') supported adhesion of any of the cell lines, even when used at high concentrations or for long assay times. GRGDS (gly-arg-gly-asp-ser) peptides inhibited cell adhesion to intact GST-OPN, as well as to fibronectin and vitronectin. In chemotaxis assays, GST-OPN promoted directed cell migration of both malignant (PAP2, MDA-MB-435) and normal (gingival fibroblast, and NIH 373) cells, while RGD-mutated OPN proteins did not. Together these results suggest that the conserved RGD sequence in OPN is required for the majority of the protein's cell attachment and migration-stimulating functions. (C) 1995 Wiley-Liss, Inc.