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 produce d in a variety of normal and pathological contexts, including tissue m ineralization and cancer. OPN contains a conserved RGD (arg-gly-asp) a mino acid sequence that has been implicated in binding of OPN to cell surface integrins. To determine whether the RGD sequence in OPN is req uired for adhesive and chemotactic functions, we have introduced two s ite-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 exp ressed control and mutated mouse OPN in E. coli as recombinant glutath ione-S-transferase (GST)-OPN fusion proteins. Control mouse GST-OPN wa s functional in cell adhesion assays, supporting attachment and spread ing of mouse (malignant PAP2 ras-transformed NIH 373, and, to a lesser extent, normal NIH 373 fibroblasts) and human (MDA-MB-435 breast canc er, and normal gingival fibroblast) cells. In contrast, neither of the RGD-mutated OPN proteins (''delRGD'' or ''RGE'') supported adhesion o f 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 bot h malignant (PAP2, MDA-MB-435) and normal (gingival fibroblast, and NI H 373) cells, while RGD-mutated OPN proteins did not. Together these r esults suggest that the conserved RGD sequence in OPN is required for the majority of the protein's cell attachment and migration-stimulatin g functions. (C) 1995 Wiley-Liss, Inc.