Analysis of the alpha 4 beta 1 integrin-osteopontin interaction

The integrin alpha 4 beta 1 is involved in mediating exfiltration of leukoc ytes from the vasculature. It interacts with a number of proteins up-regula ted during the inflammatory response including VCAM-1 and the CS-1 alternat ively spliced region of fibronectin. In addition it binds the multifunction al protein osteopontin (OPN), which can act as both a cytokine and an extra cellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via alpha 4 beta 1 using GST fusion proteins. We show that a lpha 4 beta 1 expressed in J6 cells interacts with intact OPN when the inte grin is in a high activation state, and by deletion mapping that the alpha 4 beta 1 binding region in OPN lies between amino acid residues 125 and 168 (aa125-168). This region contains the central RGD motif of OPN, which also interacts with integrins alpha v beta 3, alpha v beta 5, alpha v beta 1, a lpha 8 beta 1, and alpha 5 beta 1. Mutating the RGD motif to RAD had no eff ect on the interaction with alpha 4 beta 1. To define the binding site the region incorporating aa125-168 was divided into 5 overlapping peptides expr essed as GST fusion proteins. Two peptides supported adhesion via alpha 4 b eta 1, aa132-146, and aa153-168; of these only a synthetic peptide, SVVYGLR (aa162-168), derived from aa153-168 was able to inhibit alpha 4 beta 1 bin ding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhi bitor of alpha 4 beta 1, and the primary alpha 4 beta 1 binding site within OPN. (C) 2000 Academic Press.