Use of synthetic peptides to locate novel integrin α 2β1-binding motifs in human collagen III

Abstract

A set of 57 synthetic peptides encompassing the entire triplehelical domain of human collagen III was used to locate binding sites for the collagen-binding integrin α2β1. The capacity of the peptides to support Mg2+-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant α2 I-domain, α2β1 purified from platelet membranes, and recombinant soluble α2β1 expressed as an α2-Fos/β1-Jun heterodimer) bound well to only three peptides, two containing GXX′GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX′GEN motifs (GLKGEN and GLOGEN). Two mutant α2 I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti- α2 monoclonal antibody 6F1 and by chelation of Mg2+. We describe two novel high affinity integrin-binding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.