Identification in collagen type I of an integrin alpha(2)beta(1)-binding site containing an essential GER sequence

The collagen type I-derived fragment alpha(1)(I)CB3 is known to recognize t he platelet collagen receptor integrin alpha(2)beta(1) as effectively as th e parent collagen, although it lacks platelet-aggregatory activity. We have synthesized the fragment as seven overlapping peptides that spontaneously assemble into triple helices. On the basis of their capacity to bind purifi ed alpha(2)beta(1) and the recombinant alpha(2) A-domain, and their ability to support alpha(2)beta(1)-mediated cell adhesion, we identified two pepti des, CB3(I)-5 and -6, which contain an alpha(2)beta(1) recognition site. Sy nthesis of the peptide CB3(I)-5/6, containing the overlap sequence between peptides 5 and 6, allowed us to locate the binding site within the 15-resid ue sequence, GFP*GERGVEGPP*GPA (where P* represents hydroxyproline), corres ponding to residues 502-516 of the collagen type I alpha(1) chain. The Glu and Arg residues in the GER triplet were found to be essential for recognit ion since substitution of either residue with Ala caused a loss of alpha(2) A-domain binding. By contrast, substitution of the Glu in GVE did not redu ce binding, but rather enhanced it slightly. We were unable to detect signi ficant recognition of alpha(2)beta(1) by the peptide CB3(I)-2 containing th e putative alpha(2)beta(1) recognition sequence DGEA. Peptides CB3(I)-1 to -6, together with peptide CB3(I)-5/6, exhibited good platelet-aggregatory a ctivity, in some cases better than collagen. However, peptide CB3(I)-7 was inactive, suggesting the presence of an inhibitory element that might accou nt for the lack of aggregatory activity of the parent alpha(1)(I)CB3 fragme nt.