Identification in Collagen Type I of an Integrin α2β1-binding Site Containing an Essential GER Sequence

• Published in: The Journal of biological chemistry 1998-12, Vol.273 (50), p.33287
• Main Authors: C. Graham Knight, Laurence F. Morton, David J. Onley, Anthony R. Peachey, Anthea J. Messent, Peter A. Smethurst, Danny S. Tuckwell, Richard W. Farndale, Michael J. Barnes
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.50.33287

The collagen type I-derived fragment α 1 (I)CB3 is known to recognize the platelet collagen receptor integrin α 2 β 1 as effectively as the 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 purified α 2 β 1 and the recombinant α 2 A-domain, and their ability to support α 2 β 1 -mediated cell adhesion, we identified two peptides, CB3(I)-5 and -6, which contain an α 2 β 1 recognition site. Synthesis 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-residue sequence, GFP*GERGVEGPP*GPA (where P* represents hydroxyproline), corresponding to residues 502–516 of the collagen type I α 1 chain. The Glu and Arg residues in the GER triplet were found to be essential for recognition since substitution of either residue with Ala caused a loss of α 2 A-domain binding. By contrast, substitution of the Glu in GVE did not reduce binding, but rather enhanced it slightly. We were unable to detect significant recognition of α 2 β 1 by the peptide CB3(I)-2 containing the putative α 2 β 1 recognition sequence DGEA. Peptides CB3(I)-1 to -6, together with peptide CB3(I)-5/6, exhibited good platelet-aggregatory activity, in some cases better than collagen. However, peptide CB3(I)-7 was inactive, suggesting the presence of an inhibitory element that might account for the lack of aggregatory activity of the parent α 1 (I)CB3 fragment.