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J. Biol. Chem., Vol. 281, Issue 7, 3821-3831, February 17, 2006

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Use of Synthetic Peptides to Locate Novel Integrin ₂₁-binding Motifs in Human Collagen III^*

From the Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom

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 ₂₁. The capacity of the peptides to support Mg²⁺-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant ₂ I-domain, ₂₁ purified from platelet membranes, and recombinant soluble ₂₁ expressed as an ₂-Fos/₁-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 ₂ 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-₂ 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.

Received for publication, September 7, 2005 , and in revised form, November 22, 2005.

^* This work was supported by the Wellcome Trust, the Medical Research Council, and the British Heart Foundation. A preliminary account was presented at the XIXth Meeting of the Federation of European Connective Tissue Societies, in Giardini Naxos, Italy, July 9–13, 2004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Biochemistry, University of Cambridge, Bldg. O, Downing Site, Cambridge CB2 1QW, UK. Tel.: 44-1223-766-111; Fax: 44-1223-333-345; E-mail: rwf10{at}cam.ac.uk.

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