A Peptide Inhibiting the Collagen Binding Function of Integrin α2I Domain

• Published in: The Journal of biological chemistry 1999-02, Vol.274 (6), p.3513
• Main Authors: Johanna Ivaska, Jarmo KäpylÃ, Olli Pentikäinen, Anna-Marja Hoffrén, Jorma Hermonen, Pasi Huttunen, Mark S. Johnson, Jyrki Heino
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.6.3513

Integrin α 2 subunit forms in the complex with the β 1 subunit a cell surface receptor binding extracellular matrix molecules, such as collagens and laminin-1. It is a receptor for echovirus-1, as well. Ligands are recognized by the special “inserted” domain (I domain) in the integrin α 2 subunit. Venom from a pit viper, Bothrops jararaca , has been shown to inhibit the interaction of platelet α 2 β 1 integrin with collagen because of the action of a disintegrin/metalloproteinase named jararhagin. The finding that crude B. jararaca venom could prevent the binding of human recombinant rα 2 I domain to type I collagen led us to study jararhagin further. Synthetic peptides representing hydrophilic and charged sequences of jararhagin, including the RSECD sequence replacing the well known RGD motif in the disintegrin-like domain, were synthesized. Although the disintegrin-like domain derived peptides failed to inhibit rα 2 I domain binding to collagen, a basic peptide from the metalloproteinase domain proved to be functional. In an in vitro assay, the cyclic peptide, CTRKKHDNAQC, was shown to bind strongly to human recombinant α 2 I domain and to prevent its binding to type I and IV collagens and to laminin-1. Mutational analysis indicated that a sequence of three amino acids, arginine-lysine-lysine (RKK), is essential for rα 2 I domain binding, whereas the mutation of the other amino acids in the peptide had little if any effect on its binding function. Importantly, the peptide was functional only in the cyclic conformation and its affinity was strictly dependent on the size of the cysteine-constrained loop. Furthermore, the peptide could not bind to α 2 I domain in the absence of Mg 2+ , suggesting that the conformation of the I domain was critical, as well. Cells could attach to the peptide only if they expressed α 2 β 1 integrin, and the attachment was inhibited by anti-integrin antibodies.