A Peptide Inhibiting the Collagen Binding Function of Integrin α2I Domain

Abstract

Integrin ␣ 2 subunit forms in the complex with the ␤ 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 ␣ 2 subunit. Venom from a pit viper, Bothrops jararaca, has been shown to inhibit the interaction of platelet ␣ 2 ␤ 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 r␣ 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 disintegrinlike domain, were synthesized. Although the disintegrin-like domain derived peptides failed to inhibit r␣ 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 ␣ 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-lysinelysine (RKK), is essential for r␣ 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 ␣ 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 ␣ 2 ␤ 1 integrin, and the attachment was inhibited by anti-integrin antibodies.Integrins ␣ 1 ␤ 1 and ␣ 2 ␤ 1 are the major cellular receptors for native collagens (for review, see Refs. 1 and 2). Like all integrins their interaction with ligands is dependent on divalent cations (3). The ␣ 1 and ␣ 2 subunits contain an special inserted domain, the I domain, resembling the A domain found e.g. in von Willenbrand factor (4). It is evident that ␣ 1 I and ␣ 2 I domains are responsible for the primary recognition of collagen by the corresponding integrins (5, 6). Two other ligands for ␣ 2 ␤ 1 integrin, namely laminin-1 and echovirus-1, each bind to ␣ 2 I domain, as well. However, echovirus-1 seems to recognize a different site on the ␣ 2 I domain than the matrix proteins do (7).The binding sites of ␣ 1 ␤ 1 and ␣ 2 ␤ 1 integrins in collagens have been localized to the triple helical areas of the molecules (8, 9). One peptide sequence derived from the collagen ␣ chain has been reported to block integrin-collagen interaction (10), but in many studies it has been ineffective and it probably does not represent the actual binding site in collagen (11-13). More likely, collagen-receptor integrins recognize amino acid resi...

Evidence against a direct role of the integrin α2β1 in collagen‐induced tyrosine phosphorylation in human platelets

“…In line with this, the inhibitory effect of the metalloprotease jararhagin, which binds to the I domain of the a2-subunit and proteolytically breaks down the b1 subunit, is also overcome by high concentrations of collagen. Ivaska et al [39] have also reported that a cyclic peptide based on the active sequence of jararhagin only partially inhibits aggregation but causes a complete inhibition of binding of collagen to the a2-subunit. These data demonstrate that high concentrations of collagen are able to overcome the inhibitory effect of blockade of binding to a2b1, through interaction with a second receptor.…”

Selective Binding of Collagen Subtypes by Integrin α1I, α2I, and α10I Domains

“…Cloning and Mutagenesis of the Human Integrin ␣ 2 I Domain-Human integrin ␣ 2 I domain was generated as described earlier by Ivaska et al (24). The integrin ␣ 2 cDNA was a gift from Dr. M. Hemler, Dana Farber, Boston.…”

Gelatinase-mediated migration and invasion of cancer cells

“…Other proteases than MMPs may also interact with integrins. These include uPA/uPAR, which may interact with several integrins (Aguirre Ghiso et al, 1999;Carriero et al, 1999;Wei et al, 2001;Wei et al, 1996;Xue et al, 1997), elastase with α M β 2 integrin (Cai and Wright, 1996), snake venom disintegrin/metalloproteinase with α 2 β 1 integrin (Ivaska et al, 1999) and ADAMs with several integrins (Bax et al, 2004;Bridges et al, 2002;Nath et al, 1999).…”