Monoclonal antibodies identity residues 199-216 of the integrin alpha 2 vWFA domain as a functionally important region within alpha 2 beta 1

Integrin alpha 2 beta 1 is the major receptor for collagens in the human bo dy, and the collagen-binding site on the alpha 2 subunit von Willebrand fac tor A-type domain (vWFA domain) is now well defined. However, the biologica lly important conformational changes that are associated with collagen bind ing, and the means by which the vWFA domain is integrated into the whole in tegrin are not completely understood. We have raised monoclonal antibodies against recombinant alpha 2 vWFA domain for use as probes of function. Thre e antibodies, JA202, JA215 and JA218, inhibited binding to collagen, collag en I C-propeptide and E-cadherin, demonstrating that their function is impo rtant for structurally diverse alpha 2 beta 1 ligands. Cross-blocking studi es grouped the epitopes into two clusters: (I) JA202, the inhibitory antibo dy, Gi9, and a non-inhibitory antibody, JA208; (II) JA215 and JA218. Both c lusters were sensitive to events at the collagen binding site, as binding o f Gi9, JA202, JA215 and JA218 were inhibited by collagen peptide, JA208 bin ding was enhanced by collagen peptide, and binding of JA202 was decreased a fter mutagenesis of the cation-binding residue Thr(221) to alanine. Binding of cluster I antibodies was inhibited by the antifunctional anti-beta 1 an tibody Mab13, and binding of Gi9 and JA218 to alpha 2 beta 1 was inhibited by substituting Mn2+ for Mg2+, demonstrating that these antibodies were sen sitive to changes initiated outside the vWFA domain. Mapping of epitopes sh owed that JA202 and Gi9 bound between residues 212-216, while JA208 bound b etween residues 199-216. We have therefore identified two epitope clusters with novel properties; i.e. they are intimately associated with the collage n-binding site, responsive to conformational changes at the collagen-bindin g site and sensitive to events initiated outside the vWFA domain.