Mapping of Potent and Specific Binding Motifs, GLOGEN and GVOGEA, for Integrin α1β1 Using Collagen Toolkits II and III

Integrins are well characterized cell surface receptors for extracellular matrix proteins. Mapping integrin-binding sites within the fibrillar collagens identified GFOGER as a high affinity site recognized by alpha 2 beta 1, but with lower affinity for alpha 1 beta 1. Here, to identify specific ligands for alpha 1 beta 1, we examined binding of the recombinant human alpha 1 I domain, the rat pheochromocytoma cell line (PC12), and the rat glioma Rugli cell line to our collagen Toolkit II and III peptides using solid-phase and real-time label-free adhesion assays. We observed Mg2+-dependent binding of the alpha 1 I domain to the peptides in the following rank order: III-7 (GLOGEN), II-28 (GFOGER), II-7 and II-8 (GLOGER), II-18 (GAOGER), III-4 (GROGER). PC12 cells showed a similar profile. Using antibody blockade, we confirmed that binding of PC12 cells to peptide III-7 was mediated by integrin alpha 1 beta 1. We also identified a new alpha 1 beta 1-binding activity within peptide II-27. The sequence GVOGEA bound weakly to PC12 cells and strongly to activated Rugli cells or to an activated alpha 1 I domain, but not to the alpha 2 I domain or to C2C12 cells expressing alpha 2 beta 1 or alpha 11 beta 1. Thus, GVOGEA is specific for alpha 1 beta 1. Although recognized by both alpha 2 beta 1 and alpha 11 beta 1, GLOGEN is a better ligand for alpha 1 beta 1 compared with GFOGER. Finally, using biosensor assays, we show that although GLOGEN is able to compete for the alpha 1 I domain from collagen IV (IC50 similar to 3 mu M), GFOGER is much less potent (IC50 similar to 90 mu M), as shown previously. These data confirm the selectivity of GFOGER for alpha 2 beta 1 and establish GLOGEN as a high affinity site for alpha 1 beta 1.