Identification of a novel integrin α6β1 binding site in the angiogenic inducer CCN1 (CYR61)

The angiogenic inducer CCN1 (cysteine-rich 61, CYR61), a secreted matricellular protein of the CCN family, is a ligand of multiple integrins, including alpha(6)beta(1). Previous studies have shown that CCN1 interaction with integrin alpha(6)beta(1) mediates adhesion of fibroblasts, endothelial cells, and smooth muscle cells, as well as migration of smooth muscle cells. Recently, we have reported that CCN1-induced tubule formation of unactivated endothelial cells is also mediated through integrin alpha(6)beta(1). In this study, we demonstrate that human skin fibroblasts adhere specifically to the T1 sequence (GQKCIVQTTSWSQCSKS) within domain III of CCN1, and this process is blocked by anti-alpha(6) and anti-beta(1) monoclonal antibodies. Alanine substitution mutagenesis of the T1 sequence further defines the sequence TTSWSQC-SKS as the critical determinant for mediating alpha(6)beta(1)-dependent adhesion. Soluble T1 peptide specifically inhibits fibroblast adhesion to CCN1 in a dose-dependent manner. Furthermore, T1 also inhibits cell adhesion to other alpha(6)beta(1) ligands, including CCN2 ( CTGF), CCN3 (NOV), and laminin, but not to ligands of other integrins. In addition, T1 specifically inhibits alpha(6)beta(1)-dependent tubule formation of unactivated endothelial cells in a CCN1-containing collagen gel matrix. To confirm that T1 binds integrin alpha(6)beta(1) directly, we perform affinity chromatography and show that integrin alpha(6)beta(1) is isolated from an octylglucoside extract of fibroblasts on T1- coupled Affi-gel. Taken together, these findings define the T1 sequence in CCN1 as a novel binding motif for integrin alpha(6)beta(1), providing the basis for the development of peptide mimetics to examine the functional role of alpha(6)beta(1) in angiogenesis.