Identification of a novel integrin αvβ3 binding site in CCN1 (CYR61) critical for pro-angiogenic activities in vascular endothelial cells

CCN1 (CYR61) is a matricellular inducer of angiogenesis essential for successful vascular development. Though devoid of the canonical RGD sequence motif recognized by some integrins, CCN1 binds to, and functions through integrin alpha(v)beta(3) to promote pro-angiogenic activities in activated endothelial cells. In this study we identify a 20-residue sequence, V2 (NCKHQCTCIDGAVGCIPLCP), in domain II of CCN1 as a novel binding site for integrin alpha(v)beta(3). Immobilized synthetic V2 peptide supports alpha(v)beta(3)-mediated cell adhesion; soluble V2 peptide inhibits endothelial cell adhesion to CCN1 and the homologous family members CCN2 (connective tissue growth factor, CTGF) or CCN3 (NOV) but not to collagen. These activities are obliterated by mutation of the aspartate residue in the V2 peptide to alanine. The corresponding D125A mutation in the context of the N-terminal half of CCN1 (domains I and II) greatly diminished direct solid phase binding to purified integrin alpha(v)beta(3) and abolished alpha(v)beta(3)-mediated cell adhesion activity. Likewise, soluble full-length CCN1 with the D125A mutation is defective in binding purified alpha(v)beta(3) and impaired in alpha(v)beta(3)-mediated pro-angiogenic activities in vascular endothelial cells, including stimulation of cell migration and enhancement of DNA synthesis. In contrast, immobilized full-length CCN1-D125A mutant binds alpha(v)beta(3) and supports alpha(v)beta(3)-mediated cell adhesion similar to wild type CCN1. These results indicate that V2 is the primary alpha(v)beta(3) binding site in soluble CCN1, whereas additional cryptic alpha(v)beta(3) binding site(s) in the C-terminal half of CCN1 becomes exposed when the protein is immobilized. Together, these results identify a novel and functionally important binding site for integrin alpha(v)beta(3) and provide a new approach for dissecting alpha(v)beta(3)-specific CCN1 functions both in cultured cells and in the organism.