Targeted mutagenesis of the angiogenic protein CCN1 (CYR61) -: Selective inactivation of integrin α6β1-heparan sulfate proteoglycan coreceptor-mediated cellular functions

The matricellular protein CCN1 (CYR61) regulates multiple cellular processes and plays essential roles in embryonic vascular development. A ligand of several integrin receptors, CCN1 acts through integrin alpha(6)beta(1) and heparan sulfate proteoglycans (HSPGs) to promote specific functions in fibroblasts, smooth muscle cells, and endothelial cells. We have previously identified a novel alpha(6)beta(1) binding site, T1, in domain III of CCN1. Here we uncover two novel 16-residue sequences, H1 and H2, in domain IV that can support alpha(6)beta(1)- and HSPGs-dependent cell adhesion, suggesting that these sequences contain closely juxtaposed or overlapping sites for interaction with alpha(6)beta(1) and HSPGs. Furthermore, fibroblast adhesion to the H1 and H2 peptides is sufficient to induce prolonged MAPK activation, whereas adhesion to T1 induces transient MAPK activation. To dissect the roles of these sites in CCN1 function, we have created mutants disrupted in T1, H1, and H2 or in all three sites in the context of full-length CCN1. We show that the T1 and H1/H2 sites are functionally non-equivalent, and disruption of these sites differentially affected cell adhesion, migration, mitogen-activated protein kinase activation, and regulation of gene expression. Disruption of all three sites completely abolished alpha(6)beta(1)- HSPG-mediated cellular activities. All mutants disrupting T1, H1, and H2 fully retain alpha(v)beta(3)-mediated pro-angiogenic activities, indicating that these mutants are biologically active and are defective only in alpha(6)beta(1)- HSPG-mediated functions. Together, these findings identify and dissect the differential roles of the three sites (T1, H1, H2) required for alpha(6)beta(1)- HSPG-dependent CCN1 activities and provide a strategy to investigate these alpha(6)beta(1)-HSPG-specific activities in vivo.