Identification of a novel integrin αMβ2 binding site in CCN1 (CYR61), a matricellular protein expressed in healing wounds and atherosclerotic lesions

CCN1 (cysteine-rich 61) and CCN2 ( connective tissue growth factor) are growth factor-inducible immediate-early gene products found in atherosclerotic lesions, restenosed blood vessels, and healing cutaneous wounds. Both CCN proteins have been shown to support cell adhesion and induce cell migration through interaction with integrin receptors. Recently, we have identified integrin alpha(M)beta(2) as the major adhesion receptor mediating monocyte adhesion to CCN1 and CCN2 and have shown that the alpha(M)I domain binds specifically to both proteins. In the present study, we demonstrated that activated monocytes adhered to a synthetic peptide ( CCN1-H2, SS-VKKYRPKYCGS) derived from a conserved region within the CCN1 C-terminal domain, and this process was blocked by the anti-alpha(M) monoclonal antibody 2LPM19c. Consistently, a glutathione S-transferase (GST) fusion protein containing the alpha(M)I domain (GST-alpha(M)I) bound to immobilized CCN1-H2 as well as to the corresponding H2 sequence in CCN2 (CCN2-H2, TSVKTYRAKFCGV). By contrast, a scrambled CCN1-H2 peptide and an 18-residue peptide derived from an adjacent sequence of CCN1-H2 failed to support monocyte adhesion or alpha(M)I domain binding. To confirm that the CCN1-H2 sequence within the CCN1 protein mediates alpha(M)beta(2) interaction, we developed an anti-peptide antibody against CCN1-H2 and showed that it specifically blocked GST-alpha(M)I binding to intact CCN1. Collectively, these results identify the H2 sequence in CCN1 and CCN2 as a novel integrin alpha(M)beta(2) binding motif that bears no apparent homology to any alpha(M)beta(2) binding sequence reported to date.