Identification of a novel integrin α_Mβ₂ 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 α_Mβ₂ as the major adhesion receptor mediating monocyte adhesion to CCN1 and CCN2 and have shown that the α_MI domain binds specifically to both proteins. In the present study, we demonstrated that activated monocytes adhered to a synthetic peptide (CCN1-H2, SSVKKYRPKYCGS) derived from a conserved region within the CCN1 C-terminal domain, and this process was blocked by the anti-α_M monoclonal antibody 2LPM19c. Consistently, a glutathione S-transferase (GST) fusion protein containing the α_MI domain (GST-α_MI) 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 α_MI domain binding. To confirm that the CCN1-H2 sequence within the CCN1 protein mediates α_Mβ₂ interaction, we developed an anti-peptide antibody against CCN1-H2 and showed that it specifically blocked GST-α_MI binding to intact CCN1. Collectively, these results identify the H2 sequence in CCN1 and CCN2 as a novel integrin α_Mβ₂ binding motif that bears no apparent homology to any α_Mβ₂ binding sequence reported to date.