Identification of a novel integrin alphaMbeta2 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 alphaMbeta2 as the major adhesion receptor mediating monocyte adhesion to CCN1 and CCN2 and have shown that the alphaMI 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-alphaM monoclonal antibody 2LPM19c. Consistently, a glutathione S-transferase (GST) fusion protein containing the alphaMI domain (GST-alphaMI) 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 alphaMI domain binding. To confirm that the CCN1-H2 sequence within the CCN1 protein mediates alphaMbeta2 interaction, we developed an anti-peptide antibody against CCN1-H2 and showed that it specifically blocked GST-alphaMI binding to intact CCN1. Collectively, these results identify the H2 sequence in CCN1 and CCN2 as a novel integrin alphaMbeta2 binding motif that bears no apparent homology to any alphaMbeta2 binding sequence reported to date.
Schober
Joseph M
JM
Department of Pharmacology, University of Illinois at Chicago, College of Medicine, Chicago, Illinois 60612, USA.
Lau
Lester F
LF
Ugarova
Tatiana P
TP
Lam
Stephen C-T
SC
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001161:Arteriosclerosis / Q000378:metabolism*; D001665:Binding Sites; D002448:Cell Adhesion; D055515:Cysteine-Rich Protein 61; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000737:chemistry* / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism; D016177:Macrophage-1 Antigen / Q000737:chemistry* / Q000378:metabolism; D008345:Manganese / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014945:Wound Healing
C501848:CCN1 protein, human; D055515:Cysteine-Rich Protein 61; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D016177:Macrophage-1 Antigen; D010455:Peptides; D011993:Recombinant Fusion Proteins; D008345:Manganese; D005982:Glutathione Transferase