title: Identification of a novel integrin alpha 6 beta 1 binding site in the angiogenic inducer CCN1 (CYR61).
abstract: The angiogenic inducer CCN1 (cysteine-rich 61, CYR61), a secreted matricellular protein of the CCN family, is a ligand of multiple integrins, including alpha 6 beta 1. Previous studies have shown that CCN1 interaction with integrin alpha 6 beta 1 mediates adhesion of fibroblasts, endothelial cells, and smooth muscle cells, as well as migration of smooth muscle cells. Recently, we have reported that CCN1-induced tubule formation of unactivated endothelial cells is also mediated through integrin alpha 6 beta 1. In this study, we demonstrate that human skin fibroblasts adhere specifically to the T1 sequence (GQKCIVQTTSWSQCSKS) within domain III of CCN1, and this process is blocked by anti-alpha 6 and anti-beta 1 monoclonal antibodies. Alanine substitution mutagenesis of the T1 sequence further defines the sequence TTSWSQCSKS as the critical determinant for mediating alpha 6 beta 1-dependent adhesion. Soluble T1 peptide specifically inhibits fibroblast adhesion to CCN1 in a dose-dependent manner. Furthermore, T1 also inhibits cell adhesion to other alpha 6 beta 1 ligands, including CCN2 (CTGF), CCN3 (NOV), and laminin, but not to ligands of other integrins. In addition, T1 specifically inhibits alpha 6 beta 1-dependent tubule formation of unactivated endothelial cells in a CCN1-containing collagen gel matrix. To confirm that T1 binds integrin alpha 6 beta 1 directly, we perform affinity chromatography and show that integrin alpha 6 beta 1 is isolated from an octylglucoside extract of fibroblasts on T1-coupled Affi-gel. Taken together, these findings define the T1 sequence in CCN1 as a novel binding motif for integrin alpha 6 beta 1, providing the basis for the development of peptide mimetics to examine the functional role of alpha 6 beta 1 in angiogenesis.
authors.affiliation: Department of Molecular Genetics, University of Illinois College of Medicine, Chicago, Illinois 60607-7170, USA.
authors.forename: Shr-Jeng
authors.initials: SJ
authors.lastname: Leu
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Ningyu
authors.initials: N
authors.lastname: Chen
authors.forename: Chih-Chiun
authors.initials: CC
authors.lastname: Chen
authors.forename: Stephen C-T
authors.initials: SC
authors.lastname: Lam
authors.forename: Lester F
authors.initials: LF
authors.lastname: Lau
mesh_terms: D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002448:Cell Adhesion; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D002478:Cells, Cultured; D002845:Chromatography; D055515:Cysteine-Rich Protein 61; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology; D005347:Fibroblasts / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000737:chemistry*; D039121:Integrin alpha6beta1 / Q000737:chemistry*; D016023:Integrins / Q000737:chemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry; D014471:Umbilical Veins / Q000166:cytology
chemical_list: C501848:CCN1 protein, human; D055515:Cysteine-Rich Protein 61; D017874:Immediate-Early Proteins; D039121:Integrin alpha6beta1; D016023:Integrins; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012685:Sepharose; D005982:Glutathione Transferase; D000409:Alanine