Identification of a novel integrin alpha 6 beta 1 binding site in the angiogenic inducer CCN1 (CYR61)

J Biol Chem. 2003 Sep 5;278(36):33801-8. doi: 10.1074/jbc.M305862200. Epub 2003 Jun 24.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alanine / chemistry
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Binding Sites
  • Cell Adhesion
  • Cell Line
  • Cell Membrane / metabolism
  • Cell Movement
  • Cells, Cultured
  • Chromatography
  • Cysteine-Rich Protein 61
  • Dose-Response Relationship, Drug
  • Endothelium, Vascular / cytology
  • Fibroblasts / metabolism
  • Glutathione Transferase / metabolism
  • Humans
  • Immediate-Early Proteins / chemistry*
  • Integrin alpha6beta1 / chemistry*
  • Integrins / chemistry
  • Intercellular Signaling Peptides and Proteins / chemistry*
  • Models, Biological
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptides / chemistry
  • Precipitin Tests
  • Protein Binding
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / metabolism
  • Sepharose / chemistry
  • Umbilical Veins / cytology

Substances

  • CCN1 protein, human
  • Cysteine-Rich Protein 61
  • Immediate-Early Proteins
  • Integrin alpha6beta1
  • Integrins
  • Intercellular Signaling Peptides and Proteins
  • Peptides
  • Recombinant Fusion Proteins
  • Sepharose
  • Glutathione Transferase
  • Alanine