Transforming growth factor-beta (TGF-beta) binding to the extracellular domain of the type II TGF-beta receptor: receptor capture on a biosensor surface using a new coiled-coil capture system demonstrates that avidity contributes significantly to high affinity binding

J Mol Biol. 2003 May 16;328(5):1173-83. doi: 10.1016/s0022-2836(03)00360-7.

Abstract

Mature TGF-beta isoforms, which are covalent dimers, signal by binding to three types of cell surface receptors, the type I, II and III TGF-beta receptors. A complex composed of the TGF-beta ligand and the type I and II receptors is required for signaling. The type II receptor is responsible for recruiting TGF-beta into the heteromeric ligand/type I receptor/type II receptor complex. The purpose of this study was to test for the extent that avidity contributes to receptor affinity. Using a surface plasmon resonance (SPR)-based biosensor (the BIACORE), we captured the extracellular domain of the type II receptor (TbetaRIIED) at the biosensor surface in an oriented and stable manner by using a de novo designed coiled-coil (E/K coil) heterodimerizing system. We characterized the kinetics of binding of three TGF-beta isoforms to this immobilized TbetaRIIED. The results demonstrate that the stoichiometry of TGF-beta binding to TbetaRIIED was one dimeric ligand to two receptors. All three TGF-beta isoforms had rapid and similar association rates, but different dissociation rates, which resulted in the equilibrium dissociation constants being approximately 5pM for the TGF-beta1 and -beta3 isoforms, and 5nM for the TGF-beta2 isoform. Since these apparent affinities are at least four orders of magnitude higher than those determined when TGF-beta was immobilized, and are close to those determined for TbetaRII at the cell surface, we suggest that avidity contributes significantly to high affinity receptor binding both at the biosensor and cell surfaces. Finally, we demonstrated that the coiled-coil immobilization approach does not require the purification of the captured protein, making it an attractive tool for the rapid study of any protein-protein interaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites
  • Biosensing Techniques
  • DNA, Recombinant / genetics
  • Humans
  • In Vitro Techniques
  • Kinetics
  • Molecular Sequence Data
  • Protein Isoforms / metabolism
  • Protein Serine-Threonine Kinases
  • Protein Structure, Tertiary
  • Receptor, Transforming Growth Factor-beta Type II
  • Receptors, Transforming Growth Factor beta / chemistry*
  • Receptors, Transforming Growth Factor beta / genetics
  • Receptors, Transforming Growth Factor beta / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Surface Plasmon Resonance
  • Transforming Growth Factor beta / metabolism*
  • Transforming Growth Factor beta1
  • Transforming Growth Factor beta2
  • Transforming Growth Factor beta3

Substances

  • DNA, Recombinant
  • Protein Isoforms
  • Receptors, Transforming Growth Factor beta
  • Recombinant Fusion Proteins
  • TGFB1 protein, human
  • TGFB2 protein, human
  • Transforming Growth Factor beta
  • Transforming Growth Factor beta1
  • Transforming Growth Factor beta2
  • Transforming Growth Factor beta3
  • Protein Serine-Threonine Kinases
  • Receptor, Transforming Growth Factor-beta Type II

Associated data

  • PDB/1OXD
  • PDB/1OXE
  • PDB/1OXF