BIAcore analysis of bovine insulin-like growth factor (IGF)-binding protein-2 identifies major IGF binding site determinants in both the amino- and carboxyl-terminal domains

J Biol Chem. 2001 Jul 20;276(29):27120-8. doi: 10.1074/jbc.M101317200. Epub 2001 May 16.

Abstract

In the absence of a complete tertiary structure to define the molecular basis of the high affinity binding interaction between insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs), we have investigated binding of IGFs by discrete amino-terminal domains (amino acid residues 1-93, 1-104, 1-132, and 1-185) and carboxyl-terminal domains (amino acid residues 96-279, 136-279, and 182-284) of bovine IGFBP-2 (bIGFBP-2). Both halves of bIGFBP-2 bound IGF-I and IGF-II in BIAcore studies, albeit with different affinities ((1-132)IGFBP-2, K(D) = 36.3 and 51.8 nm; (136-279)IGFBP-2HIS, K(D) = 23.8 and 16.3 nm, respectively). The amino-terminal half appears to contain components responsible for fast association. In contrast, IGF binding by the carboxyl-terminal fragment results in a more stable complex as reflected by its K(D). Furthermore, des(1-3)IGF-I and des(1-6)IGF-II exhibited reduced binding affinity to (1-279)IGFBP-2HIS, (1-132)IGFBP-2, and (136-279)IGFBP-2HIS biosensor surfaces compared with wild-type IGF. A charge reversal at positions 3 and 6 of IGF-I and IGF-II, respectively, affects binding interactions with the amino-terminal fragment and full-length bIGFBP-2 but not the carboxyl-terminal fragment.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Binding Sites
  • Biosensing Techniques
  • COS Cells
  • Cattle
  • DNA Primers
  • Electrophoresis, Polyacrylamide Gel
  • Insulin-Like Growth Factor Binding Protein 2 / chemistry
  • Insulin-Like Growth Factor Binding Protein 2 / isolation & purification
  • Insulin-Like Growth Factor Binding Protein 2 / metabolism*
  • Kinetics
  • Molecular Sequence Data
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Somatomedins / metabolism*

Substances

  • DNA Primers
  • Insulin-Like Growth Factor Binding Protein 2
  • Recombinant Proteins
  • Somatomedins