A novel approach to identify two distinct receptor binding surfaces of insulin-like growth factor II

J Biol Chem. 2009 Mar 20;284(12):7656-64. doi: 10.1074/jbc.M808061200. Epub 2009 Jan 12.

Abstract

Very little is known about the residues important for the interaction of insulin-like growth factor II (IGF-II) with the type 1 IGF receptor (IGF-1R) and the insulin receptor (IR). Insulin, to which IGF-II is homologous, is proposed to cross-link opposite halves of the IR dimer through two receptor binding surfaces, site 1 and site 2. In the present study we have analyzed the contribution of IGF-II residues equivalent to insulin's two binding surfaces toward the interaction of IGF-II with the IGF-1R and IR. Four "site 1" and six "site 2" analogues were produced and analyzed in terms of IGF-1R and IR binding and activation. The results show that Val(43), Phe(28), and Val(14) (equivalent to site 1) are critical to IGF-1R and IR binding, whereas mutation to alanine of Gln(18) affects only IGF-1R and not IR binding. Alanine substitutions at Glu(12), Asp(15), Phe(19), Leu(53), and Glu(57) analogues resulted in significant (>2-fold) decreases in affinity for both the IGF-1R and IR. Furthermore, taking a novel approach using a monomeric, single-chain minimized IGF-1R we have defined a distinct second binding surface formed by Glu(12), Phe(19), Leu(53), and Glu(57) that potentially engages the IGF-1R at one or more of the FnIII domains.

MeSH terms

  • Amino Acid Substitution
  • Animals
  • BALB 3T3 Cells
  • Binding Sites / physiology
  • Dimerization
  • Humans
  • Insulin-Like Growth Factor II / chemistry
  • Insulin-Like Growth Factor II / genetics
  • Insulin-Like Growth Factor II / metabolism*
  • Mice
  • Mutation, Missense
  • Peptide Mapping / methods
  • Protein Binding / physiology
  • Protein Structure, Tertiary / physiology
  • Receptor, IGF Type 1 / chemistry
  • Receptor, IGF Type 1 / genetics
  • Receptor, IGF Type 1 / metabolism*
  • Receptor, Insulin / chemistry
  • Receptor, Insulin / genetics
  • Receptor, Insulin / metabolism*

Substances

  • Insulin-Like Growth Factor II
  • Receptor, IGF Type 1
  • Receptor, Insulin