Mutation of three critical amino acids of the N-terminal domain of IGF-binding protein-3 essential for high affinity IGF binding

C. K. Buckway, E. M. Wilson, M. Ahlsen, P. Bang, Y. Oh, Ronald (Ron) Rosenfeld

Research output: Contribution to journalArticle

56 Citations (Scopus)

Abstract

The N-terminal domain is conserved in all members of the IGF-binding protein superfamily. Most recently, studies have demonstrated the importance of an IGF-binding protein N-terminal hydrophobic pocket for IGF binding. To examine more critically the amino acids important for IGF binding within the full-length IGF-binding protein-3 protein while minimizing changes in the tertiary structure, we targeted residues I56, L80, and L81 within the proposed hydrophobic pocket for mutation. With a single change at these sites to the nonconserved glycine there was a notable decrease in binding. A greater reduction was seen when both L80 and L81 were substituted with glycine, and complete loss of affinity for IGF-I and IGF-II occurred when all three targeted amino acids were changed to glycine. Furthermore, the ability of the IGF-binding protein-3 mutants to inhibit IGF-I-stimulated phosphorylation of its receptor was a reflection of their affinity for IGF, with the lowest affinity mutants having the least inhibitory effect. These studies, thus, support the hypothesis that an N-terminal hydrophobic pocket is the primary site of high affinity binding of IGF to IGF-binding protein-3. The mutants provide a tool for future studies directed at IGF-dependent and IGF-independent actions of IGF-binding protein-3.

Original languageEnglish (US)
Pages (from-to)4943-4950
Number of pages8
JournalJournal of Clinical Endocrinology and Metabolism
Volume86
Issue number10
DOIs
StatePublished - 2001

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Insulin-Like Growth Factor Binding Protein 3
Glycine
Amino Acids
Insulin-Like Growth Factor Binding Proteins
Mutation
Insulin-Like Growth Factor I
Phosphorylation
Insulin-Like Growth Factor II
Proteins

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology, Diabetes and Metabolism

Cite this

Mutation of three critical amino acids of the N-terminal domain of IGF-binding protein-3 essential for high affinity IGF binding. / Buckway, C. K.; Wilson, E. M.; Ahlsen, M.; Bang, P.; Oh, Y.; Rosenfeld, Ronald (Ron).

In: Journal of Clinical Endocrinology and Metabolism, Vol. 86, No. 10, 2001, p. 4943-4950.

Research output: Contribution to journalArticle

Buckway, C. K. ; Wilson, E. M. ; Ahlsen, M. ; Bang, P. ; Oh, Y. ; Rosenfeld, Ronald (Ron). / Mutation of three critical amino acids of the N-terminal domain of IGF-binding protein-3 essential for high affinity IGF binding. In: Journal of Clinical Endocrinology and Metabolism. 2001 ; Vol. 86, No. 10. pp. 4943-4950.
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AU - Bang, P.

AU - Oh, Y.

AU - Rosenfeld, Ronald (Ron)

PY - 2001

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AB - The N-terminal domain is conserved in all members of the IGF-binding protein superfamily. Most recently, studies have demonstrated the importance of an IGF-binding protein N-terminal hydrophobic pocket for IGF binding. To examine more critically the amino acids important for IGF binding within the full-length IGF-binding protein-3 protein while minimizing changes in the tertiary structure, we targeted residues I56, L80, and L81 within the proposed hydrophobic pocket for mutation. With a single change at these sites to the nonconserved glycine there was a notable decrease in binding. A greater reduction was seen when both L80 and L81 were substituted with glycine, and complete loss of affinity for IGF-I and IGF-II occurred when all three targeted amino acids were changed to glycine. Furthermore, the ability of the IGF-binding protein-3 mutants to inhibit IGF-I-stimulated phosphorylation of its receptor was a reflection of their affinity for IGF, with the lowest affinity mutants having the least inhibitory effect. These studies, thus, support the hypothesis that an N-terminal hydrophobic pocket is the primary site of high affinity binding of IGF to IGF-binding protein-3. The mutants provide a tool for future studies directed at IGF-dependent and IGF-independent actions of IGF-binding protein-3.

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