An insulin-like growth factor II (IGF-II) affinity-enhancing domain localized within extracytoplasmic repeat 13 of the IGF-II/Mannose 6-phosphate receptor

Gayathri R. Devi, James C. Byrd, Dorothy H. Slentz, Richard G MacDonald

Research output: Contribution to journalArticle

52 Citations (Scopus)

Abstract

Insulin-like growth factor II (IGF-II) and phosphomannosylated glycoproteins bind to distinct sites on the same receptor, the IGF- II/mannose 6-phosphate receptor (IGF2R). Analysis of truncated receptors (minireceptors) has been used to map the IGF-II binding site within the receptor's extracytoplasmic domain, which consists of 15 homologous repeats. A minireceptor consisting of repeat 11 contained the minimal elements for binding IGF-II, but with 5- to 10-fold lower relative binding affinity than the full-length receptor. We hypothesized that the complete, high-affinity IGF-II binding site is formed by interaction between the primary site in repeat 11 and a putative affinity-enhancing domain. To determine the minimum portion of the IGF2R's extracytoplasmic domain needed for expression of high- affinity IGF-II binding, a nested set of FLAG epitope-tagged minireceptors encompassing repeats 11 through 15 was prepared and transiently expressed in 293T cells. Minireceptors containing repeats 11-13 or 11-15 exhibited high affinity, comparable to the full-length receptor (IC50 = 1-2 nM), whereas constructs containing repeat 11 only or repeats 11-12 did not (IC50 = 10- 20 nM). These data suggested that the affinity-enhancing domain is located within repeat 13, which contains a unique 43-residue insert that has ~50% sequence identity to the type II repeat of fibronectin. Although a repeat 13 minireceptor did not bind IGF-II on its own, an 11-13 minireceptor containing a deletion of the 43-residue insert exhibited low IGF-II binding affinity (IC50 = 10-20 nM). Expression of mutant receptors from a full-length IGF2R construct bearing a deletion of the 43-residue insert was very low relative to wild type. Depletion assays using IGF-II-Sepharose showed that the mutant receptor had lower affinity for IGF-II than the wild-type receptor. This study reveals that two independent receptor domains are involved in the formation of a high-affinity binding site for IGF-II, and that a complete repeat 13 is required for high-affinity IGF-II binding.

Original languageEnglish (US)
Pages (from-to)1661-1672
Number of pages12
JournalMolecular Endocrinology
Volume12
Issue number11
DOIs
StatePublished - Jan 1 1998

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IGF Type 2 Receptor
Insulin-Like Growth Factor II
Inhibitory Concentration 50
Binding Sites
HEK293 Cells
Fibronectins
Sepharose
Epitopes
Glycoproteins

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology

Cite this

An insulin-like growth factor II (IGF-II) affinity-enhancing domain localized within extracytoplasmic repeat 13 of the IGF-II/Mannose 6-phosphate receptor. / Devi, Gayathri R.; Byrd, James C.; Slentz, Dorothy H.; MacDonald, Richard G.

In: Molecular Endocrinology, Vol. 12, No. 11, 01.01.1998, p. 1661-1672.

Research output: Contribution to journalArticle

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abstract = "Insulin-like growth factor II (IGF-II) and phosphomannosylated glycoproteins bind to distinct sites on the same receptor, the IGF- II/mannose 6-phosphate receptor (IGF2R). Analysis of truncated receptors (minireceptors) has been used to map the IGF-II binding site within the receptor's extracytoplasmic domain, which consists of 15 homologous repeats. A minireceptor consisting of repeat 11 contained the minimal elements for binding IGF-II, but with 5- to 10-fold lower relative binding affinity than the full-length receptor. We hypothesized that the complete, high-affinity IGF-II binding site is formed by interaction between the primary site in repeat 11 and a putative affinity-enhancing domain. To determine the minimum portion of the IGF2R's extracytoplasmic domain needed for expression of high- affinity IGF-II binding, a nested set of FLAG epitope-tagged minireceptors encompassing repeats 11 through 15 was prepared and transiently expressed in 293T cells. Minireceptors containing repeats 11-13 or 11-15 exhibited high affinity, comparable to the full-length receptor (IC50 = 1-2 nM), whereas constructs containing repeat 11 only or repeats 11-12 did not (IC50 = 10- 20 nM). These data suggested that the affinity-enhancing domain is located within repeat 13, which contains a unique 43-residue insert that has ~50{\%} sequence identity to the type II repeat of fibronectin. Although a repeat 13 minireceptor did not bind IGF-II on its own, an 11-13 minireceptor containing a deletion of the 43-residue insert exhibited low IGF-II binding affinity (IC50 = 10-20 nM). Expression of mutant receptors from a full-length IGF2R construct bearing a deletion of the 43-residue insert was very low relative to wild type. Depletion assays using IGF-II-Sepharose showed that the mutant receptor had lower affinity for IGF-II than the wild-type receptor. This study reveals that two independent receptor domains are involved in the formation of a high-affinity binding site for IGF-II, and that a complete repeat 13 is required for high-affinity IGF-II binding.",
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