High-affinity ligand binding by wild-type/mutant heteromeric complexes of the mannose 6-phosphate/insulin-like growth factor II receptor

Michelle A. Hartman, Jodi L. Kreiling, James C. Byrd, Richard G MacDonald

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

The mannose 6-phosphate/insulin-like growth factor II receptor has diverse ligand-binding properties contributing to its roles in lysosome biogenesis and growth suppression. Optimal receptor binding and internalization of mannose 6-phosphate (Man-6-P)-bearing ligands requires a dimeric structure leading to bivalent high-affinity binding, presumably mediated by cooperation between sites on both subunits. Insulin-like growth factor II (IGF-II) binds to a single site on each monomer. It is hypothesized that IGF-II binding to cognate sites on each monomer occurs independently, but bivalent Man-6-P ligand binding requires cooperative contributions from sites on both monomers. To test this hypothesis, we co-immunoprecipitated differentially epitope-tagged soluble mini-receptors and assessed ligand binding. Pairing of wild-type and point-mutated IGF-II binding sites between two dimerized mini-receptors had no effect on the function of the contralateral binding site, indicating IGF-II binding to each side of the dimer is independent and manifests no intersubunit effects. As expected, heterodimeric receptors composed of a wild-type monomer and a mutant bearing two Man-6-P-binding knockout mutations form functional IGF-II binding sites. By contrast to prediction, such heterodimeric receptors also bind Man-6-P-based ligands with high affinity, and the amount of binding can be attributed entirely to the immunoprecipitated wild-type receptors. Anchoring of both C-terminal ends of the heterodimer produces optimal binding of both IGF-II and Man-6-P ligands. Thus, IGF-II binds independently to both subunits of the dimeric mannose 6-phosphate/insulin-like growth factor II receptor. Although wild-type/mutant hetero-oligomers form readily when mixed, it appears that multivalent Man-6-P ligands bind preferentially to wild-type sites, possibly by cross-bridging receptors within clusters of immobilized receptors.

Original languageEnglish (US)
Pages (from-to)1915-1929
Number of pages15
JournalFEBS Journal
Volume276
Issue number7
DOIs
StatePublished - Apr 1 2009

Fingerprint

IGF Type 2 Receptor
Insulin-Like Growth Factor II
Ligands
Bearings (structural)
Monomers
Binding Sites
mannose-6-phosphate
Lysosomes
Oligomers
Dimers
Epitopes
Mutation

Keywords

  • Insulin-like growth factor II
  • Ligand binding
  • Mannose 6-phosphate
  • Mannose 6-phosphate/insulin-like growth factor II receptor
  • Oligomerization

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

High-affinity ligand binding by wild-type/mutant heteromeric complexes of the mannose 6-phosphate/insulin-like growth factor II receptor. / Hartman, Michelle A.; Kreiling, Jodi L.; Byrd, James C.; MacDonald, Richard G.

In: FEBS Journal, Vol. 276, No. 7, 01.04.2009, p. 1915-1929.

Research output: Contribution to journalArticle

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AB - The mannose 6-phosphate/insulin-like growth factor II receptor has diverse ligand-binding properties contributing to its roles in lysosome biogenesis and growth suppression. Optimal receptor binding and internalization of mannose 6-phosphate (Man-6-P)-bearing ligands requires a dimeric structure leading to bivalent high-affinity binding, presumably mediated by cooperation between sites on both subunits. Insulin-like growth factor II (IGF-II) binds to a single site on each monomer. It is hypothesized that IGF-II binding to cognate sites on each monomer occurs independently, but bivalent Man-6-P ligand binding requires cooperative contributions from sites on both monomers. To test this hypothesis, we co-immunoprecipitated differentially epitope-tagged soluble mini-receptors and assessed ligand binding. Pairing of wild-type and point-mutated IGF-II binding sites between two dimerized mini-receptors had no effect on the function of the contralateral binding site, indicating IGF-II binding to each side of the dimer is independent and manifests no intersubunit effects. As expected, heterodimeric receptors composed of a wild-type monomer and a mutant bearing two Man-6-P-binding knockout mutations form functional IGF-II binding sites. By contrast to prediction, such heterodimeric receptors also bind Man-6-P-based ligands with high affinity, and the amount of binding can be attributed entirely to the immunoprecipitated wild-type receptors. Anchoring of both C-terminal ends of the heterodimer produces optimal binding of both IGF-II and Man-6-P ligands. Thus, IGF-II binds independently to both subunits of the dimeric mannose 6-phosphate/insulin-like growth factor II receptor. Although wild-type/mutant hetero-oligomers form readily when mixed, it appears that multivalent Man-6-P ligands bind preferentially to wild-type sites, possibly by cross-bridging receptors within clusters of immobilized receptors.

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