Mechanisms for high affinity mannose 6-phosphate ligand binding to the insulin-like growth factor II/mannose 6-phosphate receptor. Negative cooperativity and receptor oligomerization

James C. Byrd, Richard G MacDonald

Research output: Contribution to journalArticle

38 Scopus citations

Abstract

The two mannose 6-phosphate (Man-6-P) binding domains of the insulin- like growth factor II/mannose 6-phosphate receptor (Man-6-P/IGF2R), located in extra-cytoplasmic repeats 1-3 and 7-9, are capable of binding Man-6-P with low affinity and glycoproteins that contain more than one Man-6-P residue with high affinity. High affinity multivalent ligand binding sites could be formed through two possible mechanisms: the interaction of two Man-6-P binding domains within one Man-6-P/IGF2R molecule or by receptor oligomerization. To discriminate between these mechanisms, truncated FLAG epitope-tagged Man-6-P/IGF2R constructs, containing one or both of the Man-6- P binding domains, were expressed in 293T cells, and characterized for binding of pentamannose phosphate-bovine serum albumin (PMP-BSA), a pseudoglycoprotein bearing multiple Man-6-P residues. A construct containing all 15 repeats of the Man-6-P/IGF2R extracytoplasmic domain bound PMP-BSA with the same affinity as the full-length receptor (K(d) = 0.54 nM) with a curvilinear Scatchard plot. The presence of excess unlabeled PMP-BSA increased the dissociation rate of pre-formed 125I-PMP-BSA/receptor complexes, suggesting negative cooperativity in multivalent ligand binding and affirming the role of multiple Man-6-P/IGF2R binding domains in forming high affinity binding sites. Truncated receptors containing only one Man-6-P binding domain and mutant receptor constructs, containing an Arg1325 → Ala mutation that eliminates binding to the repeats 7-9 binding domain, formed high affinity PMP-BSA binding, but with reduced stoichiometries. Collectively, these observations suggest that alignment of Man-6-P binding domains of separate Man-6-P/IGF2R molecules is responsible for the formation of high affinity Man-6-P binding sites and provide functional evidence for Man-6-P/IGF2R oligomerization.

Original languageEnglish (US)
Pages (from-to)18638-18646
Number of pages9
JournalJournal of Biological Chemistry
Volume275
Issue number25
DOIs
Publication statusPublished - Jun 23 2000

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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