Divalent cation-dependent stimulation of ligand binding to the 46-kDa mannose 6-phosphate receptor correlates with divalent cation-dependent tetramerization.
The quaternary structure and binding activity of the murine 46-kDa mannose 6-phosphate receptor (46MPR) were studied in semi-intact murine cells that overexpress the murine receptor. Chemical cross-linking studies showed that the murine 46MPR exists in monomer, dimer, and tetramer forms in membranes of overexpressing murine cells. Treatment of permeabilized cells with Mn2+ increased the tetramer form of 46MPR, and this tetramerization was reversed by removal of Mn2+. Thus, the divalent cations affected the distribution of receptor among the three forms, favoring tetramerization at the expense of dimer and monomer. Low temperature (4 degrees C) also increases the fraction present as tetramer. The binding assay results show that Mn2+ is required for the 46MPR to achieve and retain the ability to bind ligand at 37 degrees C but not at 4 degrees C. Preincubation with Mn2+ produced a 3-fold increase in Man-6-P-specific binding of beta-glucuronidase which paralleled the 3-fold increase in tetramer seen during preincubation with Mn2+. The similarity of the effects of addition and removal of Mn2+ on enzyme binding to the effects of Mn2+ on favoring tetramer formation suggests that divalent cation-dependent tetramerization of the 46MPR contributes to the stimulation of ligand binding to the 46MPR by divalent cations.
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Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, Missouri 63104.
Grubb
J H
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Sly
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D000818:Animals; D002413:Cations, Divalent; D002460:Cell Line; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005966:Glucuronidase / Q000378:metabolism; D008024:Ligands; D046911:Macromolecular Substances; D008345:Manganese / Q000737:chemistry; D008360:Mannosephosphates / Q000378:metabolism*; D051379:Mice; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*
D002413:Cations, Divalent; D003432:Cross-Linking Reagents; D008024:Ligands; D046911:Macromolecular Substances; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D008345:Manganese; D005966:Glucuronidase