The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of beta-glucuronidase.
We studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human beta-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the presence of EDTA but was enhanced by added divalent cations. Up to 2.3% of the total functional receptor could be detected on the cell surface by enzyme binding. We present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of beta-glucuronidase. At pH 7.5, the rate of endocytosis was only 14% the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized beta-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized beta-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.
Watanabe
H
H
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, MO 63104.
Grubb
J H
JH
Sly
W S
WS
D000818:Animals; D002413:Cations, Divalent; D002463:Cell Membrane Permeability; D002846:Chromatography, Affinity; D004705:Endocytosis; D005966:Glucuronidase / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007739:L Cells / Q000378:metabolism; D008360:Mannosephosphates / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D011485:Protein Binding; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D002413:Cations, Divalent; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D005966:Glucuronidase