The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of beta-glucuronidase.

Abstract

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 ,3-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 P-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 ,B-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized P-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.

PAQR10 and PAQR11 mediate Ras signaling in the Golgi apparatus

“…**P < 0.01 between control group and PAQR10/PAQR11 siRNA group. Figure S6C), which was previously reported to traffic through the Golgi apparatus [39]. In MDCK cells, PAQR10 also elevated Golgi localization of HRas, NRas and KRas4A, but not KRas4B (Supplementary information, Figure S7).…”

Structural Insights into the Mechanism of pH-dependent Ligand Binding and Release by the Cation-dependent Mannose 6-Phosphate Receptor

“…The CD-MPR displays a bell-shaped carbohydrate binding profile that is influenced by pH, with optimum binding occurring at pH 6.4 and little or no binding at pH values above pH 7.5 or below pH 5.5 (5,7,28). To identify those regions of the CD-MPR that may undergo structural perturbations dependent upon pH, the receptor was crystallized under different pH conditions, and comparative analyses of all structures were performed.…”

“…Both MPRs display optimal ligand binding at pH ϳ6.5 and no detectable binding below pH ϳ5, which is consistent with their function of binding their cargo in the trans Golgi network and subsequently releasing their ligands in the acidic environment of endosomes. The lysosomal enzymes are packaged into lysosomes, whereas the MPRs recycle back to the trans Golgi network to retrieve additional hydrolytic enzymes from the secretory pathway (1)(2)(3) or move to the cell surface where the CI-MPR, but not the CD-MPR, binds and internalizes lysosomal enzymes (4,5). In vitro binding studies support these observations, demonstrating that the CI-MPR retains phosphomannosyl binding capabilities at neutral pH, whereas the CD-MPR displays a bell-shaped pH-sensitive binding profile, with optimum binding occurring at pH ϳ6.…”

Twists and Turns of the Cation-dependent Mannose 6-Phosphate Receptor

“…Thus, it is not unreasonable to assume that His 105 , the only residue in which a side chain is involved in the binding of the phosphate moiety of the ligand, is accountable for the basic side of the maximum of the pH dependence. It is interesting to note that residues 102-105 (Tyr-Asp-AsnHis) are missing in the sequences of the two Man-6-P recognition sites of the IGF-II/CI-MPR (5), which, unlike the CD-MPR, binds the Man-6-P ligand at the cell surface where the pH is around 7.4 (38,40). The combination of the two pH effects are responsible for the optimal binding of the CD-MPR to Man-6-P at a pH value around 6.5.…”