The Two Mannose 6-Phosphate Receptors Transport Distinct Complements of Lysosomal Proteins

Abstract

Mammalian cells express two different mannose 6-phosphate receptors (MPR 46 and MPR 300), which both mediate targeting of Man-6-P-containing lysosomal proteins to lysosomes. To assess the contribution of either and both MPRs to the transport of lysosomal proteins, fibroblasts were established from mouse embryos that were homozygous for disrupted alleles of either MPR 46 or MPR 300 or both MPRs. Fibroblasts missing both MPRs secreted most of the newly synthesized lysosomal proteins and were unable to maintain the catabolic function of lysosomes. The intracellular levels of lysosomal proteins decreased to <20%, and undigested material accumulated in the lysosomal compartment. Fibroblasts lacking either MPR exhibited only a partial missorting and maintained, in general, half-normal to normal levels of lysosomal proteins. The same species of lysosomal proteins were found in secretions of double MPR-deficient fibroblasts as in secretions of single MPR-deficient fibroblasts, but at different ratios. This clearly indicates that neither MPR has an exclusive affinity for one or several lysosomal proteins. Furthermore, neither MPR can substitute in vivo for the loss of the other. It is proposed that the heterogeneity of the Man-6-P recognition marker within a lysosomal protein and among different lysosomal proteins has necessitated the evolution of two MPRs with complementary binding properties to ensure an efficient targeting of lysosomal proteins.

Serine phosphorylation site of the 46-kDa mannose 6-phosphate receptor is required for transport to the plasma membrane in Madin-Darby canine kidney and mouse fibroblast cells.

“…The cells were solubilized in 1 N NaOH, and the cell-associated radioactivity was determined with respect to cell protein (Lowry et al, 1951). Immunoprecipitation of cathepsin D of pulse-labeled-transfected, MPR-/ -MEFs were carried out as described (Hemer et al, 1993;Pohlmann et al, 1995 Due to the low MPR46 expression at the cell surface, the cycling of receptors via the plasma membrane was measured by the more sensitive endocytosis assay in which the uptake of the 125I-labeled 21D3 antibodies during an incubation at 37°C is measured. Figure 4 shows that the amount of endocytosed 125I-labeled antibodies in MDCK cells expressing the WT MPR46 increased with the time of incubation.…”

The mannose 6‐phosphate/insulin‐like growth factor II receptor restricts the tumourigenicity and invasiveness of squamous cell carcinoma cells

“…[8][9][10] Lysosomal sorting via M6P/IGF2R is generally far more efficient than by MPR46, demonstrating that the former is the main lysosomal targeting receptor in mammalian cells. 11,12 However, M6P/IGF2R also binds a variety of other factors that impinge on the proliferation, migration and invasiveness of tumour cells, including insulin-like growth factor II (IGF-II), 13 transforming growth factor b, 14 urokinase-type plasminogen activator receptor 15 and plasminogen. 16 Hence, it is of high relevance that the M6P/IGF2R gene is frequently inactivated in human and animal tumours.…”

A replacement of the active-site aspartic acid residue 293 in mouse cathepsin D affects its intracellular stability, processing and transport in HEK-293 cells

“…Immunoblotting experiments using mCTSD-specific antibodies indicated the predominance of a 45 kDa form with a minor band of 43 kDa in the overexpressing HEK-293 cells. The proteolytically processed 30 kDa mature mCTSD, which was earlier shown to be present in small amounts in mouse splenocytes, hepatocytes or fibroblasts [11,36], was never observed in the overexpressing HEK-293 cells. The mutant D293N mCTSD was inactive in HEK-293 cells, as expected, thus being in agreement with the earlier observations on the cathepsin D-deficient sheep [31], and the mutant D295N human CTSD [32].…”