46-kDa Mannose 6-phosphate Receptor Research Articles

The 46-kDa mannose 6-phosphate receptor does not depend on endosomal acidification for delivery of hydrolases to lysosomes

In mammalian cells, the mannose 6-phosphate receptor pathway accounts for the transport of most soluble acid hydrolases to lysosomes. It is believed that dissociation of mannose 6-phosphate receptors and their ligands is entirely driven by the acidic environment in endosomal compartments. Indeed, pH-perturbing substances such as ammonium chloride and monensin have been shown to inhibit lysosomal enzyme targeting in cells that express both known mannose 6-phosphate receptors. We now demonstrate that ammonium chloride and monensin exert modest effects on the intracellular retention of lysosomal hydrolases in murine cells that synthesize only the 46-kDa mannose 6-phosphate receptor. Neither ammonium chloride nor monensin induces changes to the subcellular localization of lysosomal hydrolases and the 46-kDa mannose 6-phosphate receptor in these cells. This suggests that endosomal dissociation of the receptor and its ligands still occurs in the presence of these agents. We conclude that the murine 46-kDa mannose 6-phosphate receptor has the capacity to deliver its cargo proteins to lysosomes even in the absence of endosomal acidification.

Identification of residues essential for carbohydrate recognition and cation dependence of the 46-kDa mannose 6-phosphate receptor

The 46 kDa cation-dependent mannose 6-phosphate receptor (CD-MPR) plays an essential role in the biogenesis of lysosomes by diverting newly synthesized mannose 6-phosphate (Man-6-P)-containing lysosomal enzymes from the secretory pathway to acidified endosomes. Previous crystallographic studies of the CD-MPR have identified 11 amino acids within its carbohydrate binding pocket. These residues were evaluated quantitatively by assaying the binding affinity of mutant receptors containing a single amino acid substitution toward a lysosomal enzyme. The results show that substitution of Gln-66, Arg-111, Glu-133, or Tyr-143 results in a >800-fold decrease in affinity, demonstrating these four amino acids are essential for carbohydrate recognition by the CD-MPR. Solution binding and surface plasmon resonance analyses demonstrated that the presence of Mn2+ enhanced the affinity of the CD-MPR for a lysosomal enzyme by 2- to 4-fold and increased the stoichiometry of the interaction between a heterogeneous population of a lysosomal enzyme and the receptor by approximately 3-fold. In contrast, substitution of Asp-103 results in a protein that no longer exhibits enhanced binding affinities or altered stoichiometry in the presence of cations, and electron spin resonance demonstrated that the D103S mutant exhibits a 6-fold lower affinity for Mn2+ than the wild-type receptor (Kd = 3.7 6 1.4 mM versus 0.6 6 0.1 mM). Chemical cross-linking revealed that Mn2+ influences the stoichiometry of interaction between the CD-MPR and lysosomal enzymes by increasing the oligomeric state of the receptor from dimer to higher order oligomers. Taken together, these studies provide the molecular basis for high affinity carbohydrate recognition by the CD-MPR. Furthermore, Asp-103 has been identified as the key residue which mediates the effects of divalent cations on the binding properties of the CD-MPR.

Role of LAMP-2 in lysosome biogenesis and autophagy.

In LAMP-2-deficient mice autophagic vacuoles accumulate in many tissues, including liver, pancreas, muscle, and heart. Here we extend the phenotype analysis using cultured hepatocytes. In LAMP-2-deficient hepatocytes the half-life of both early and late autophagic vacuoles was prolonged as evaluated by quantitative electron microscopy. However, an endocytic tracer reached the autophagic vacuoles, indicating delivery of endo/lysosomal constituents to autophagic vacuoles. Enzyme activity measurements showed that the trafficking of some lysosomal enzymes to lysosomes was impaired. Immunoprecipitation of metabolically labeled cathepsin D indicated reduced intracellular retention and processing in the knockout cells. The steady-state level of 300-kDa mannose 6-phosphate receptor was slightly lower in LAMP-2-deficient hepatocytes, whereas that of 46-kDa mannose 6-phosphate receptor was decreased to 30% of controls due to a shorter half-life. Less receptor was found in the Golgi region and in vesicles and tubules surrounding multivesicular endosomes, suggesting impaired recycling from endosomes to the Golgi. More receptor was found in autophagic vacuoles, which may explain its shorter half-life. Our data indicate that in hepatocytes LAMP-2 deficiency either directly or indirectly leads to impaired recycling of 46-kDa mannose 6-phosphate receptors and partial mistargeting of a subset of lysosomal enzymes. Autophagic vacuoles may accumulate due to impaired capacity for lysosomal degradation.

Multiple C-terminal Motifs of the 46-kDa Mannose 6-Phosphate Receptor Tail Contribute to Efficient Binding of Medium Chains of AP-2 and AP-3

The interaction of adaptor protein (AP) complexes with signal structures in the cytoplasmic domains of membrane proteins is required for intracellular sorting. Tyrosine- or dileucine-based motifs have been reported to bind to medium chain subunits (mu) of AP-1, AP-2, or AP-3. In the present study, we have examined the interaction of the entire 67-amino acid cytoplasmic domain of the 46-kDa mannose 6-phosphate receptor (MPR46-CT) containing tyrosine- as well as dileucine-based motifs with mu2 and mu3A chains using the yeast two-hybrid system. Both mu2 and mu3A bind specifically to the MPR46-CT. In contrast, mu3A fails to bind to the cytoplasmic domain of the 300-kDa mannose 6-phosphate receptor. Mutational analysis of the MPR46-CT revealed that the tyrosine-based motif and distal sequences rich in acidic amino acid residues are sufficient for effective binding to mu2. However, the dileucine motif was found to be one part of a consecutive complex C-terminal structure comprising tyrosine and dileucine motifs as well as clusters of acidic residues necessary for efficient binding of mu3A. Alanine substitution of 2 or 4 acidic amino acid residues of this cluster reduces the binding to mu3A much more than to mu2. The data suggest that the MPR46 is capable of interacting with different AP complexes using multiple partially overlapping sorting signals, which might depend on posttranslational modifications or subcellular localization of the receptor.

Stabilization of Mutant 46-kDa Mannose 6-Phosphate Receptors by Proteasomal Inhibitor Lactacystin

Palmitoylation of cysteine residue 34 within the 67-amino acid cytoplasmic domain of the 46-kDa mannose 6-phosphate receptor (MPR 46), which may be anchored to the lipid bilayer, prevents the receptor from entering lysosomes (Schweizer, A., Kornfeld, S., and Rohrer, J. (1996) J. Cell Biol. 132, 577-584). In the present study, we examined the importance of the spacing between the transmembrane domain and the palmitoylation anchor site in the cytoplasmic domain for stability and trafficking of MPR 46. MPR 46 mutants with deletions of residues 20-23 and 24-29 expressed in baby hamster kidney cells were rapidly degraded with half-lives of less than 10 h. The replacement of residues 24-29 by alanine resulted in prolongation of receptor stability (t(1)/(2) approximately 20 h). Whereas mutant MPR 46 could not be detected in lysosomal fractions and inhibitors of lysosomal proteases failed to prevent degradation, treatment with the proteasome inhibitor lactacystin resulted in increased stability of mutant MPR 46. Pulse-chase experiments at low temperature and the acquirement of endoglucosaminidase H-resistant oligosaccharides indicate that the majority of mutant MPR 46 is degraded after leaving the Golgi compartment. Altered trafficking of mutant MPR 46 may be the result of decreased palmitoylation reaching 40% of wild type receptors. The data suggest that the spacing between the transmembrane domain and the proposed palmitoylation anchor site in the cytoplasmic domain of MPR 46 is important for a post Golgi sorting step preventing receptor degradation by multiple proteolytic systems including the proteasome.

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.

Up to 4% of the human 46-kDa mannose 6-phosphate receptor (MPR46) expressed in Madin-Darby canine kidney (MDCK) cells are localized at the cell surface. At steady state, the expression of MPR46 on the apical surface of filter-grown MDCK cells is about sixfold lower than on the basolateral surface. The cytoplasmic domain of the MPR46 is phosphorylated on serine 56 at low stoichiometry. By expressing mutant MPR46 we have shown that the MPR46 phosphorylation site is required for delivery to the plasma membrane. In addition, mutant MPR46 expressed in MPR-deficient mouse embryonic fibroblasts were not detected at the cell surface and their ability to sort newly synthesized cathepsin D was not altered. Since the loss of MPR46 phosphorylation correlates with the lack of cell surface expression, phosphorylation of serine 56 may either function as a direct plasma membrane targeting signal or inhibit MPR46 recycling from endosomes to Golgi, resulting in trafficking to the cell surface.

The oligomeric state of 46-kDa mannose 6-phosphate receptor does not change upon intracellular recycling and binding of ligands.

46-kDa mannose-6-phosphate receptor forms homooligomers in cell membranes and in detergent solution. The quaternary structure of detergent-solubilized 46-kDa mannose 6-phosphate receptor is regulated by the presence of ligands, pH and receptor concentration [Waheed, A. & von Figura, K. (1990) Eur. J. Biochem. 193, 47-54). To find out whether the intracellular recycling of 46-kDa mannose 6-phosphate receptor is accompanied by changes in its quaternary structure, we have performed chemical cross-linking in membranes of intact cells. In all conditions tested, the dimer was the predominating form (more than 67% of total 46-kDa mannose 6-phosphate receptor). The amount of trimeric and tetrameric forms varied among cell lines and contributed up to 20% of total endogenous 46-kDa mannose 6-phosphate receptor in human and mouse fibroblasts. Within a given cell line, the ratio of the oligomers was not significantly changed upon elevating endosomal pH by bafilomycin A1, upon changes in receptor occupancy (treatment of cells with tunicamycin or use of mouse fibroblasts deficient in 300-kDa mannose 6-phosphate receptor), nor upon depletion of adaptors from clathrin-coated vesicles of the trans Golgi network by brefeldin A. At the cell surface, where 46-kDa mannose 6-phosphate receptor does not bind ligands, the percentage of dimer was similar to that observed intracellularly. Thus, the oligomeric state of 46-kDa mannose 6-phosphate receptor apparently does not change during recycling as well as binding and dissociation of ligands. In view of the abundance of the dimer of 46-kDa mannose 6-phosphate receptor in situ, our data suggest that it represents the main physiologically active form of the receptor, and therefore present indirect evidence that binding of ligands to 46-kDa mannose 6-phosphate receptor is probably regulated by conformational changes of receptor or ligand rather than by changes in the quaternary structure.

Role of mannose-6-phosphate receptors in herpes simplex virus entry into cells and cell-to-cell transmission.

Herpes simplex virus (HSV) glycoprotein D (gD) is essential for virus entry into cells, is modified with mannose-6-phosphate (M-6-P), and binds to both the 275-kDa M-6-P receptor (MPR) and the 46-kDa MPR (C. R. Brunetti, R. L. Burke, S. Kornfeld, W. Gregory, K. S. Dingwell, F. Masiarz, and D. C. Johnson, J. Biol. Chem. 269:17067-17074, 1994). Since MPRs are found on the surfaces of mammalian cells, we tested the hypothesis that MPRs could serve as receptors for HSV during virus entry into cells. A soluble form of the 275-kDa MPR, derived from fetal bovine serum, inhibited HSV plaques on monkey Vero cells, as did polyclonal rabbit anti-MPR antibodies. In addition, the number and size of HSV plaques were reduced when cells were treated with bovine serum albumin conjugated with pentamannose-phosphate (PM-PO4-BSA), a bulky ligand which can serve as a high-affinity ligand for MPRs. These data imply that HSV can use MPRs to enter cells; however, other molecules must also serve as receptors for HSV because a reasonable fraction of virus could enter cells treated with even the highest concentrations of these inhibitors. Consistent with the possibility that there are other receptors, HSV produced the same number of plaques on MPR-deficient mouse fibroblasts as were produced on normal mouse fibroblasts, but there was no inhibition with PM-PO4-BSA with either of these embryonic mouse cells. Together, these results demonstrate that HSV does not rely solely on MPRs to enter cells, although MPRs apparently play some role in virus entry into some cell types and, perhaps, act as one of a number of cell surface molecules that can facilitate entry. We also found that HSV produced small plaques on human fibroblasts derived from patients with pseudo-Hurler's polydystrophy, cells in which glycoproteins are not modified with M-6-P residues and yet production of infectious HSV particles was not altered in the pseudo-Hurler cells. In addition, HSV plaque size was reduced by PM-PO4-BSA; therefore, it appears that M-6-P residues and MPRs are required for efficient transmission of HSV between cells, a process which differs in some respects from entry of exogenous virus particles.

Localization of the Insulin-like Growth Factor II Binding Site to Amino Acids 1508–1566 in Repeat 11 of the Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) binds insulin-like growth factor II (IGF-II) with high affinity. To localize the IGF-II binding site within the 15 repeating units that form the extracytoplasmic domain of the receptor, purified human M6P/IGF-II receptor was digested with thermolysin, and the fragments were analyzed for their ability to bind 125I-IGF-II in a cross-linking assay. Two IGF-II-binding receptor fragments of 23 and 37 kDa were purified. Sequence analysis revealed that the fragments consist of disulfide connected peptides comprising amino acids 1331-1566 and 1331-1697 of the receptor repeats 9-12. In a second approach we expressed truncated forms of the M6P/IGF-II receptor fused to the C terminus of the extracytoplasmic domain of the 46-kDa mannose 6-phosphate receptor. Fusion proteins containing M6P/IGF-II receptor repeats 10-15, 10-11, or 11-15 bound IGF-II, whereas a fusion protein containing the single repeat 10 failed to bind. This result indicates that repeat 11 (amino acids 1508-1650) is sufficient for binding of IGF-II. Residues 1508-1566, which are shared by the 23-kDa IGF-II-binding fragment and repeat 11, are proposed to form the IGF-II binding site of the M6P/IGF-II receptor.

In vitro phosphorylation of the 46-kDa mannose 6-phosphate receptor by casein kinase II. Structural requirements for efficient phosphorylation

Some steps in the receptor-mediated transport of newly synthesized mannose 6-phosphate-containing lysosomal enzymes are assumed to be accompanied by changes in the phosphorylation state of receptors. In vitro, the metabolically phosphorylated 46-kDa mannose 6-phosphate receptor (MPR 46) was dephosphorylated by protein phosphatase 2A. The synthetic cytoplasmic domain of MPR 46 was phosphorylated in vitro by casein kinase II. Tryptic phosphopeptide mapping showed that casein kinase II phosphorylates MPR 46 in vitro at the same site that is phosphorylated in vivo. Inhibition studies using synthetic peptides corresponding to different amino acid sequences of the cytoplasmic tail of MPR 46 revealed that the sequence 26-32 (ADGCDFV) contribute to efficient phosphorylation of serine 56. Baby hamster kidney cells were transfected with wild type human MPR 46 cDNA or cDNAs containing mutations in the cytoplasmic tail and assayed for their phosphorylation state in vivo. The phosphorylation of mutant receptors with deleted residues 23-28 (NLVADG) was strongly reduced. These data indicate that residues on the N-terminal side of the phosphorylatable serine 56 may influence the efficiency with which a casein-kinase II-like kinase phosphorylates MPR 46.

Herpes simplex virus glycoprotein D acquires mannose 6-phosphate residues and binds to mannose 6-phosphate receptors.

Herpes simplex viruses (HSV) use multiple and sequential receptors to enter host cells. HSV glycoprotein D (gD) has been implicated in binding to cellular receptors that facilitate virus penetration into cells. We used soluble forms of gD that were expressed in Chinese hamster ovary cells to characterize and identify a putative cellular receptor for HSV as the 275-kDa mannose 6-phosphate/insulin-like growth factor II receptor. Soluble gD also bound to the 46-kDa cation-dependent mannose 6-phosphate (Man-6-P) receptor and was extensively modified with Man-6-P residues on its Asn-linked oligosaccharides. Additionally, soluble gD was a high affinity substrate for N-acetylglucosamine-1-phosphotransferase, the first enzyme in the biosynthetic pathway for the addition of Man-6-P residues to lysosomal enzymes. The membrane form of gD immunoprecipitated from HSV-infected cells also bound to the 275-kDa mannose 6-phosphate/insulin-like growth factor II receptor, albeit poorly, and only a small fraction of the membrane gD was modified with Man-6-P. Notwithstanding this low level of mannose phosphorylation, the interaction between gD and Man-6-P receptors may play a role in some aspect of virus entry or egress.

Phosphorylation of the human 46-kDa mannose 6-phosphate receptor in the cytoplasmic domain at serine 56

The human 46-kDa mannose 6-phosphate receptor (MPR46) is phosphorylated in its cytoplasmic domain at serine residues. Substitution of cytoplasmic serines (at positions 35 and 56) with alanine, expression of mutant receptors in baby hamster kidney cells, and phosphopeptide mapping revealed that serine 56 is phosphorylated. Mutant MPR46 and wild-type MPR46 were found to be similarly distributed between the cell surface and intracellular membranes. Phosphate incorporation in the presence of cycloheximide indicates that phosphorylation occurred on pre-existing MPR46. Similar half-lives for the wild-type and mutant receptor proteins (approximately 43 h) and the receptor-associated phosphate (1.4 h) were found. The mutant receptors were internalized at the same rate as the wild-type receptors. Expression of mutant MPR46 and wild-type MPR46 in mouse L-cells deficient in 300-kDa mannose 6-phosphate receptors did not affect the sorting of newly synthesized cathepsin D to lysosomes. Phosphorylation of cytoplasmic serine 56 is therefore essential neither for stability nor for cell-surface expression and transport activities of MPR46.

In vitro binding of plasma membrane-coated vesicle adaptors to the cytoplasmic domain of lysosomal acid phosphatase

Sorting of the newly synthesized membrane-bound precursor of lysosomal acid phosphatase (LAP) involves internalization from the plasma membrane via clathrin-coated pits. Using an in vitro system, we present direct evidence for high affinity interaction of the cytoplasmic domain of LAP with the amino-terminal trunk portion of plasma membrane-coated vesicle adaptors. Coated vesicle adaptors of the trans-Golgi network displayed poor binding to LAP, but high affinity binding to the cytoplasmic tail of the 46-kDa mannose 6-phosphate receptor, which is included in clathrin-coated pits of the trans-Golgi network. Binding of plasma membrane adaptors to the tail peptide of LAP required an internalization signal that contains either tyrosine or phenylalanine.

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.

Cloning, sequencing, and functional characterization of the murine 46-kDa mannose 6-phosphate receptor

We have cloned and sequenced the 2175-nucleotide, full-length cDNA for the mouse 46-kDa Man 6-P receptor (46MPR) and studied its functional properties in stably transfected mouse L cells which do not express the insulin-like growth factor-II receptor/mannose 6-phosphate receptor (IGF-IIR/MPR). The 278-amino acid sequence deduced from the cDNA for the murine 46MPR shows 19 amino acid differences from that of the human 46MPR, none of which are found in the 68-amino acid cytoplasmic tail. Binding of ligand to the murine 46MPR in permeabilized cells showed a pH optimum of 6.5, was completely inhibited by Man 6-P, and was stimulated by divalent cations. Mn2+ was more effective than Ca2+ or Mg2+. Endocytosis was demonstrated at pH 6.5 and was stimulated 4-7-fold by Mn2+. In its responsiveness to divalent cations and its preference for Mn2+, the murine 46MPR resembled the bovine 46MPR more than the human 46MPR. It was even less efficient than the human receptor in its ability to mediate endocytosis in transfected murine cells. It was also no more efficient than the human 46MPR in correcting the sorting defect of IGF-IIR/MPR-deficient mouse L cells. We conclude that the previously observed relative inefficiency of the human 46MPR in sorting enzymes to lysosomes in murine cells is a property of the 46MPR itself and not a manifestation of studying its expression in a heterologous cell line.

Isolation and analysis of the human 46‐kDa mannose 6‐phosphate receptor gene

From a genomic library in EMBL 3, two overlapping clones for the human 46-kDa mannose 6-phosphate receptor (MPR46) were isolated, which span the entire coding sequence. The human MPR46 gene is distributed over 12 kb and is divided into seven exons (110-1573 bp). All the intron/exon borders agree with the consensus sequences of splice junctions. Exon 1 codes for a 5' untranslated sequence. The ATG initiation codon begins with the second nucleotide in exon 2. A signal sequence of 26 amino acid residues is followed by the extracytoplasmic (luminal) domain, which extends to exon 5. The transmembrane domain of the receptor spans exons 5 and 6 and the cytoplasmic domain is encoded by exons 6 and 7. The latter domain also codes for an extended 3' untranslated sequence. The transcription-initiation site was defined by primer extension. The sequence upstream of the cap site has strong promoter activity and contains structural elements characteristic of promoters found in housekeeping genes. No correlation between the genomic organization and known protein domains of the MPR46 was apparent. Moreover, the sequence of about 150 amino acids within the luminal domain of MPR46, which is homologous to the 15 repeats that constitute the luminal domain of the 300-kDa mannose 6-phosphate receptor (MPR300), does not correlate with intron/exon borders. MPR46 and MPR300 have therefore diverged from a common ancestral gene before introduction of the present intron sequences.

Molecular Cloning of the Mouse 46-kDa Mannose 6-Phosphate Receptor (MPR 46)

A cDNA clone for the mouse 46-kDa mannose 6-phosphate receptor (MPR 46) was isolated from an embryonic mouse cDNA library. Its single open reading frame codes for a protein of 278 residues. It shows an over-all amino-acid identity of 93% with the human receptor. Nine non-conservative amino-acid exchanges are found in the luminal domain, one non-conservative exchange of hydrophobic amino acids is in the transmembrane domain, while the cytoplasmic receptor tails are identical. All five potential N-glycosylation sites are conserved as well as amino acids that are important for ligand binding (Arg 137 and His 131) and disulfide pairing (Cys 32 and 78, Cys 132 and Cys 167, Cys 145 and Cys 179). The absolute identity in the cytoplasmic MPR 46 tail suggests the importance of this amino-acid sequence for the intracellular routing of the MPR 46.

Rapid equilibrium between monomeric, dimeric and tetrameric forms of the 46‐kDa mannose 6‐phosphate receptor at 37°C

At 4 degrees C, detergent-solubilized 46-kDa mannose 6-phosphate receptor (MPR 46) exists as a mixture of dimeric and tetrameric forms [A. Waheed, A. Hille, U. Junghans & K. von Figura (1990) Biochemistry 29, 2449-2455]. At 37 degrees C, MPR 46 exists as a mixture of monomeric, dimeric and tetrameric forms, which can be separated by sucrose density centrifugation or chromatography on a mannose-6-phosphate affinity matrix. Monomeric MPR 46 did not bind to the affinity matrix, while dimeric and tetrameric receptors were eluted with increasing concentrations of mannose 6-phosphate. Depending on the incubation temperature, dimeric MPR 46 preferentially associates to tetramers (less than or equal to 16 degrees C) or dissociates to monomers (greater than or equal to 25 degrees C). The presence of mannose 6-phosphate shifts the equilibrium to higher quaternary structure, with a maximal effect at 37 degrees C. Incubation of dimeric or tetrameric receptor at pH 4.0 induces a rapid dissociation of about a third of the receptor with t1/2 of less than 2 min, followed by a phase of slow dissociation. Readjusting the pH to 7.5 induces a rapid formation of dimers and tetramers, which is promoted by the presence of mannose 6-phosphate or an increase of the receptor concentration. These observations may indicate that the recycling of the receptor between the Golgi apparatus, where it binds ligands at near-neutral pH, and the acidic prelysosomes, where it releases the ligands, is associated with a change of its quaternary structure, which in turn may affect the recycling kinetics. In keeping with this assumption, we observed in baby hamster kidney cells over-expressing MPR 46 and in membrane prepared from these cells monomeric, dimeric and tetrameric forms of the receptor.

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.