Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P20645 (mannose-6-phosphate receptor)
 320 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human lysosomal alpha-galactosidase A (alpha-Gal A) was stably overexpressed in CHO cells and its biosynthesis and targeting were investigated. Clone AGA5.3-1000Mx, which was the highest enzyme overexpressor, produced intracellular alpha-Gal A levels of 20,900 U/mg (approximately 100 micrograms of enzyme/10(7) cells) and secreted approximately 13,000 U (or 75 micrograms/10(7) cells) per day. Ultrastructural examination of these cells revealed numerous 0.25-1.5 microns crystalline structures in dilated trans-Golgi network (TGN) and in lysosomes which stained with immunogold particles using affinity-purified anti-human alpha-Gal A antibodies. Pulse-chase studies revealed that approximately 65% of the total enzyme synthesized was secreted, while endogenous CHO lysosomal enzymes were not, indicating that the alpha-Gal A secretion was specific. The recombinant intracellular and secreted enzyme forms were normally processed and phosphorylated; the secreted enzyme had mannose-6-phosphate moieties and bound the immobilized 215-kD mannose-6-phosphate receptor (M6PR). Thus, the overexpressed enzyme's selective secretion did not result from oversaturation of the M6PR-mediated pathway or abnormal binding to the M6PR. Of note, the secreted alpha-Gal A was sulfated and the percent of enzyme sulfation decreased with increasing amplification, presumably due to the inaccessibility of the enzyme's tyrosine residues for the sulfotransferase in the TGN. Overexpression of human lysosomal alpha-N-acetylgalactosaminidase and acid sphingomyelinase in CHO cell lines also resulted in their respective selective secretion. In vitro studies revealed that purified secreted alpha-Gal A was precipitated as a function of enzyme concentration and pH, with 30% of the soluble enzyme being precipitated when 10 mg/ml of enzyme was incubated at pH 5.0. Thus, it is hypothesized that these overexpressed lysosomal enzymes are normally modified until they reach the TGN where the more acidic environment of this compartment causes the formation of soluble and particulate enzyme aggregates. A significant proportion of these enzyme aggregates are unable to bind the M6PR and are selectively secreted via the constitutive secretory pathway, while endogenous lysosomal enzymes bind the M6PRs and are transported to lysosomes.
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PMID:Overexpression of human alpha-galactosidase A results in its intracellular aggregation, crystallization in lysosomes, and selective secretion. 133 79

The internalization signals of several constitutively recycling receptors have recently been identified as regions of four or six amino acids that include an aromatic residue, usually tyrosine. Here, we show that transplanted signals from the low density lipoprotein receptor (LDLR) and cation-independent mannose-6-phosphate receptor (Man-6-PR) promote rapid internalization of the transferrin receptor (TR), directly establishing that recognition signals are interchangeable, self-determined structural motifs and that signals from type I membrane proteins are active in a type II receptor. We also show that the chemical and spatial patterns of critical residues in both four- and six-residue internalization motifs are consistent with a tight turn structure. A six-residue LDLR signal is needed for activity in TR, suggesting that an amino-terminal aromatic side chain is obligatory. In contrast, the carboxy-terminal aromatic side chain in the TR signal can be replaced by a large hydrophobic residue. Thus, internalization signals apparently require an aromatic amino-terminal residue and either an aromatic or large hydrophobic carboxy-terminal residue rather than a conserved tyrosine per se. Consistent with this conclusion, predicted internalization signals from the poly-Ig receptor, YSAF, and asialoglycoprotein receptor (ASGPR) subunit H1, YQDL, also promote internalization of TR.
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PMID:Transplanted LDL and mannose-6-phosphate receptor internalization signals promote high-efficiency endocytosis of the transferrin receptor. 165 15

An affinity matrix of LDL receptor cytoplasmic tails binds the HA-II 100/50/16 kd complexes found in plasma membrane coated pits. Other receptors (or their cytoplasmic domains), which are localized in coated pits during endocytosis, inhibit this binding. This includes an 8 residue peptide containing tyrosine, corresponding to the cytoplasmic portion of a mutant influenza haemagglutinin. In contrast, the equivalent peptide lacking tyrosine (like the tail of the native haemagglutinin, a protein excluded from coated pits) does not compete. These results imply that the HA-II complex has a recognition site for a common signal, probably involving a tyrosine residue, carried by the LDL receptor and competing receptors also found in plasma membrane coated pits. The HA-II complex therefore fulfils the role of an 'adaptor', the name proposed for the structural units which mediate the binding of clathrin to receptors in coated vesicles. Another related complex, the HA-I adaptor, which is restricted to Golgi coated pits, probably does not recognize the 'tyrosine signal' on the LDL receptor tail. The HA-I adaptor is likely to contain a recognition site for a different signal carried by receptors, e.g. the mannose-6-phosphate receptor, which are found in Golgi coated pits.
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PMID:Receptors compete for adaptors found in plasma membrane coated pits. 290 61

VPS10 (Vacuolar Protein Sorting) encodes a large type I transmembrane protein (Vps10p), involved in the sorting of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) to the Saccharomyces cerevisiae lysosome-like vacuole. Cells lacking Vps10p missorted greater than 90% CPY and 50% of another vacuolar hydrolase, PrA, to the cell surface. In vitro equilibrium binding studies established that the 1,380-amino acid lumenal domain of Vps10p binds CPY precursor in a 1:1 stoichiometry, further supporting the assignment of Vps10p as the CPY sorting receptor. Vps10p has been immunolocalized to the late-Golgi compartment where CPY is sorted away from the secretory pathway. Vps10p is synthesized at a rate 20-fold lower that that of its ligand CPY, which in light of the 1:1 binding stoichiometry, requires that Vps10p must recycle and perform multiple rounds of CPY sorting. The 164-amino acid Vps10p cytosolic domain is involved in receptor trafficking, as deletion of this domain resulted in delivery of the mutant Vps10p to the vacuole, the default destination for membrane proteins in yeast. A tyrosine-based signal (YSSL80) within the cytosolic domain enables Vps10p to cycle between the late-Golgi and prevacuolar/endosomal compartments. This tyrosine-based signal is homologous to the recycling signal of the mammalian mannose-6-phosphate receptor. A second yeast gene, VTH2, encodes a protein highly homologous to Vps10p which, when over-produced, is capable of suppressing the CPY and PrA missorting defects of a vps10 delta strain. These results indicate that a family of related receptors act to target soluble hydrolases to the vacuole.
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PMID:Vps10p cycles between the late-Golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases. 863 29

Tyrosinase-related protein (TRP)-1 is one of the most abundant melanosomal glycoproteins involved in melanogenesis. This report summarizes our recent research efforts related to the biological role and biosynthesis of TRP-1 and its transport from TGN (trans-Golgi network) to the stage I melanosome. Our UV irradiation and tyrosinase and TRP-1 cDNA co-transfection studies indicated that human TRP-1 is involved in not only melanogenesis but also prevention of melanocyte death, which may occur during biosynthesis of melanin pigment in the presence of tyrosinase. Furthermore, a coordinated gene interaction was indicated between tyrosinase and TRP-1, resulting in upregulation of mRNA and protein expression of LAMP (lysosome-associated membrane protein)-1 that would directly prevent the tyrosinase-mediated programmed cell death of melanocytes. Similar to tyrosinase, however, TRP-1 appears to require a molecular chaperone, calnexin, which we have cloned recently. Our cDNA transfection study of tyrosinase with calnexin showed clearly the necessity of calnexin in order to have efficient, functional activity of melanosomal glycoprotein, especially tyrosinase. Once glycosylation is completed, TRP-1 will be transported from TGN to the stage I melanosome. At this stage, TRP-1 will have its own target signal, in particular, tyrosine-rich leucine residues in cytoplasmic tail. Our TRP-1 cDNA transfection and immunoelectron microscopy study shows that TRP-1 will be transported through small vesicles, probably non-clathrin-coated type, to large vacuoles, identical to the MPR (mannose-6-phosphate receptor)-positive, late endosomes. In this transport process a low molecular weight G-protein, rab-7, was isolated from the purified melanosomal protein on 2D-PAGE and identified by subsequent sequencing and PCR amplification. Confocal microscopy with double immunostaining and immunoelectron microscopy confirmed the co-localization of rab-7 and TRP-1 in the melanosomes with early stages of maturation (I-HI). Furthermore, this process will also be regulated by phosphatidylinositol 3-kinase (PI-3 kinase).
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PMID:Biological role of tyrosinase related protein and its biosynthesis and transport from TGN to stage I melanosome, late endosome, through gene transfection study. 926 27

The cytoplasmic tail of the human 46 kDa mannose 6-phosphate receptor (MPR 46) is necessary for rapid internalization of the receptor and sufficient to mediate internalization of a resident plasma membrane protein. To localize the internalization sequences within the 67 amino acids of the cytoplasmic tail, the tail was progressively shortened from its C-terminus, internal deletions of between four and eight amino acids were introduced into the tail, and individual residues were substituted by alanine, glycine or serine. Three sequences were identified that contribute to the internalization of MPR 46. The first is located within the 23 juxtamembrane cytoplasmic residues of the tail. It contains four essential residues within a heptapeptide and does not resemble known internalization signals. The second sequence contains as a critical residue Tyr-45. The third region is located within the C-terminal seven residues and contains a di-leucine pair as essential residues. The first and third sequences were shown to function as autonomous internalization sequences. Substitution of critically important residues within a single internalization sequence was tolerated, with no or only a moderate decrease in the internalization rate. When essential residues from two or all three internalization sequences were substituted, however, the internalization rate was decreased by more than 60% and 90% respectively. This indicates that the autonomous internalization signals in the cytoplasmic tail of MPR 46 function in an additive manner, but are partly redundant.
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PMID:Identification of three internalization sequences in the cytoplasmic tail of the 46 kDa mannose 6-phosphate receptor. 929 Nov 24

Endocytosis and intracellular transport of ricin were studied in stable transfected HeLa cells where overexpression of wild-type (WT) or mutant dynamin is regulated by tetracycline. Overexpression of the temperature-sensitive mutant dynG273D at the nonpermissive temperature or the dynK44A mutant inhibits clathrin-dependent endocytosis (Damke, H., T. Baba, A.M. van der Blieck, and S.L. Schmid. 1995. J. Cell Biol. 131: 69-80; Damke, H., T. Baba, D.E. Warnock, and S.L. Schmid. 1994. J. Cell Biol. 127:915-934). Under these conditions, ricin was endocytosed at a normal level. Surprisingly, overexpression of both mutants made the cells less sensitive to ricin. Butyric acid and trichostatin A treatment enhanced dynamin overexpression and increased the difference in toxin sensitivity between cells with normal and mutant dynamin. Intoxication with ricin seems to require toxin transport to the Golgi apparatus (Sandirg, K., and B. van Deurs. 1996. Physiol. Rev. 76:949-966), and this process was monitored by measuring the incorporation of radioactive sulfate into a modified ricin molecule containing a tyrosine sulfation site. The sulfation of ricin was much greater in cells expressing dynWT than in cells expressing dynK44A. Ultrastructural analysis using a ricin-HRP conjugate confirmed that transport to the Golgi apparatus was severely inhibited in cells expressing dynK44A. In contrast, ricin transport to lysosomes as measured by degradation of 125I-ricin was essentially unchanged in cells expressing dynK44A. These data demonstrate that although ricin is internalized by clathrin-independent endocytosis in cells expressing mutant dynamin, there is a strong and apparently selective inhibition of ricin transport to the Golgi apparatus. Also, in cells with mutant dynamin, there is a redistribution of the mannose-6-phosphate receptor.
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PMID:Expression of mutant dynamin inhibits toxicity and transport of endocytosed ricin to the Golgi apparatus. 945 16

The role of clathrin in intracellular sorting was investigated by expression of a dominant-negative mutant form of clathrin, termed the hub fragment. Hub inhibition of clathrin-mediated membrane transport was established by demonstrating a block of transferrin internalization and an alteration in the intracellular distribution of the cation-independent mannose-6-phosphate receptor. Hubs had no effect on uptake of FITC-dextran, adaptor distribution, organelle integrity in the secretory pathway, or cell surface expression of constitutively secreted molecules. Hub expression blocked lysosomal delivery of chimeric molecules containing either the tyrosine-based sorting signal of H2M or the dileucine-based sorting signal of CD3gamma, confirming a role for clathrin-coated vesicles (CCVs) in recognizing these signals and sorting them to the endocytic pathway. Hub expression was then used to probe the role of CCVs in targeting native molecules bearing these sorting signals in the context of HLA-DM and the invariant chain (I chain) complexed to HLA-DR. The distribution of these molecules was differentially affected. Accumulation of hubs before expression of the DM dimer blocked DM export from the TGN, whereas hubs had no effect on direct targeting of the DR-I chain complex from the TGN to the endocytic pathway. However, concurrent expression of hubs, such that hubs were building to inhibitory concentrations during DM or DR-I chain expression, caused cell surface accumulation of both complexes. These observations suggest that both DM and DR-I chain are directly transported to the endocytic pathway from the TGN, DM in CCVs, and DR-I chain independent of CCVs. Subsequently, both complexes can appear at the cell surface from where they are both internalized by CCVs. Differential packaging in CCVs in the TGN, mediated by tyrosine- and dileucine-based sorting signals, could be a mechanism for functional segregation of DM from DR-I chain until their intended rendezvous in late endocytic compartments.
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PMID:A dominant-negative clathrin mutant differentially affects trafficking of molecules with distinct sorting motifs in the class II major histocompatibility complex (MHC) pathway. 949 Jul 17

The cytosolic domain of the 46 kDa mannose-6-phosphate receptor (MPR 46) contains a signal that mediates sorting of the receptor and of a reporter protein to the basolateral surface domain of Madin-Darby canine kidney cells. Progressive truncation of the 67 cytosolic residues indicated that the 19 juxtamembrane residues are sufficient for basolateral sorting. Alanine/glycine-scanning mutagenesis identified Glu-11 and Ala-17 as the critical residues between residues 7 and 19. Glu-11 is also of critical importance for the one of the three internalization signals in the cytosolic tail of the receptor [Denzer, Weber, Hille-Rehfeld, von Figura and Pohlmann (1997) Biochem. J. 326, 497-505]. Although overlapping, the signals for basolateral sorting and internalization depend on different residues. The basolateral sorting signal of MPR 46 is distinct from tyrosine- or dileucine-based basolateral sorting signals and also lacks similarity to the few other basolateral signals that do not fall into these two classes.
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PMID:The 46 kDa mannose-6-phosphate receptor contains a signal for basolateral sorting within the 19 juxtamembrane cytosolic residues. 958 60

Elevated expression of insulin-like growth factor-II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon, and liver cancer. As IGF-II can deliver a mitogenic signal through both IGF-IR and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is a potential alternative option to IGF-IR-directed agents. Using a Fab-displaying phage library and a biotinylated precursor form of IGF-II (1-104 amino acids) as a target, we isolated Fabs specific for the E-domain COOH-terminal extension form of IGF-II and for mature IGF-II. One of these Fabs that bound to both forms of IGF-II was reformatted into a full-length IgG, expressed, purified, and subjected to further analysis. This antibody (DX-2647) displayed a very high affinity for IGF-II/IGF-IIE (K(D) value of 49 and 10 pmol/L, respectively) compared with IGF-I (approximately 10 nmol/L) and blocked binding of IGF-II to IGF-IR, IR-A, a panel of insulin-like growth factor-binding proteins, and the mannose-6-phosphate receptor. A crystal complex of the parental Fab of DX-2647 bound to IGF-II was resolved to 2.2 A. DX-2647 inhibited IGF-II and, to a lesser extent, IGF-I-induced receptor tyrosine phosphorylation, cellular proliferation, and both anchorage-dependent and anchorage-independent colony formation in various cell lines. In addition, DX-2647 slowed tumor progression in the Hep3B xenograft model, causing decreased tumoral CD31 staining as well as reduced IGF-IIE and IGF-IR phosphorylation levels. Therefore, DX-2647 offers an alternative approach to targeting IGF-IR, blocking IGF-II signaling through both IGF-IR and IR-A.
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PMID:A human monoclonal antibody against insulin-like growth factor-II blocks the growth of human hepatocellular carcinoma cell lines in vitro and in vivo. 2051 53


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