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Query: UNIPROT:P20645 (mannose-6-phosphate receptor)
 320 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Wild-type and mutant human transferrin receptors (TR) have been expressed in chicken embryo fibroblasts using a helper-independent retroviral vector. By functional studies of the mutant TRs, we have identified the tetrapeptide sequence, YXRF, in the cytoplasmic tail of the receptor as the internalization signal required for high efficiency endocytosis and shown that transplanted internalization signals from the low density lipoprotein receptor (LDLR) and the cation-independent mannose-6-phosphate receptor (Man-6-PR) are able to promote rapid internalization of the human TR. A six-residue LDLR signal, FDNPVY, is required for activity in TR, whereas a four-residue Man-6-PR signal, YSKV, is sufficient. These data indicate that internalization signals are interchangeable self-determined structural motifs and that signals from type I membrane proteins are active in a type II receptor. Putative internalization signals in the cytoplasmic tails of other receptors and membrane proteins can be identified based on the sequence patterns of the LDLR, Man-6-PR, and TR signals. Two such putative four-residue internalization signals, one from the poly-Ig receptor and one from the asialoglycoprotein receptor, were tested for activity by transplantation into TR and were found to promote high efficiency internalization. These results suggest that an exposed tight turn is the conformational motif for high efficiency endocytosis.
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PMID:Structural requirements for high efficiency endocytosis of the human transferrin receptor. 143 81

Epithelial cells in situ can internalize their desmosomes. This can be induced in cell cultures after removal of calcium ions from the cell medium. To study this endocytic process, a nontumorigenic human breast epithelial cell line, HMT-3522, was used. HMT-3522 cells were grown in serum-free, chemically defined medium, containing epidermal growth factor (EGF). Removal of EGF from the medium led to growth arrest and a kind of epithelial differentiation process in which adjacent cells interdigitated and formed more desmosomes than in the proliferating state. Growth-inhibited HMT-3522 cells dissociated following EGTA treatment, the desmosomes divided in a symmetrical fashion, and the desmosomal plaques (half-desmosomes) on the cell surface became internalized. The internalization was independent of clathrin, since immunogold labeling of ultracryosections never showed clathrin on desmosomal plaque-associated membrane domains. Moreover, cytosol acidification, which selectively inhibits endocytosis from clathrin-coated pits, practically blocked the uptake of transferrin, whereas internalization of desmosomal plaques continued. In contrast, actin filaments appeared to be involved in the desmosomal internalization. Thus, depolymerization of actin filaments by cytochalasin D significantly reduced endocytosis of half-desmosomes. Immunogold labeling showed that the vesicles with desmosomal plaques were not enriched in MPR (cation-independent mannose-6-phosphate receptor), cathepsin D or the lysosome-associated membrane protein lamp-1. In addition, the morphology was different. Thus, the endocytic vesicles with desmosomal plaques represent a special compartment, distinct from typical endosomes and lysosomes.
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PMID:Endocytosis of desmosomal plaques depends on intact actin filaments and leads to a nondegradative compartment. 792 92

We have followed the transfer of EGF-EGF receptor (EGFR) complexes from endosomal vacuoles that contain transferrin receptors (TfR) to lysosome vacuoles identified by their content of HRP loaded as a 15-min pulse 4 h previously. We show that the HRP-loaded lysosomes are lysosomal-associated membrane protein-1 (LAMP-1) positive, mannose-6-phosphate receptor (M6PR) negative. and contain active acid hydrolase. EGF-EGFR complexes are delivered to these lysosomes intact and are then rapidly degraded. Preactivating the HRP contained within the preloaded lysosomes inhibits the delivery of EGFR and degradation of EGF, and results in the accumulation of EGFR-containing multivesicular bodies (MVB). With time these accumulating MVB undergo a series of maturation changes that include the loss of TfR, the continued recruitment of EGFR, and the accumulation of internal vesicles, but they remain LAMP-1 and M6PR negative. The mature MVB are often seen to make direct contact with lysosomes containing preactivated HRP, but their perimeter membranes remain intact. Together our observations suggest that the transfer of EGF-EGFR complexes from the TfR-containing endosome compartment to the lysosomes that degrade them employs a single vacuolar intermediate, the maturing MVB, and can be achieved by a single heterotypic fusion step.
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PMID:Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes. 860 81

The translocation of a unique facilitative glucose transporter isoform (GLUT4) from an intracellular site to the plasma membrane accounts for the large insulin-dependent increase in glucose transport observed in muscle and adipose tissue. The intracellular location of GLUT4 in the basal state and the pathway by which it reaches the cell surface upon insulin stimulation are unclear. Here, we have examined the colocalization of GLUT4 with the transferrin receptor, a protein which is known to recycle through the endosomal system. Using an anti-GLUT4 monoclonal antibody we immunoisolated a vesicular fraction from an intracellular membrane fraction of 3T3-L1 adipocytes that contained > 90% of the immunoreactive GLUT4 found in this fraction, but only 40% of the transferrin receptor (TfR). These results suggest only a limited degree of colocalization of these proteins. Using a technique to cross-link and render insoluble ("ablate') intracellular compartments containing the TfR by means of a transferrin-horseradish peroxidase conjugate (Tf-HRP), we further examined the relationship between the endosomal recycling pathway and the intracellular compartment containing GLUT4 in these cells. Incubation of non-stimulated cells with Tf-HRP for 3 h at 37 degrees C resulted in quantitative ablation of the intracellular TfR, GLUT1 and mannose-6-phosphate receptor and a shift in the density of Rab5-positive membranes. In contrast, only 40% of intracellular GLUT4 was ablated under the same conditions. Ablation was specific for the endosomal system as there was no significant ablation of either TGN38 or lgp120, which are markers for the trans Golgi reticulum and lysosomes respectively. Subcellular fractionation analysis revealed that most of the ablated pools of GLUT4 and TfR were found in the intracellular membrane fraction. The extent of ablation of GLUT4 from the intracellular fraction was unchanged in cells which were insulin-stimulated prior to ablation, whereas GLUT1 exhibited increased ablation in insulin-stimulated cells. Pretreatment of adipocytes with okadaic acid, an inhibitor of Type-I and -IIa phosphatases, increased GLUT4 ablation in the presence of insulin, consistent with okadaic acid increasing the internalization of GLUT4 from the plasma membrane under these conditions. Using a combination of subcellular fractionation, vesicle immunoadsorption and compartment ablation using the Tf-HRP conjugate we have been able to resolve overlapping but distinct intracellular distributions of the TfR and GLUT4 in adipocytes. At least three separate compartments were identified: TfR-positive/GLUT4-negative. TfR-negative/GLUT4-positive, and TfR-positive/GLUT4-positive, as defined by the relative abundance of these two markers. We propose that the TfR-negative/GLUT4-positive compartment, which contains approximately 60% of the intracellular GLUT4, represents a specialized intracellular compartment that is withdrawn from the endosomal system. The biosynthesis and characteristics of this compartment may be fundamental to the unique insulin regulation of GLUT4.
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PMID:Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes. 861 19

Polarized cells such as epithelial cells and neurons have distinct endosomal compartments associated with different plasma membrane domains. The endosomes of the neuronal cell body and the basolateral cytoplasm of epithelial cells are thought to perform cellular "housekeeping" functions such as the uptake of nutrients and metabolites, while the endosomes in the apical cytoplasm or axons are thought to be specialized for the sorting and transcytosis of cell type-specific ligands and receptors. However, it is not known if nonpolarized cells such as fibroblasts contain a specialized endosomal compartment analogous to the specialized endosomes found in neurons and epithelia. We have expressed a protein that is normally found in the apical early endosomes of developing intestinal epithelial cells in normal rat kidney fibroblasts. This apical endosomal marker, called endotubin, is targeted to early endosomes in transfected fibroblasts, and is present in peripheral as well as perinuclear endosomes. The peripheral endosomes that contain endotubin appear to exclude transferrin, fluid phase markers, and the mannose-6-phosphate receptor, although in the perinuclear region colocalization of endotubin and these markers is present. In addition, endotubin positive structures do not tubulate in response to brefeldin A and instead redistribute to a diffuse perinuclear location. Since this endosomal compartment has many of the characteristics of an apical or axonal endosomal compartment, our results indicate that nonpolarized cells also contain a specialized early endosomal compartment.
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PMID:Targeting of an intestinal apical endosomal protein to endosomes in nonpolarized cells. 901 3

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 clathrin assembly lymphoid myeloid leukemia (CALM) gene encodes a putative homologue of the clathrin assembly synaptic protein AP180. Hence the biochemical properties, the subcellular localization, and the role in endocytosis of a CALM protein were studied. In vitro binding and coimmunoprecipitation demonstrated that the clathrin heavy chain is the major binding partner of CALM. The bulk of cellular CALM was associated with the membrane fractions of the cell and localized to clathrin-coated areas of the plasma membrane. In the membrane fraction, CALM was present at near stoichiometric amounts relative to clathrin. To perform structure-function analysis of CALM, we engineered chimeric fusion proteins of CALM and its fragments with the green fluorescent protein (GFP). GFP-CALM was targeted to the plasma membrane-coated pits and also found colocalized with clathrin in the Golgi area. High levels of expression of GFP-CALM or its fragments with clathrin-binding activity inhibited the endocytosis of transferrin and epidermal growth factor receptors and altered the steady-state distribution of the mannose-6-phosphate receptor in the cell. In addition, GFP-CALM overexpression caused the loss of clathrin accumulation in the trans-Golgi network area, whereas the localization of the clathrin adaptor protein complex 1 in the trans-Golgi network remained unaffected. The ability of the GFP-tagged fragments of CALM to affect clathrin-mediated processes correlated with the targeting of the fragments to clathrin-coated areas and their clathrin-binding capacities. Clathrin-CALM interaction seems to be regulated by multiple contact interfaces. The C-terminal part of CALM binds clathrin heavy chain, although the full-length protein exhibited maximal ability for interaction. Altogether, the data suggest that CALM is an important component of coated pit internalization machinery, possibly involved in the regulation of clathrin recruitment to the membrane and/or the formation of the coated pit.
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PMID:Clathrin assembly lymphoid myeloid leukemia (CALM) protein: localization in endocytic-coated pits, interactions with clathrin, and the impact of overexpression on clathrin-mediated traffic. 1043 22

Antigen presentation to CD4(+) T lymphocytes requires transport of newly synthesized major histocompatibility complex (MHC) class II molecules to the endocytic pathway, where peptide loading occurs. This step is mediated by a signal located in the cytoplasmic tail of the MHC class II-associated Ii chain, which directs the MHC class II-Ii complexes from the trans-Golgi network (TGN) to endosomes. The subcellular machinery responsible for the specific targeting of MHC class II molecules to the endocytic pathway, as well as the first compartments these molecules enter after exit from the TGN, remain unclear. We have designed an original experimental approach to selectively analyze this step of MHC class II transport. Newly synthesized MHC class II molecules were caused to accumulate in the Golgi apparatus and TGN by incubating the cells at 19 degrees C, and early endosomes were functionally inactivated by in vivo cross-linking of transferrin (Tf) receptor-containing endosomes using Tf-HRP complexes and the HRP-insoluble substrate diaminobenzidine. Inactivation of Tf-containing endosomes caused a marked delay in Ii chain degradation, peptide loading, and MHC class II transport to the cell surface. Thus, early endosomes appear to be required for delivery of MHC class II molecules to the endocytic pathway. Under cross-linking conditions, most alphabetaIi complexes accumulated in tubules and vesicles devoid of gamma-adaptin and/or mannose-6-phosphate receptor, suggesting an AP1-independent pathway for the delivery of newly synthesized MHC class II molecules from the TGN to endosomes.
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PMID:Early endosomes are required for major histocompatiblity complex class II transport to peptide-loading compartments. 1047 34

We report here detection of novel intracellular clathrin-coated structures revealed by continuous high-speed imaging of cells expressing green fluorescent protein fusion proteins. These structures, which we operationally term 'gyrating clathrin' (G-clathrin), are characterized by localized but extremely rapid movement, leading to the hypothesis that they are coated buds on waving membrane tubules. G-clathrin structures have structurally and functionally distinct features. They lack detectable adaptor proteins AP-1 and AP-2 but contain GGA1 [Golgi-localized, gamma-ear-containing, Arf (ADP-ribosylation factor)-binding protein] as well as the cation-dependent mannose-6-phosphate receptor. While they accumulate internalized transferrin (Tf), they do not contain detectable levels of cargos targeted for the late endosome/lysosome pathway such as EGF and dextran. Pulse-chase studies indicate that Tf appears in G-clathrin structures in the cell periphery after sorting endosomes (SEs), but before filling of the perinuclear endocytic recycling compartment. Furthermore, the inhibitors LY294002 and wortmannin, which inhibit direct recycling of Tf from SEs to the plasma membrane, also block its appearance in G-clathrin. These observations suggest that peripheral G-clathrin contributes to rapid recycling, a kinetically defined compartment that has largely eluded structural identification. More generally, the rapid continuous live cell imaging reported here reveals new aspects of membrane trafficking.
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PMID:Gyrating clathrin: highly dynamic clathrin structures involved in rapid receptor recycling. 1881 26

Hereditary hemochromatosis is most frequently associated with mutations in HFE, which encodes a class Ib histocompatibility protein. HFE binds to the transferrin receptor-1 (TfR1) in competition with iron-loaded transferrin (Fe-Tf). HFE is released from TfR1 by increasing concentrations of Fe-Tf, and free HFE may then regulate iron homeostasis by binding other ligands. To search for new HFE ligands we expressed recombinant forms of HFE in the human cell line 293T. HFE protein was purified, biotinylated and made into fluorescently labelled tetramers. HFE tetramers bound to TfR1 in competition with Tf, but in addition we detected a binding activity on some cell types that was not blocked by Fe-Tf or by mutations in HFE that prevent binding to TfR1. We identified this second HFE ligand as the cation independent mannose-6-phosphate receptor (CI-MPR, also known as the insulin-like growth factor-2 receptor, IGF2R). HFE:CI-MPR binding was mediated through phosphorylated mannose residues on HFE. Recombinant murine Hfe also bound to CI-MPR. HFE bound to TfR1 was prevented from binding CI-MPR until released by increasing concentrations of Fe-Tf, a feature consistent with an iron sensing mechanism. However, it remains to be determined whether endogenous HFE in vivo also acquires the mannose-6 phosphate modification and binds to CI-MPR.
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PMID:In vitro binding of HFE to the cation-independent mannose-6 phosphate receptor. 1948 39


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