Receptor-mediated endocytosis and intracellular trafficking of lipoproteins and transferrin in insect cells

While the intracellular pathways of ligands after receptor-mediated endocytosis have been studied extensively in mammalian cells, in insect cells these pathways are largely unknown. We transfected Drosophila Schneider line 2 (S2) cells with the human low-density lipoprotein (LDL) receptor (LDLR) and transferrin (Tf) receptor (TfR), and used endocytosis of LDL and Tf as markers. After endocytosis in mammalian cells, LDL is degraded in lysosomes, whereas Tf is recycled. Fluorescence microscopy analysis revealed that LDL and Tf are internalized by S2 cells transfected with LDLR or TfR, respectively. In transfectants simultaneously expressing LDLR and TfR, both ligands colocalize in endosomes immediately after endocytic uptake, and their location remained unchanged after a chase. Similar results were obtained with Spodoptera frugiperda Sf9 cells that were transfected with TfR, suggesting that Tf is retained intracellularly by both cell lines. The insect lipoprotein, lipophorin, is recycled upon lipophorin receptor (LpR)-mediated endocytosis by mammalian cells, however, not after endocytosis by LpR-expressing S2 transfectants, suggesting that this recycling mechanism is cell-type specific. LpR is endogenously expressed by fat body tissue of Locusta migratoria for a limited period after an ecdysis. A chase following endocytosis of labeled lipophorin by isolated fat body tissue at this developmental stage resulted in a significant decrease of lipophorin-containing vesicles, indicative of recycling of the ligand.     

Parvovirus infection of cells by using variants of the feline transferrin receptor altering clathrin-mediated endocytosis, membrane domain localization, and capsid-binding domains

The feline and canine transferrin receptors (TfRs) bind canine parvovirus to host cells and mediate rapid capsid uptake and infection. The TfR and its ligand transferrin have well-described pathways of endocytosis and recycling. Here we tested several receptor-dependent steps in infection for their role in virus infection of cells. Deletions of cytoplasmic sequences or mutations of the Tyr-Thr-Arg-Phe internalization motif reduced the rate of receptor uptake from the cell surface, while polar residues introduced into the transmembrane sequence resulted in increased degradation of transferrin. However, the mutant receptors still mediated efficient virus infection. In contrast, replacing the cytoplasmic and transmembrane sequences of the feline TfR with those of the influenza virus neuraminidase (NA) resulted in a receptor that bound and endocytosed the capsid but did not mediate viral infection. This chimeric receptor became localized to detergent-insoluble membrane domains. To test the effect of structural virus receptor interaction on infection, two chimeric receptors were prepared which contained antibody-variable domains that bound the capsid in place of the TfR ectodomain. These chimeric receptors bound CPV capsids and mediated uptake but did not result in cell infection. Adding soluble feline TfR ectodomain to the virus during that uptake did not allow infection.     

Characterization of the Interaction between Diferric Transferrin and Transferrin Receptor 2 by Functional Assays and Atomic Force Microscopy

Transferrin receptor 2 (TfR2), a homologue of the classical transferrin receptor 1 (TfR1), is found in two isoforms, α and β. Like TfR1, TfR2α is a type II membrane protein, but the β form lacks transmembrane portions and therefore is likely to be an intracellular protein. To investigate the functional properties of TfR2α, we expressed the protein with FLAG tagging in transferrin-receptor-deficient Chinese hamster ovary cells. The association constant for the binding of diferric transferrin (Tf) to TfR2α is 5.6 × 10⁶ M^− 1, which is about 50 times lower than that for the binding of Tf to TfR1, with correspondingly reduced rates of iron uptake. Evidence for Tf internalization and recycling via TfR2α without degradation, as in the TfR1 pathway, was also found. The interaction of TfR2α with Tf was further investigated using atomic force microscopy, a powerful tool used for investigating the interaction between a ligand and its receptor at the single-molecule level on the living cell surface. Dynamic force microscopy reveals a difference in the interactions of Tf with TfR2α and TfR1, with Tf-TfR1 unbinding characterized by two energy barriers, while only one is present for Tf-TfR2. We speculate that this difference may reflect Tf binding to TfR2α by a single lobe, whereas two lobes of Tf participate in binding to TfR1. The difference in the binding properties of Tf to TfR1 and TfR2α may help account for the different physiological roles of the two receptors.     

Unconventional endocytosis and trafficking of transferrin receptor induced by iron

The posttranslational regulation of transferrin receptor (TfR1) is largely unknown. We investigated whether iron availability affects TfR1 endocytic cycle and protein stability in HepG2 hepatoma cells exposed to ferric ammonium citrate (FAC). NH₄Cl and bafilomycin A1, but not the proteasomal inhibitor MG132, prevented the FAC-mediated decrease in TfR1 protein levels, thus indicating lysosomal involvement. Knockdown experiments showed that TfR1 lysosomal degradation is independent of 1) endocytosis mediated by the clathrin adaptor AP2; 2) Tf, which was suggested to facilitate TfR1 internalization; 3) H-ferritin; and 4) MARCH8, previously implicated in TfR1 degradation. Notably, FAC decreased the number of TfR1 molecules at the cell surface and increased the Tf endocytic rate. Colocalization experiments confirmed that, upon FAC treatment, TfR1 was endocytosed in an AP2- and Tf-independent pathway and trafficked to the lysosome for degradation. This unconventional endocytic regulatory mechanism aimed at reducing surface TfR1 may represent an additional posttranslational control to prevent iron overload. Our results show that iron is a key regulator of the trafficking of TfR1, which has been widely used to study endocytosis, often not considering its function in iron homeostasis.     

Two mechanisms of iron uptake from transferrin by melanoma cells. The effect of desferrioxamine and ferric ammonium citrate.

The effects of ferric ammonium citrate (FAC) and desferrioxamine (DFO) on iron (Fe), and transferrin (Tf) uptake have been investigated using SK-MEL-28 human melanoma cells, which express the Tf homologue, melanotransferrin, in high concentrations. Previously we demonstrated two separate Fe uptake mechanisms from Tf, viz. a specific process mediated by the transferrin receptor (TfR) and a nonspecific process (Richardson, D. R., and Baker, E. (1990) Biochim. Biophys. Acta 1053, 1-12). Cells exposed to DFO demonstrated up-regulation of the TfR with a concurrent increase in the rate of Fe uptake. Desferrioxamine also stimulated the nonspecific process of Fe uptake, resulting in a further increase in accumulation of Fe over Tf after saturation of the specific TfR. Ferric ammonium citrate had two effects. First, it resulted in down-regulation of the TfR. Second, and paradoxically, it markedly stimulated the rate of Fe uptake from Tf by the nonspecific process without increasing the rate of nonspecific Tf uptake. These data conclusively demonstrate that two entirely different mechanisms of iron uptake from Tf exist in melanoma cells and that ferric ammonium citrate may be a useful experimental tool to further characterize the specific and nonspecific mechanisms of Fe uptake from Tf.     

Oligomerized transferrin receptors are selectively retained by a lumenal sorting signal in a long-lived endocytic recycling compartment

Cross-linking of surface receptors results in altered receptor trafficking in the endocytic system. To better understand the cellular and molecular mechanisms by which receptor cross-linking affects the intracellular trafficking of both ligand and receptor, we studied the intracellular trafficking of the transferrin receptor (TfR) bound to multivalent-transferrin (Tf10) which was prepared by chemical cross- linking of transferrin (Tf). Tf10 was internalized about two times slower than Tf and was retained four times longer than Tf, without being degraded in CHO cells. The intracellular localization of Tf10 was investigated using fluorescence and electron microscopy. Tf10 was not delivered to the lysosomal pathway followed by low density lipoprotein but remained accessible to Tf in the pericentriolar endocytic recycling compartment for at least 60 min. The retained Tf10 was TfR-associated as demonstrated by a reduction in surface TfR number when cells were incubated with Tf10. The presence of Tf10 within the recycling compartment did not affect trafficking of subsequently endocytosed Tf. Retention of Tf10 within the recycling compartment did not require the cytoplasmic domain of the TfR since Tf10 exited cells with the same rate when bound to the wild-type TfR or a mutated receptor with only four amino acids in the cytoplasmic tail. Thus, cross-linking of surface receptors by a multivalent ligand acts as a lumenal retention signal within the recycling compartment. The data presented here show that the recycling compartment labeled by Tf10 is a long-lived organelle along the early endosome recycling pathway that remains fusion accessible to subsequently endocytosed Tf.     

Impairment of Protein Trafficking upon Overexpression and Mutation of Optineurin

Background

Glaucoma is a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and axons. Optineurin is one of the candidate genes identified so far. A mutation of Glu⁵⁰ to Lys (E50K) has been reported to be associated with a more progressive and severe disease. Optineurin, known to interact with Rab8, myosin VI and transferrin receptor (TfR), was speculated to have a role in protein trafficking. Here we determined whether, and how optineurin overexpression and E50K mutation affect the internalization of transferrin (Tf), widely used as a marker for receptor-mediated endocytosis.

Methodology/Principal Findings

Human retinal pigment epithelial (RPE) and rat RGC5 cells transfected to overexpress wild type optineurin were incubated with Texas Red-Tf to evaluate Tf uptake. Granular structures or dots referred to as foci formed in perinuclear regions after transfection. An impairment of the Tf uptake was in addition observed in transfected cells. Compared to overexpression of the wild type, E50K mutation yielded an increased foci formation and a more pronounced defect in Tf uptake. Co-transfection with TfR, but not Rab8 or myosin VI, construct rescued the optineurin inhibitory effect, suggesting that TfR was the factor involved in the trafficking phenotype. Forced expression of both wild type and E50K optineurin rendered TfR to colocalize with the foci. Surface biotinylation experiments showed that the surface level of TfR was also reduced, leading presumably to an impeded Tf uptake. A non-consequential Leu¹⁵⁷ to Ala (L157A) mutation that displayed much reduced foci formation and TfR binding had normal TfR distribution, normal surface TfR level and normal Tf internalization.

Conclusions/Significance

The present study demonstrates that overexpression of wild type optineurin results in impairment of the Tf uptake in RPE and RGC5 cells. The phenotype is related to the optineurin interaction with TfR. Our results further indicate that E50K induces more dramatic effects than the wild type optineurin, and is thus a gain-of-function mutation. The defective protein trafficking may be one of the underlying bases why glaucoma pathology develops in patients with E50K mutation.     

Comparison of the interactions of transferrin receptor and transferrin receptor 2 with transferrin and the hereditary hemochromatosis protein HFE

The transferrin receptor (TfR) interacts with two proteins important for iron metabolism, transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. A second receptor for Tf, TfR2, was recently identified and found to be functional for iron uptake in transfected cells (Kawabata, H., Germain, R. S., Vuong, P. T., Nakamaki, T., Said, J. W., and Koeffler, H. P. (2000) J. Biol. Chem. 275, 16618-16625). TfR2 has a pattern of expression and regulation that is distinct from TfR, and mutations in TfR2 have been recognized as the cause of a non-HFE linked form of hemochromatosis (Camaschella, C., Roetto, A., Cali, A., De Gobbi, M., Garozzo, G., Carella, M., Majorano, N., Totaro, A., and Gasparini, P. (2000) Nat. Genet. 25, 14-15). To investigate the relationship between TfR, TfR2, Tf, and HFE, we performed a series of binding experiments using soluble forms of these proteins. We find no detectable binding between TfR2 and HFE by co-immunoprecipitation or using a surface plasmon resonance-based assay. The affinity of TfR2 for iron-loaded Tf was determined to be 27 nm, 25-fold lower than the affinity of TfR for Tf. These results imply that HFE regulates Tf-mediated iron uptake only from the classical TfR and that TfR2 does not compete for HFE binding in cells expressing both forms of TfR.     

Characterization of glyceraldehyde-3-phosphate dehydrogenase as a novel transferrin receptor

A majority of cells obtain of transferrin (Tf) bound iron via transferrin receptor 1 (TfR1) or by transferrin receptor 2 (TfR2) in hepatocytes. Our study establishes that cells are capable of acquiring transferrin iron by an alternate pathway via GAPDH.These findings demonstrate that upon iron depletion, GAPDH functions as a preferred receptor for transferrin rather than TfR1 in some but not all cell types. We utilized CHO-TRVb cells that do not express TfR1 or TfR2 as a model system. A knockdown of GAPDH in these cells resulted in a decrease of not only transferrin binding but also associated iron uptake. The current study also demonstrates that, unlike TfR1 and TfR2 which are localized to a specific membrane fraction, GAPDH is located in both the detergent soluble and lipid raft fractions of the cell membrane. Further, transferrin uptake by GAPDH occurs by more than one mechanism namely clathrin mediated endocytosis, lipid raft endocytosis and macropinocytosis. By determining the kinetics of this pathway it appears that GAPDH-Tf uptake is a low affinity, high capacity, recycling pathway wherein transferrin is catabolised. Our findings provide an explanation for the detailed role of GAPDH mediated transferrin uptake as an alternate route by which cells acquire iron.     

Transferrin-bound and transferrin free iron uptake by cultured rat astrocytes.

Previously we had demonstrated the presence of transferrin receptor (TfR) on the plasma membrane of cultured rat cortical astrocytes. In this study, we investigated the roles of TfR in transferrin-bound iron (Tf-Fe) as well as transferrin-free iron (Fe II) uptake by the cells. The cultured rat astrocytes were incubated with 1 microM of double-labelled transferrin (125I-Tf-59Fe) in serum- free DMEM F12 medium or 59Fe II in isotonic sucrose solution at 37 degrees C or 4 degrees C for varying times. The cellular Tf-Fe, Tf and Fe II uptake was analyzed by measuring the intracellular radioactivity with gamma counter. The result showed that Tf-Fe uptake kept increasing in a linear manner at least in the first 30-min. In contrast to Tf-Fe uptake, the internalization of Tf into the cells was rapid initially but then slowed to a plateau level after 10 min. of incubation. The addition of either NH4Cl or CH3NH2, the blockers of Tf-Fe uptake via inhibiting iron release from Tf within endosomes, decreased the cellular Tf-Fe uptake but had no significant effect on Tf uptake. Pre-treated cells with trypsin inhibited significantly the cellular uptake of Tf-Fe as well as Tf. These findings suggested that Tf-Fe transport across the membrane of astrocytes is mediated by Tf-TfR endocytosis. The results of transferrin-free iron uptake indicated that the cultured rat cortical astrocytes had the capacity to acquire Fe II. The highest uptake of Fe II occurred at pH 6.5. The Fe II uptake was time and temperature dependent, iron concentration saturable, inhibited by several divalent metal ions, such as Co2+, Zn2+, Mn2+ and Ni2+ and not significantly affected by phenylarsine oxide treatment. These characteristics of Fe II uptake by the cultured astrocytes suggested that Fe II uptake is not mediated by TfR and implied that a carrier-mediated iron transport system might be present on the membrane of the cultured cells.     

Transferrin receptor 2 mediates a biphasic pattern of transferrin uptake associated with ligand delivery to multivesicular bodies

The physiological role of transferrin (Tf) receptor 2 (TfR2), a homolog of the well-characterized TfR1, is unclear. Mutations in TfR2 result in hemochromatosis, indicating that this receptor has a unique role in iron metabolism. We report that HepG2 cells, which endogenously express TfR2, display a biphasic pattern of Tf uptake when presented with ligand concentrations up to 2 µM. The apparently nonsaturating pathway of Tf endocytosis resembles TfR1-independent Tf uptake, a process previously characterized in some liver cell types. Exogenous expression of TfR2 but not TfR1 induces a similar biphasic pattern of Tf uptake in HeLa cells, supporting a role for TfR2 in this process. Immunoelectron microscopy reveals that while Tf, TfR1, and TfR2 are localized in the plasma membrane and tubulovesicular endosomes, TfR2 expression is associated with the additional appearance of Tf in multivesicular bodies. These combined results imply that unlike TfR1, which recycles apo-Tf back to the cell surface after the release of iron, TfR2 promotes the intracellular deposition of ligand. Tf delivered by TfR2 does not appear to be degraded, which suggests that its delivery to this organelle may be functionally relevant to the storage of iron in overloaded states. iron transport; HepG2 cells     

Transferrin receptor 2: evidence for ligand-induced stabilization and redirection to a recycling pathway

Transferrin receptor 2 (TfR2) is a homologue of transferrin receptor 1 (TfR1), the protein that delivers iron to cells through receptor-mediated endocytosis of diferric transferrin (Fe(2)Tf). TfR2 also binds Fe(2)Tf, but it seems to function primarily in the regulation of systemic iron homeostasis. In contrast to TfR1, the trafficking of TfR2 within the cell has not been extensively characterized. Previously, we showed that Fe(2)Tf increases TfR2 stability, suggesting that trafficking of TfR2 may be regulated by interaction with its ligand. In the present study, therefore, we sought to identify the mode of TfR2 degradation, to characterize TfR2 trafficking, and to determine how Fe(2)Tf stabilizes TfR2. Stabilization of TfR2 by bafilomycin implies that TfR2 traffics to the lysosome for degradation. Confocal microscopy reveals that treatment of cells with Fe(2)Tf increases the fraction of TfR2 localizing to recycling endosomes and decreases the fraction of TfR2 localizing to late endosomes. Mutational analysis of TfR2 shows that the mutation G679A, which blocks TfR2 binding to Fe(2)Tf, increases the rate of receptor turnover and prevents stabilization by Fe(2)Tf, indicating a direct role of Fe(2)Tf in TfR2 stabilization. The mutation Y23A in the cytoplasmic domain of TfR2 inhibits its internalization and degradation, implicating YQRV as an endocytic motif.     

Aluminum exposure affects transferrin-dependent and -independent iron uptake by K562 cells

Aluminum (Al) and iron (Fe) share several physicochemical characteristics and they both bind to transferrin (Tf), entering the cell via Tf receptors (TfR). Previously, we found similar values of affinity constant for the binding of TfR to Tf carrying either Al or Fe. The competitive interaction between both metals prevented normal Fe incorporation into K562 cells and triggered the upregulation of Fe transport. In the present work we demonstrated that Al modified Fe uptake without affecting the expression of Tf receptors. Both TfR and TfR2 mRNA levels, evaluated by RT-PCR, and TfR antigenic sites, analyzed by flow cytometry, were found unchanged after Al exposure. In turn, Al did induce upregulation of non-Tf bound Fe (NTBI) uptake. This modulation was not due to intracellular Fe decrease since NTBI transport proved not to be regulated by Fe depletion. Unlike its behavior in the presence of Tf, Al was unable to compete with NTBI uptake, suggesting that both metals do not share the same alternative transport pathway. We propose that Al interference with TfR-mediated Fe incorporation might trigger the upregulation of NTBI uptake, an adaptation aimed at incorporating the essential metal required for cellular metabolism without allowing the simultaneous access of a potentially toxic metal.     

Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.     

Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump

Endocytosed proteins are sorted in early endosomes to be recycled to the plasma membrane or transported further into the degradative pathway. We studied the role of endosomes acidification on the endocytic trafficking of the transferrin receptor (TfR) as a representative for the recycling pathway, the cation-dependent mannose 6-phosphate receptor (MPR) as a prototype for transport to late endosomes, and fluid-phase endocytosed HRP as a marker for transport to lysosomes. Toward this purpose, bafilomycin A1 (Baf), a specific inhibitor of the vacuolar proton pump, was used to inhibit acidification of the vacuolar system. Microspectrofluorometric measurement of the pH of fluorescein-rhodamine-conjugated transferrin (Tf)-containing endocytic compartments in living cells revealed elevated endosomal pH values (pH > 7.0) within 2 min after addition of Baf. Although recycling of endocytosed Tf to the plasma membrane continued in the presence of Baf, recycled Tf did not dissociate from its receptor, indicating failure of Fe3+ release due to a neutral endosomal pH. In the presence of Baf, the rates of internalization and recycling of Tf were reduced by a factor of 1.40 +/- 0.08 and 1.57 +/- 0.25, respectively. Consequently, little if any in TfR expression at the cell surface was measured during Baf treatment. Sorting between endocytosed TfR and MPR was analyzed by the HRP-catalyzed 3,3'- diaminobenzidine cross-linking technique, using transferrin conjugated to HRP to label the endocytic pathway of the TfR. In the absence of Baf, endocytosed surface 125I-labeled MPR was sorted from the TfR pathway starting at 10 min after uptake, reaching a plateau of 40% after 45 min. In the presence of Baf, sorting was initiated after 20 min of uptake, reaching approximately 40% after 60 min. Transport of fluid-phase endocytosed HRP to late endosomes and lysosomes was measured using cell fractionation and immunogold electron microscopy. Baf did not interfere with transport of HRP to MPR-labeled late endosomes, but nearly completely abrogated transport to cathepsin D- labeled lysosomes. From these results, we conclude that trafficking through early and late endosomes, but not to lysosomes, continued upon inactivation of the vacuolar proton pump.     

Relations between the intracellular pathways of the receptors for transferrin, asialoglycoprotein, and mannose 6-phosphate in human hepatoma cells

We compared the intracellular pathways of the transferrin receptor (TfR) with those of the asialoglycoprotein receptor (ASGPR) and the cation-independent mannose 6-phosphate receptor (MPR)/insulin-like growth factor II receptor during endocytosis in Hep G2 cells. Cells were allowed to endocytose a conjugate of horseradish peroxidase and transferrin (Tf/HRP) via the TfR system. Postnuclear supernatants of homogenized cells were incubated with 3,3'-diaminobenzidine (DAB) and H2O2. Peroxidase-catalyzed oxidation of DAB within Tf/HRP-containing endosomes cross-linked their contents to DAB polymer. The cross-linking efficiency was dependent on the intravesicular Tf/HRP concentration. The loss of detectable receptors from samples of cell homogenates treated with DAB/H2O2 was used as a measure of colocalization with Tf/HRP. To compare the distribution of internalized plasma membrane receptors with Tf/HRP, cells were first surface-labeled with 125I at 0 degrees C. After uptake of surface 125I-labeled receptors at 37 degrees C in the presence of Tf/HRP, proteinase K was used at 0 degrees C to remove receptors remaining at the plasma membrane. Endocytosed receptors were isolated by means of immunoprecipitation. 125I-TfR and 125I-ASGPR were not sorted from endocytosed Tf/HRP. 125I-MPR initially also resided in Tf/HRP-containing compartments, however 70% was sorted from the Tf/HRP pathway between 20 and 45 min after uptake. To study the accessibility of total intracellular receptor pools to endocytosed Tf/HRP, nonlabeled cells were used, and the receptors were detected by means of Western blotting. The entire intracellular TfR population, but only 70 and 50% of ASGPR and MPR, respectively, were accessible to endocytosed Tf/HRP. These steady-state levels were reached by 10 min of continuous Tf/HRP uptake at 37 degrees C. We conclude that 30% of the intracellular ASGPR pool is not involved in endocytosis (i.e., is silent). Double-labeling immunoelectron microscopy on DAB-labeled cells showed a considerable pool of ASGPR in secretory albumin-positive, Tf/HRP-negative, trans-Golgi reticulum. We suggest that this pool represents the silent ASGPR that has been biochemically determined. A model of receptor transport routes is presented and discussed.     

Effects of epidermal growth factor on transferrin receptor phosphorylation and surface expression in malignant epithelial cells

The transferrin (Tf) receptor is a major transmembrane protein which provides iron for normal and malignant cell growth. Epidermal growth factor (EGF) has been reported to rapidly and transiently alter the number of surface Tf receptors in normal and transformed epithelial cells. To investigate mechanisms of EGF-induced changes in surface Tf display, EGF effects on surface Tf receptors were compared in two cell lines which differ in their number of EGF receptors and growth responses to EGF. In cloned A431 cells with high receptor numbers which are growth-inhibited by EGF, EGF caused a 50% decrease in Tf receptor expression after 30 min. In contrast, EGF induced a rapid, transitory increase (within 5 min) in the number of surface Tf receptors on KB carcinoma cells which returned to basal levels by 15 min. The observed changes in Tf receptor display were due to altered receptor distribution and not changes in ligand affinity or total cellular transferrin receptor pools. Anti-EGF receptor monoclonal antibody blocked effects of EGF on transferrin receptor expression. Since the antibody is internalized and causes EGF receptor down-regulation, effects on transferrin receptor expression were independent of these events. EGF-induced alterations in Tf receptor display occurred even when cells were pretreated with colchicine, suggesting that changes in surface Tf binding were not mediated by cytoskeletal components. Na orthovanadate, which mimics some early cellular effects of EGF, duplicated EGF's effects on A431 Tf receptors, but had no effect on KB cells, suggesting these responses occur by differing mechanisms. To determine whether EGF caused changes in Tf receptor phosphorylation, 32P-labelled Tf receptors were immunoprecipitated after EGF treatment. After exposure to EGF, A431 cells showed no change in Tf phosphorylation, but KB cells showed a transient, 6-fold increase in transferrin receptor phosphorylation on serine residues. In both A431 and KB cells, phorbol ester (PMA) also increased phosphorylation on transferrin receptors, but had little effect on surface Tf receptor expression. In malignant cell lines, EGE induces rapid, variable changes in transferrin receptor expression and phosphorylation which differ from the effects of PMA. These early responses to EGF appear to differ with the cell type and correlate poorly with alterations in Tf receptor phosphorylation. These results suggest Tf receptor phosphorylation does not regulate Tf receptor display in all cells.     

Iron uptake by melanoma cells from the soluble form of the transferrin homologue,melanotransferrin

Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue that can also exist in a soluble form (sMTf). Considering the high homology of MTf to Tf, it is possible to suggest that sMTf could bind to the high affinity transferrin receptor 1 (TfR1) or lower affinity TfR2. We have used sMTf labelled with 59Fe to examine its ability to donate Fe to cells. Our experiments demonstrate that sMTf is far less effective than Tf at donating Fe to cells and this does not occur via specific receptors. Indeed, the uptake of sMTf by cells occurred via a non-specific process (e.g. adsorptive pinocytosis).     

Expression of iron transport proteins divalent metal transporter-1, Ferroportin-1, HFE and transferrin receptor-1 in human monocyte-derived dendritic cells

Iron is essential for cell survival and regulates many cell functions. In the context of the immune response, iron-related metabolism is tightly controlled in activated lymphocytes as well as in cells of the innate immunity. More precisely, for dendritic cells (DCs), which are the key cell type in the development of a specific immune response, the importance of iron absorption was recently unravelled by showing that depletion of iron inhibits the maturation of DCs. On this basis, we studied in detail the expression of iron transport proteins and HFE in DCs. We found that iron uptake in this cell type is mediated by divalent-metal transporter 1 (DMT1) and transferrin receptor-1 (TfR) whereas Ferroportin-1 is very weakly expressed. HFE that regulates TfR's activity is also detected at the mRNA level. The expression of DMT1 and HFE barely varies upon endotoxin-induced maturation but TfR is up-regulated and the iron export molecule Ferroportin-1 is down-regulated. As opposed to MHC class II molecules, the intracellular localization of TfR is not changed during maturation. Our results indicate that the uptake of iron during DCs development and maturation is mediated by a strong expression of iron-uptake molecules such as DMT1 and TfR as well as a down-regulation of iron export molecules such as Ferroportin-1.