Endogenous Transferrin Receptor Research Articles

Overexpression of the MRI Reporter Genes Ferritin and Transferrin Receptor Affect Iron Homeostasis and Produce Limited Contrast in Mesenchymal Stem Cells.

Imaging technologies that allow the non-invasive monitoring of stem cells in vivo play a vital role in cell-based regenerative therapies. Recently, much interest has been generated in reporter genes that enable simultaneous monitoring of the anatomical location and viability of cells using magnetic resonance imaging (MRI). Here, we investigate the efficacy of ferritin heavy chain-1 (Fth1) and transferrin receptor-1 (TfR1) as reporters for tracking mesenchymal stem cells. The overexpression of TfR1 was well tolerated by the cells but Fth1 was found to affect the cell’s iron homeostasis, leading to phenotypic changes in the absence of iron supplementation and an upregulation in transcript and protein levels of the cell’s endogenous transferrin receptor. Neither the sole overexpression of Fth1 nor TfR1 resulted in significant increases in intracellular iron content, although significant differences were seen when the two reporter genes were used in combination, in the presence of high concentrations of iron. The supplementation of the culture medium with iron sources was a more efficient means to obtain contrast than the use of reporter genes, where high levels of intracellular iron were reflected in transverse (T2) relaxation. The feasibility of imaging iron-supplemented cells by MRI is shown using a 3R-compliant chick embryo model.

The kinesin KIF16B mediates apical transcytosis of transferrin receptor in AP-1B-deficient epithelia

Polarized epithelial cells take up nutrients from the blood through receptors that are endocytosed and recycle back to the basolateral plasma membrane (PM) utilizing the epithelial-specific clathrin adaptor AP-1B. Some native epithelia lack AP-1B and therefore recycle cognate basolateral receptors to the apical PM, where they carry out important functions for the host organ. Here, we report a novel transcytotic pathway employed by AP-1B-deficient epithelia to relocate AP-1B cargo, such as transferrin receptor (TfR), to the apical PM. Lack of AP-1B inhibited basolateral recycling of TfR from common recycling endosomes (CRE), the site of function of AP-1B, and promoted its transfer to apical recycling endosomes (ARE) mediated by the plus-end kinesin KIF16B and non-centrosomal microtubules, and its delivery to the apical membrane mediated by the small GTPase rab11a. Hence, our experiments suggest that the apical recycling pathway of epithelial cells is functionally equivalent to the rab11a-dependent TfR recycling pathway of non-polarized cells. They define a transcytotic pathway important for the physiology of native AP-1B-deficient epithelia and report the first microtubule motor involved in transcytosis.

Delivery of a Peptide Radiopharmaceutical to Brain with an IgG-Avidin Fusion Protein

The genetic engineering, host cell expression, purity, identity, and in vivo brain drug targeting properties are described for a new IgG-fusion protein, designated the cTfRMAb-AV fusion protein. Avidin (AV) is fused to the carboxyl terminus of the heavy chain of the genetically engineered chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR). The TfRMAb binds the endogenous TfR on the blood-brain barrier (BBB), which triggers transport into brain from blood. The cTfRMAb-AV fusion protein is produced in stably transfected Chinese hamster ovary cells, which are grown in serum free medium under conditions of biotin starvation. Following affinity purification, the purity and identity of the cTfRMAb-AV fusion protein were verified by electrophoresis and Western blotting. The affinity of the cTfRMAb for the murine TfR is high, K(I) = 4.6 ± 0.5 nM, despite fusion of avidin to the antibody heavy chain. The model peptide radiopharmaceutical used in this study is the Aβ(1-40) amyloid peptide of Alzheimer's disease (AD), which in a brain-penetrating form could be used to image the amyloid plaque in brain in AD. The BBB transport and brain uptake of the [(125)I]-Aβ(1-40) peptide was measured in mice injected intravenously (IV) with the peptide either free or conjugated to the cTfRMAb-AV fusion protein. The brain uptake of the free Aβ(1-40) peptide was very low, 0.1% of injected dose (ID)/gram brain following i.v. injection, and is comparable to the brain uptake of a brain blood volume marker. However, the brain uptake of the Aβ(1-40) peptide was high, 2.1 ± 0.2% ID/gram brain, following attachment of the biotinylated peptide to the cTfRMAb-AV fusion protein. Capillary depletion analysis showed the peptide penetrated the brain parenchyma from blood. The cTfRMAb-AV fusion protein is a new drug delivery system that can target to mouse brain monobiotinylated peptide or antisense radiopharmaceuticals.

Proteome of ubiquitin/MVB pathway: possible involvement of iron-induced ubiquitylation of transferrin receptor in lysosomal degradation

Ubiquitylation of membrane proteins triggers their endocytosis at the plasma membrane and subsequent lysosomal degradation through multivesicular bodies (MVBs). A dominant-negative mutant SKD1/Vps4B caused an accumulation of ubiquitylated membrane proteins in MVBs. We have identified 22 membrane proteins whose trafficking is potentially regulated by ubiquitylation. Nine of them, including transferrin receptor (TfR), are indeed ubiquitylated and/or accumulated in MVBs in the cells expressing mutant Vps4. While the recycling route and iron-regulated expression of TfR are well characterized, the mechanism by which the degradation of TfR is regulated is largely unknown. We show that an excess of iron enhances both TfR's ubiquitylation and degradation in lysosomes. Probably, the up-regulated expression of ferritin, an endogenous iron-chelating molecule, attenuated the iron-induced degradation of TfR. Exogenously introduced lysine-less TfR, compared to the wild-type one, showed resistance to the iron-induced ubiquitylation and degradation, when endogenous TfR, which most certainly heterodimerizes with exogenous ones, was depleted with siRNA. These data suggest that the iron-induced ubiquitylation and degradation of TfR along with MVB pathway physiologically plays an important role in iron homeostasis.

Insulin-stimulated Phosphorylation of the Rab GTPase-activating Protein TBC1D1 Regulates GLUT4 Translocation

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular locations to the plasma membrane in adipose and muscle cells. Prior studies have shown that Akt phosphorylation of the Rab GTPase-activating protein, AS160 (160-kDa Akt substrate; also known as TBC1D4), triggers GLUT4 translocation, most likely by suppressing its Rab GTPase-activating protein activity. However, the regulation of a very similar protein, TBC1D1 (TBC domain family, member 1), which is mainly found in muscle, in insulin-stimulated GLUT4 translocation has been unclear. In the present study, we have identified likely Akt sites of insulin-stimulated phosphorylation of TBC1D1 in C2C12 myotubes. We show that a mutant of TBC1D1, in which several Akt sites have been converted to alanine, is considerably more inhibitory to insulin-stimulated GLUT4 translocation than wild-type TBC1D1. This result thus indicates that similar to AS160, Akt phosphorylation of TBC1D1 enables GLUT4 translocation. We also show that in addition to Akt activation, activation of the AMP-dependent protein kinase partially relieves the inhibition of GLUT4 translocation by TBC1D1. Finally, we show that the R125W variant of TBC1D1, which has been genetically associated with obesity, is equally inhibitory to insulin-stimulated GLUT4 translocation, as is wild-type TBC1D1, and that healthy and type 2 diabetic individuals express approximately the same level of TBC1D1 in biopsies of vastus lateralis muscle. In conclusion, phosphorylation of TBC1D1 is required for GLUT4 translocation. Thus, the regulation of TBC1D1 resembles that of its paralog, AS160.

Interaction of the Hereditary Hemochromatosis Protein HFE with Transferrin Receptor 2 Is Required for Transferrin-Induced Hepcidin Expression

The mechanisms that allow the body to sense iron levels in order to maintain iron homeostasis are unknown. Patients with the most common form of hereditary iron overload have mutations in the hereditary hemochromatosis protein HFE. They have lower levels of hepcidin than unaffected individuals. Hepcidin, a hepatic peptide hormone, negatively regulates iron efflux from the intestines into the blood. We report two hepatic cell lines, WIF-B cells and HepG2 cells transfected with HFE, where hepcidin expression responded to iron-loaded transferrin. The response was abolished when endogenous transferrin receptor 2 (TfR2) was suppressed or in primary hepatocytes lacking either functional TfR2 or HFE. Furthermore, transferrin-treated HepG2 cells transfected with HFE chimeras containing only the alpha3 and cytoplasmic domains could upregulate hepcidin expression. Since the HFE alpha3 domain interacts with TfR2, these results supported our finding that TfR2/HFE complex is required for transcriptional regulation of hepcidin by holo-Tf.

βPIX controls cell motility and neurite extension by regulating the distribution of GIT1

Cell motility entails the reorganization of the cytoskeleton and membrane trafficking for effective protrusion. GIT1/p95-APP1 is a member of a family of GTPase-activating proteins for ARF GTPases that affect endocytosis, adhesion and migration. GIT1 associates with paxillin and a complex including the Rac/Cdc42 exchanging factors PIX/Cool and the kinase PAK. In this study, we show that overexpression of betaPIX induces the accumulation of endogenous and overexpressed GIT1 at large structures similar to those induced by an ArfGAP-defective mutant of GIT1 (p95-C2). Immunohistochemical analysis and immunoelectron microscopy reveal that these structures include the endogenous transferrin receptor. Time-lapse analysis during motogenic stimuli shows that the formation and perinuclear accumulation of the p95-C2-positive structures is paralleled by inhibition of lamellipodium formation and cell retraction. Both dimerization and a functional SH3 domain of betaPIX are required for the accumulation of GIT1 in fibroblasts, which is prevented by the monomeric PIX-PG-DeltaLZ. This mutant also prevents the formation of endocytic aggregates and inhibition of neurite outgrowth in retinal neurons expressing p95-C2. Our results indicate that betaPIX is an important regulator of the subcellular distribution of GIT1, and suggest that alteration in the level of expression of the complex affects the endocytic compartment and cell motility.

Lack of Tyrosine 320 Impairs Spontaneous Endocytosis and Enhances Release of HLA-B27 Molecules

Several lines of evidence suggest that endocytosis of MHC class I molecules requires conserved motifs within the cytoplasmic domain. In this study, we show, in the C58 rat thymoma cell line transfected with HLA-B27 molecules, that replacement of the highly conserved tyrosine (Tyr320) in the cytoplasmic domain of HLA-B27 does not hamper cell surface expression of beta2-microglobulin H chain heterodimers or formation of misfolded molecules. However, Tyr320 replacement markedly impairs spontaneous endocytosis of HLA-B27. Although wild-type molecules are mostly internalized via endosomal compartments, Tyr320-mutated molecules remain at the plasma membrane in which partial colocalization with endogenous transferrin receptors can be observed, also impairing their endocytosis. Finally, we show that Tyr320 substitution enhances release of cleaved forms of HLA-B27 from the cell surface. These studies show for the first time that Tyr320 is most likely part of a cytoplasmic sorting motif involved in spontaneous endocytosis and shedding of MHC class I molecules.

GPI valence and the fate of secretory membrane proteins in African trypanosomes

Progression of GPI-anchored proteins in bloodstream African trypanosomes correlates with GPI-valence: homodimeric VSG (2 GPI) is a surface protein; heterodimeric transferrin receptor (1 GPI) localizes in the flagellar pocket; homodimeric GPI-minus VSG (0 GPI) is rapidly degraded in the lysosome. We test this relationship using three native secretory/endocytic proteins as monomeric GPI-plus and -minus reporters. GPI-minus procyclin trafficks to the lysosome and is degraded. GPI-plus procyclin trafficks to the flagellar pocket/cell surface and is released (approximately 50%) with an intact anchor, the remainder (approximately 50%) is degraded in the lysosome. GPI-plus BiPNHP, derived from the ER marker BiP, is released quantitatively (>80%), while GPI-plus p67HP, derived from the lysosomal marker p67, turns over by both release (approximately 15%) and lysosomal degradation (>50%). Turnover of endogenous transferrin receptor occurs primarily by lysosomal degradation (>90%). Thus shedding of monovalent GPI reporters correlates inversely with lysosomal targeting. We propose that mono-GPI reporters cycle through the flagellar pocket and endosome until they are disposed of by either shedding or lysosomal targeting. Partitioning between these fates may be a function of individual physical properties. Release is likely due to the exclusive use of C-14:0 myristate in the bloodstream stage GPI anchor. Up-regulation of transferrin receptor by culture in dog serum resulted in prominent cell surface localization, but not in elevated release. Surface receptor was non-functional for ligand binding suggesting that it may be bivalent homodimers of the GPI-anchored ESAG6 receptor subunit.

The hereditary hemochromatosis protein, HFE, lowers intracellular iron levels independently of transferrin receptor 1 in TRVb cells

AbstractHereditary hemochromatosis (HH) is an autosomal recessive disease that leads to parenchymal iron accumulation. The most common form of HH is caused by a single amino acid substitution in the HH protein, HFE, but the mechanism by which HFE regulates iron homeostasis is not known. In the absence of transferrin (Tf), HFE interacts with transferrin receptor 1 (TfR1) and the 2 proteins co-internalize, and in vitro studies have shown that HFE and Tf compete for TfR1 binding. Using a cell line lacking endogenous transferrin receptors (TRVb cells) transfected with different forms of HFE and TfR1, we demonstrate that even at low concentrations Tf competes effectively with HFE for binding to TfR1 on living cells. Transfection of TRVb cells or the derivative line TRVb1 (which stably expresses human TfR1) with HFE resulted in lower ferritin levels and decreased Fe2+ uptake. These data indicate that HFE can regulate intracellular iron storage independently of its interaction with TfR1. Earlier studies found that in HeLa cells, HFE expression lowers Tf-mediated iron uptake; here we show that HFE lowers non–Tf-bound iron in TRVb cells and add to a growing body of evidence that HFE may play different roles in different cell types.

The Transferrin Receptor: A Potential Molecular Imaging Marker for Human Cancer

Noninvasive imaging of differences between the molecular properties of cancer and normal tissue has the potential to enhance the detection of tumors. Because overexpression of endogenous transferrin receptor (TfR) has been qualitatively described for various cancers and is presumably due to malignant transformation of cells, TfR may represent a suitable target for application of molecular imaging technologies to increase detection of smaller tumors. In the work reported here, investigation into the biology of this receptor using electron microscopy has demonstrated that iron oxide particles targeted to TfR are internalized and accumulate in lysosomal vesicles within cells. Biochemical analysis of the interaction of imaging probes with cells overexpressing the TfR demonstrated that the extent of accumulation, and therefore probe efficacy, is dependent on the nature of the chemical cross-link between transferrin and the iron oxide particle. These data were utilized to design and synthesize an improved imaging probe. Experiments demonstrate that the novel magnetic resonance imaging (MRI) probe is sensitive enough to detect small differences in endogenous TfR expression in human cancer cell lines. Quantitative measurement of TfR overexpression in a panel of 27 human breast cancer patients demonstrated that 74% of patient cancer tissues overexpressed the TfR and that the sensitivity of the new imaging agent was suitable to detect TfR overexpression in greater than 40% of these cases. Based on a biochemical and cell biological approach, these studies have resulted in the synthesis and development of an improved MRI probe with the best in vitro and in vivo imaging properties reported to date.

Association of human transferrin receptor with GABARAP

A yeast two-hybrid screen identified a specific interaction between the cytoplasmic domain of transferrin receptor (TfR) and GABARAP, a 14 kDa protein that binds to the γ2 subunit of neuronal GABA A receptors. The specificity of the TfR–GABARAP interaction was demonstrated by in vitro binding assays with purified proteins and by co-immunoprecipitation of GABARAP with endogenous TfR from HeLa cell lysates. Replacement of the YTRF internalization motif with ATRA within the cytoplasmic domain of TfR reduced interaction with GABARAP in the yeast two-hybrid screen and in vitro binding assays. The intracellular location of GABARAP using chimeric GABARAP–GFP showed that the majority of GABARAP was located in perinuclear vesicles. Our results show that GABARAP plays a more general role outside the confines of neuronal cells and GABA A receptors.

Iron deficiency can upregulate expression of transferrin receptor at both the mRNA and protein level

Expression of the transferrin receptor (TfR) is controlled essentially by post-transcriptional regulation, which is mediated by the interaction between iron regulatory proteins (IRPs) and iron-responsive elements (IREs) located in the 3'-untranslated region (UTR) of TfR mRNA. We examined whether additional controls of expression might occur by investigating the regulation of TfR in the absence of its' IREs by cloning 3'-UTR truncated TfR cDNA (TfRDeltaIRE) into pcDNA3.1(+) and stably expressing it in CHO-TRVb (Chinese Hamster Ovary, TfR variant cells, clone b) cells which have no endogenous TfR. Both the surface and the total cell TfR protein levels increased significantly after treating the stably transfected cells with the iron chelator desferrioxamine (DFO) compared with the control cells. In contrast, expression of the transferrin receptor 2 (TfR2), which was also cloned in the same vector and stably expressed in the TRVb-CHO cells, was not regulated by iron chelation. In addition, TfR2 fused to the 3'-UTR of TfR (TfR2-IRE) was cloned into the same vector and stably expressed in the CHO-TRVb cells. A comparison showed that expression of full-length TfR and TfR2-IRE increased by fivefold and twofold, respectively, after addition of DFO. Further experiments showed that TRVb-CHO cells, which were stably transfected with TfRDeltaIRE and exposed to either normal medium or DFO, resulted in similar levels of TfR mRNA, but the TfR protein biosynthetic rate increased with iron chelation. In summary, expression of TfR in cells placed in an iron-deficient milieu can increase by a mechanism which is independent of the IREs of TfR.

Overexpression of hemochromatosis protein, HFE, alters transferrin recycling process in human hepatoma cells

HFE is a MHC class 1-like protein that is mutated in hereditary hemochromatosis. In order to elucidate the role of HFE protein on cellular iron metabolism, functional studies were carried out in human hepatoma cells (HLF) overexpressing a fusion gene of HFE and green fluorescent protein (GFP). The expression of HFE-GFP was found to be localized on cell membrane and perinuclear compartment by fluorescent microscopy. By co-immunoprecipitation and Western blotting, HFE-GFP protein formed a complex with endogenous transferrin receptor and β 2-microglobulin, suggesting that this fusion protein has the function of HFE reported previously. We then examined the 59Fe uptake and release, and internalization and recycling of 125I-labeled transferrin in order to elucidate the functional roles of HFE in the cell system. In the transfectants, HFE protein decreased the rate of transferrin receptor-dependent iron ( 59Fe) uptake by the cells, but did not change the rate of iron release, indicating that HFE protein decreased the rate of iron influx. Scatchard analysis of transferrin binding to HFE-transfected cells showed an elevation of the dissociation constant from 1.9 to 4.3 nM transferrin, indicating that HFE protein decreased the affinity of transferrin receptor for transferrin, while the number of transferrin receptors decreased from 1.5×10 5/cell to 1.2×10 5/cell. In addition, the rate of transferrin recycling, especially return from endosome to surface, was decreased in the HFE-transfected cells by pulse-chase study with 125I-labeled transferrin. Our results strongly suggest an additional role of HFE on transferrin receptor recycling in addition to the decrease of receptor affinity, resulting in the reduced cellular iron.

Protein targeting by tyrosine- and di-leucine-based signals: evidence for distinct saturable components.

Targeting of transmembrane proteins to lysosomes, endosomal compartments, or the trans-Golgi network is largely dependent upon cytoplasmically exposed sorting signals. Among the most widely used signals are those that conform to the tyrosine-based motif, YXXO (where Y is tyrosine, X is any amino acid, and O is an amino acid with a bulky hydrophobic group), and to the di-leucine (or LL) motif. Signals conforming to both motifs have been implicated in protein localization to similar post-Golgi compartments. We have exploited the saturability of sorting to ask whether different YXXO or LL signals use shared components of the targeting machinery. Chimeric proteins containing various cytoplasmic domains and/or targeting signals were overexpressed in HeLa cells by transient transfection. Endogenous transferrin receptor and lysosomal proteins accumulated at the cell surface upon overexpression of chimeric proteins containing functional YXXO targeting signals, regardless of the compartmental destination imparted by the signal. Furthermore, overexpression of these chimeric proteins compromised YXXO-mediated endocytosis and lysosomal delivery. These activities were ablated by mutating the signals or by appending sequences that conformed to the YXXO motif but lacked targeting activity. Interestingly, overexpression of chimeric proteins containing cytoplasmic LL signals failed to induce surface displacement of endogenous YXXO-containing proteins, but did displace other proteins containing LL motifs. Our data demonstrate that: (a) Protein targeting and internalization mediated by either YXXO or LL motifs are saturable processes; (b) common saturable components are used in YXXO-mediated protein internalization and targeting to different post-Golgi compartments; and (c) YXXO- and LL-mediated targeting mechanisms use distinct saturable components.

MafB Is an Interaction Partner and Repressor of Ets-1 That Inhibits Erythroid Differentiation

Using a yeast one-hybrid screen with a DNA-bound Ets-1 protein, we have identified MafB, an AP-1 like protein, as a direct interaction partner. MafB is specifically expressed in myelomonocytic cells and binds to the DNA-binding domain of Ets-1 via its basic region or leucine–zipper domain. Furthermore, it represses Ets-1 transactivation of synthetic promoters containing Ets binding sites and inhibits Ets-1–mediated transactivation of the transferrin receptor, which is known to be essential for erythroid differentiation. Accordingly, overexpression of MafB in an erythroblast cell line down-regulates the endogenous transferrin receptor gene and inhibits differentiation without affecting cell proliferation. These results highlight the importance of inhibitory interactions between transcription factors in regulating lineage-specific gene expression.

Generation of the soluble transferrin receptor requires cycling through an endosomal compartment.

The transmembrane protein, transferrin receptor (TfR), is found in a soluble form in human serum and in the medium of cell lines grown in tissue culture. The soluble form is generated by proteolytic cleavage between Arg-100 and Leu-101. We used two mutant human TfRs expressed in Chinese hamster ovary (CHO) cells lacking endogenous transferrin receptor to characterize the protease that cleaves the TfR and determine its location in the cell. The T104D mutant TfR lacks the O-linked carbohydrate at position 104, and is more susceptible to proteolytic cleavage at Arg-100 than the wildtype human TfR in these cells. We find that the protease is not a component of the serum in the growth medium, and it is not secreted by the cells. Cleavage does not occur during biosynthesis of the TfR, and occurs after the TfR has reached the cell surface. Expression of the T104D TfR in a temperature-sensitive acidification defective CHO cell line, G.7.1, shows that cleavage of the TfR is not dependent on acidification of endosomes. The C20A23 TfR is an endocytosis deficient mutant lacking an internalization signal. This mutant TfR, which is mainly localized to the cell surface, is cleaved less efficiently than the wild-type TfR, indicating that the protease is localized to an intracellular compartment.

Elimination of the O-linked glycosylation site at Thr 104 results in the generation of a soluble human-transferrin receptor

The transferrin receptor (TfR) is the plasma membrane protein responsible for the binding and internalization of the major iron-transport protein, transferrin. The function of the single O-linked oligosaccharide near the transmembrane domain of the TfR at amino acid Thr 104 is unknown. To elucidate the effect of the O-linked carbohydrate on TfR function, the oligosaccharide was eliminated by replacing Thr 104 with Asp and the mutated cDNA was expressed in a cell line lacking endogenous TfR. Elimination of the oligosaccharide at Thr 104 results in a form of the receptor that is susceptible to cleavage. A 78-kD soluble TfR that can bind transferrin is released into the growth medium. The intact mutant TfR is not grossly altered in its structure and does not differ significantly from the wild-type human receptor in many respects: (1) It shows the same distribution between the plasma membrane and intracellular compartments; (2) the binding constant for transferrin is similar to that of the wild-type TfR; and (3) it is not rapidly degraded. Protein-sequence analysis of the soluble form indicates that the sequence begins at amino acid 101 of the intact receptor. This is the same cleavage site reported for a soluble form of normal receptor found in human serum. Substitution of Gly, Glu, or Met at position 104 also results in increased cleavage of the TfR and suggests that elimination of the O-linked carbohydrate at position 104 enhances the susceptibility of TfR to cleavage and may mimic a naturally occurring process previously described as being related to erythropoiesis.

Elimination of the O-Linked Glycosylation Site at Thr 104 Results in the Generation of a Soluble Human-Transferrin Receptor

AbstractThe transferrin receptor (TfR) is the plasma membrane protein responsible for the binding and internalization of the major iron-transport protein, transferrin. The function of the single O-linked oligosaccharide near the transmembrane domain of the TfR at amino acid Thr 104 is unknown. To elucidate the effect of the O-linked carbohydrate on TfR function, the oligosaccharide was eliminated by replacing Thr 104 with Asp and the mutated cDNA was expressed in a cell line lacking endogenous TfR. Elimination of the oligosaccharide at Thr 104 results in a form of the receptor that is susceptible to cleavage. A 78-kD soluble TfR that can bind transferrin is released into the growth medium. The intact mutant TfR is not grossly altered in its structure and does not differ significantly from the wild-type human receptor in many respects: (1) It shows the same distribution between the plasma membrane and intracellular compartments; (2) the binding constant for transferrin is similar to that of the wild-type TfR; and (3) it is not rapidly degraded. Protein-sequence analysis of the soluble form indicates that the sequence begins at amino acid 101 of the intact receptor. This is the same cleavage site reported for a soluble form of normal receptor found in human serum. Substitution of Gly, Glu, or Met at position 104 also results in increased cleavage of the TfR and suggests that elimination of the O-linked carbohydrate at position 104 enhances the susceptibility of TfR to cleavage and may mimic a naturally occurring process previously described as being related to erythropoiesis.

A region of the C-terminal portion of the human transferrin receptor contains an asparagine-linked glycosylation site critical for receptor structure and function

The transferrin receptor is a cell surface protein and is responsible for the uptake of iron into many eukaryotic cells. In its mature form, the receptor possesses three asparagine-linked oligosaccharides. The effect of asparagine-linked glycosylation on the processing and cell surface localization of the human transferrin receptor is examined here by site-directed mutagenesis. Each of the extracellular consensus sequences (Asn-X-Ser/Thr) for asparagine-linked glycosylation was mutated individually and in all possible combinations. The constructs were transfected stably into NIH-3T3 cells and a Chinese hamster ovary cell line lacking endogenous transferrin receptors. Of the seven possible combinations of glycosylation sites, single mutations eliminating glycosylation at either Asn251 or Asn317 do not affect the processing and surface localization of the receptor. Eliminating both of these sites together has a small effect on the behavior of the receptor. However, mutation of the C-terminal glycosylation site (Asn727) has the most profound negative effect on the appearance of the receptor at the cell surface. The mutants lacking glycosylation at Asn727 appear to be retained in the endoplasmic reticulum as an increased association with binding immunoglobulin protein (BiP) is observed. Addition of a new glycosylation site in the C-terminal region of the unglycosylated mutated transferrin receptor restores the cell surface localization and the transferrin binding of the transferrin receptor, indicating that glycosylation in this region is critical for the correct transport of this receptor to the cell surface.