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.     

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.     

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     

The iron regulatory hormone hepcidin inhibits expression of iron release as well as iron uptake proteins in J774 cells

The mechanism by which hepcidin controls cellular iron release protein ferroportin 1 (Fpn1) in macrophages has been well established. However, little is known about the effects of hepcidin on cellular iron uptake proteins. Here, we demonstrated for the first time that hepcidin can significantly inhibit the expression of transferrin receptor 1 (TfR1) and divalent metal transporter 1 in addition to Fpn1, and therefore reduce transferrin-bound iron and non-transferrin-bound iron uptake and also iron release in J774 macrophages. Analysis of mechanisms using the iron-depleted cells showed that hepcidin has a direct inhibitory effect on all iron transport proteins we examined. Further studies demonstrated that the down-regulation of TfR1 induced by hepcidin is associated with cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA), probably being mediated by the cAMP–PKA pathway in J774 macrophages.     

Intracellular movement of cell surface receptors after endocytosis: resialylation of asialo-transferrin receptor in human erythroleukemia cells

The intracellular movement of cell surface transferrin receptor (TfR) after internalization was studied in K562 cultured human erythroleukemia cells. The sialic acid residues of the TfR glycoprotein were used to monitor transport to the Golgi complex, the site of sialyltransferases. Surface-labeled cells were treated with neuraminidase, and readdition of sialic acid residues, monitored by isoelectric focusing of immunoprecipitated TfR, was used to assess the movement of receptor to sialyltransferase-containing compartments. Asialo-TfR was resialylated by the cells with a half-time of 2-3 h. Resialylation occurred in an intracellular organelle, since it was inhibited by treatments that allow internalization of surface components but block transfer out of the endosomal compartment. Moreover, roughly half of the resialylated molecules were cleaved when cells were retreated with neuraminidase after culturing, indicating that this fraction of the molecules had returned to the cell surface. These results suggest that TfR is transported from the cell surface to the Golgi complex, the intracellular site of sialyltransferases, and then returns to the cell surface. This pathway, which has not been previously described for a cell surface receptor, may be different from the route followed by TfR in iron uptake, since reported rates of transferrin uptake and release are significantly more rapid than the resialylation of asialo-TfR.     

Ferristatin II Promotes Degradation of Transferrin Receptor-1 In Vitro and In Vivo

Previous studies have shown that the small molecule iron transport inhibitor ferristatin (NSC30611) acts by down-regulating transferrin receptor-1 (TfR1) via receptor degradation. In this investigation, we show that another small molecule, ferristatin II (NSC8679), acts in a similar manner to degrade the receptor through a nystatin-sensitive lipid raft pathway. Structural domains of the receptor necessary for interactions with the clathrin pathway do not appear to be necessary for ferristatin II induced degradation of TfR1. While TfR1 constitutively traffics through clathrin-mediated endocytosis, with or without ligand, the presence of Tf blocked ferristatin II induced degradation of TfR1. This effect of Tf was lost in a ligand binding receptor mutant G647A TfR1, suggesting that Tf binding to its receptor interferes with the drug’s activity. Rats treated with ferristatin II have lower TfR1 in liver. These effects are associated with reduced intestinal ⁵⁹Fe uptake, lower serum iron and transferrin saturation, but no change in liver non-heme iron stores. The observed hypoferremia promoted by degradation of TfR1 by ferristatin II appears to be due to induced hepcidin gene expression.     

Transferrin receptor 2: a new molecule in iron metabolism

Transferrin receptor 1 (TfR1) which mediates uptake of transferrin-bound iron, is essential for life in mammals. Recently, a close homologue of human transferrin receptor 1 was cloned and called transferrin receptor 2 (TfR2). A similar molecule has been identified in the mouse. Human transferrin receptor 2 is 45% identical with transferrin receptor 1 in the extracellular domain, but contains no iron responsive element in its mRNA and is apparently not regulated by intracellular iron concentration nor by interaction with HFE. Transferrin receptor 2, like transferrin receptor 1, binds transferrin in a pH-dependent manner (but with 25 times lower affinity) and delivers iron to cells. However, transferrin receptor 2 distribution differs from transferrin receptor 1, increasing in differentiating hepatocytes and decreasing in differentiating erythroblasts. Expression of both receptors is cell cycle dependent. Mutations in the human transferrin receptor 2 gene cause iron overload disease, suggesting it has a role in iron homeostasis.     

Src regulates Tyr(20) phosphorylation of transferrin receptor-1 and potentiates breast cancer cell survival

Transferrin receptor 1 (TfR1) is a ubiquitous type II membrane receptor with 61 amino acids in the N-terminal cytoplasmic region. TfR1 is highly expressed in cancer cells, particularly under iron deficient conditions. Overexpression of TfR1 is thought to meet the increased requirement of iron uptake necessary for cell growth. In the present study, we used transferrin (Tf), a known ligand of TfR1, and gambogic acid (GA), an apoptosis-inducing agent and newly identified TfR1 ligand to investigate the signaling role of TfR1 in breast cancer cells. We found that GA but not Tf induced apoptosis in a TfR1-dependent manner in breast cancer MDA-MB-231 cells. Estrogen receptor-positive MCF-7 cells lack caspase-3 and were not responsive to GA treatment. GA activated the three major signaling pathways of the MAPK family, as well as caspase-3, -8, and Poly(ADP-ribose)polymerase apoptotic pathway. Interestingly, only Src inhibitor PP2 greatly sensitized the cells to GA-mediated apoptosis. Further investigations by confocal fluorescence microscopy and immunoprecipitation revealed that Src and TfR1 are constitutively bound. Using TfR1-deficient CHO TRVB cells, point mutation studies showed that Tyr(20) within the (20)YTRF(23) motif of the cytoplasmic region of TfR1 is the phosphorylation site by Src. TfR1 Tyr(20) phosphomutants were more sensitive to GA-mediated apoptosis. Our results indicate that, albeit its iron uptake function, TfR1 is a signaling molecule and tyrosine phosphorylation at position 20 by Src enhances anti-apoptosis and potentiates breast cancer cell survival.     

Transferrin-receptor-independent but iron-dependent proliferation of variant Chinese hamster ovary cells

The purpose of this study is to clarify the role of iron, transferrin, an iron-binding protein in vertebrate plasma, and transferrin receptors in cell proliferation. Transferrin, which is indispensable for most cells growing in tissue culture, is frequently referred to as a "growth factor". Proliferating cells express high numbers of transferrin receptors, and the binding of transferrin to their receptors that is needed for cells to initiate and maintain their DNA synthesis is sometimes regarded as analogous to other growth factor-receptor interactions. Although numerous previous experiments strongly indicate that the only function of transferrin in supporting cell proliferation is supplying cells with iron, they did not completely rule out some direct or signaling role transferrin receptors could play in cell proliferation. To address this issue, we exploited transferrin-receptor-deficient mutant Chinese hamster ovary (CHO) cells (McGraw, T. E., Greenfield, L., and Maxfield, F. R., 1987, J. Cell. Biol. 105, 207-214) in which various aspects of iron and transferrin metabolism in relation to their capacity to proliferate were investigated. Variant cells neither specifically bind transferrin nor do their extracts contain any detectable functional transferrin receptors, yet they proliferate and synthesize DNA with rates comparable to those observed with parent CHO cells. Desferrioxamine, an iron chelating agent, inhibits growth and DNA synthesis of both variant and control CHO cells. This inhibition can be fully alleviated, in both cell types, by ferric pyridoxal isonicotinoyl hydrazone, which can supply cells with a utilizable form of iron by a pathway not requiring transferrin and their receptors. Studies of 59Fe uptake and 125I-transferrin binding revealed that parent cells can take up iron by at least three mechanisms: from transferrin by receptor-dependent and -independent (nonspecific, nonsaturable, not requiring acidification) pathways and from inorganic iron salts (initially present in the medium as FeSO4). Although variant CHO cells are unable to acquire transferrin iron via the receptor pathway, two remaining mechanisms provide these cells with sufficient amounts of iron for DNA synthesis and cell proliferation. In conclusion, although transferrin receptors are dispensable in terms of their absolute requirement for proliferating cells, a supply of iron is still needed for their DNA synthesis. Transferrin-receptor-deficient CHO cells may be a useful model for investigating receptor-independent iron uptake from transferrin and nontransferrin iron sources.     

Release of the soluble transferrin receptor is directly regulated by binding of its ligand ferritransferrin

The human transferrin receptor (TfR) is shed by an integral metalloprotease releasing a soluble form (sTfR) into serum. The sTfR reflects the iron demand of the body and is postulated as a regulator of iron homeostasis via binding to the hereditary hemochromatosis protein HFE. To study the role of transferrin in this process, we investigated TfR shedding in HL60 cells and TfR-deficient Chinese hamster ovary cells transfected with human TfR. Independent of TfR expression, sTfR release decreases with increasing ferritransferrin concentrations, whereas apo-transferrin exhibits no inhibitory effect. To investigate the underlying mechanism, we generated several TfR mutants with different binding affinities for transferrin. Shedding of TfR mutants in transfected cells correlates exactly with their binding affinity, implying that the effect of ferritransferrin on TfR shedding is mediated by a direct molecular interaction. Analysis of sTfR release from purified microsomal membranes revealed that the regulation is independent from intracellular trafficking or cellular signaling events. Our results clearly demonstrated that sTfR does not only reflect the iron demand of the cells but also the iron availability in the bloodstream, mirrored by iron saturation of transferrin, corroborating the important potential function of sTfR as a regulator of iron homeostasis.     

The molecular mechanism for receptor-stimulated iron release from the plasma iron transport protein transferrin

Human transferrin receptor 1 (TfR) binds iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes where iron is released in a TfR-facilitated process. Consistent with our hypothesis that TfR binding stimulates iron release from Fe-Tf at acidic pH by stabilizing the apo-Tf conformation, a TfR mutant (W641A/F760A-TfR) that binds Fe-Tf, but not apo-Tf, cannot stimulate iron release from Fe-Tf, and less iron is released from Fe-Tf inside cells expressing W641A/F760A-TfR than cells expressing wild-type TfR (wtTfR). Electron paramagnetic resonance spectroscopy shows that binding at acidic pH to wtTfR, but not W641A/F760A-TfR, changes the Tf iron binding site > or =30 A from the TfR W641/F760 patch. Mutation of Tf histidine residues predicted to interact with the W641/F760 patch eliminates TfR-dependent acceleration of iron release. Identification of TfR and Tf residues critical for TfR-facilitated iron release, yet distant from a Tf iron binding site, demonstrates that TfR transmits long-range conformational changes and stabilizes the conformation of apo-Tf to accelerate iron release from Fe-Tf.     

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.     

A novel iron responsive element in the 3'UTR of human MRCKalpha

Human untranslated region (UTR) databases were searched to identify novel proteins potentially regulated by an iron responsive element (IRE), and found two candidates-cell cycle phosphatase Cdc14A variant 1 and myotonic dystrophy kinase-related Cdc42-binding kinase alpha (MRCKalpha), both possessing a putative IRE in their 3'UTR. In further experiments, we focused on MRCKalpha. Biochemical analyses of the MRCKalpha IRE revealed that it was functional and mediated the response to iron level in the same way as transferrin receptor 1 IREs (TfR) did. Similarly to TfR mRNA, MRCKalpha mRNA is stabilized, when iron supply is low, while it is destabilized under iron-rich conditions. The expression of MRCKalpha mRNA was found to be ubiquitous; the highest levels were noted in testes, the lowest in skeletal muscle. The level of MRCKalpha mRNA in various tissues strongly positively correlates with the level of TfR mRNA, indicating its possible role in the transferrin iron uptake pathway.     

转铁蛋白受体2及其功能与相关疾病

转铁蛋白受体2(transferrin receptor 2, TfR2) 是最近发现的一种重要铁代谢蛋白.研究显示它不仅是一种介导肝脏细胞铁摄取的主要蛋白,而且在调节小肠铁吸收方面起着极其关键的作用,是控制肝脏铁调素合成和释放的关键成分.已经证实,TfR2基因突变是遗传性血色素沉着病的重要原因之一.     

CD81 Promotes Both the Degradation of Transferrin Receptor 2 (TfR2) and the Tfr2-mediated Maintenance of Hepcidin Expression

Mutations in transferrin receptor 2 (TfR2) cause a rare form of the hereditary hemochromatosis, resulting in iron overload predominantly in the liver. TfR2 is primarily expressed in hepatocytes and is hypothesized to sense iron levels in the blood to positively regulate the expression of hepcidin through activation of the BMP signaling pathway. Hepcidin is a peptide hormone that negatively regulates iron egress from cells and thus limits intestinal iron uptake. In this study, a yeast two-hybrid approach using the cytoplasmic domain of TfR2 identified CD81 as an interacting protein. CD81 is an abundant tetraspanin in the liver. Co-precipitations of CD81 with different TfR2 constructs demonstrated that both the cytoplasmic and ecto-transmembrane domains of TfR2 interact with CD81. Knockdown of CD81 using siRNA significantly increased TfR2 levels by increasing the half-life of TfR2, indicating that CD81 promotes degradation of TfR2. Previous studies showed that CD81 is targeted for degradation by GRAIL, an ubiquitin E3 ligase. Knockdown of GRAIL in Hep3B-TfR2 cells increased TfR2 levels, consistent with inhibition of CD81 ubiquitination. These results suggest that down-regulation of CD81 by GRAIL targets TfR2 for degradation. Surprisingly, knockdown of CD81 decreased hepcidin expression, implying that the TfR2/CD81 complex is involved in the maintenance of hepcidin mRNA. Moreover, knockdown of CD81 did not affect the stimulation of hepcidin expression by BMP6 but increased both the expression of ID1 and SMAD7, direct targets of BMP signaling pathway, and the phosphorylation of ERK1/2, indicating that the CD81 regulates hepcidin expression differently from the BMP and ERK1/2 signaling pathways.     

Calmodulin dependence of transferrin receptor recycling in rat reticulocytes.

Kinetic analysis of transferrin receptor properties in 6-8 day rat reticulocytes showed the existence of a single class of high-affinity receptors (Kd 3-10 nM), of which 20-25% were located at the cell surface and the remainder within an intracellular pool. Total transferrin receptor cycling time was 3.9 min. These studies examined the effects of various inhibitors on receptor-mediated transferrin iron delivery in order to define critical steps and events necessary to maintain the functional integrity of the pathway. Dansylcadaverine inhibited iron uptake by blocking exocytic release of transferrin and return of receptors to the cell surface, but did not affect transferrin endocytosis; this action served to deplete the surface pool of transferrin receptors, leading to shutdown of iron uptake. Calmidazolium and other putative calmodulin antagonists exerted an identical action on iron uptake and receptor recycling. The inhibitory effects of these agents on receptor recycling were overcome by the timely addition of Ca2+/ionomycin. From correlative analyses of the effects of these and other inhibitors, it was concluded that: (1) dansylcadaverine and calmodulin antagonists inhibit iron uptake by suppression of receptor recycling and exocytic transferrin release, (2) protein kinase C, transglutaminase, protein synthesis and release of transferrin-bound iron are not necessary for the functional integrity of the iron delivery pathway, (3) exocytic transferrin release and concomitant receptor recycling in rat reticulocytes is dependent upon Ca2+/calmodulin, (4) dansylcadaverine, dimethyldansylcadaverine and calmidazolium act on iron uptake by interfering with calmodulin function, and (5) the endocytotic and exocytotic arms of the iron delivery pathway are under separate regulatory control.     

Transferrin receptor 2-alpha supports cell growth both in iron-chelated cultured cells and in vivo

In most cells, transferrin receptor (TfR1)-mediated endocytosis is a major pathway for cellular iron uptake. We recently cloned the human transferrin receptor 2 (TfR2) gene, which encodes a second receptor for transferrin (Kawabata, H., Yang, R., Hirama, T., Vuong, P. T., Kawano, S., Gombart, A. F., and Koeffler, H. P. (1999) J. Biol. Chem. 274, 20826-20832). In the present study, the regulation of TfR2 expression and function was investigated. A select Chinese hamster ovary (CHO)-TRVb cell line that does not express either TfR1 or TfR2 was stably transfected with either TfR1 or TfR2-alpha cDNA. TfR2-alpha-expressing cells had considerably lower affinity for holotransferrin when compared with TfR1-expressing CHO cells. Interestingly, in contrast to TfR1, expression of TfR2 mRNA in K562 cells was not up-regulated by desferrioxamine (DFO), a cell membrane-permeable iron chelator. In MG63 cells, expression of TfR2 mRNA was regulated in the cell cycle with the highest expression in late G(1) phase and no expression in G(0)/G(1). DFO reduced cell proliferation and DNA synthesis of CHO-TRVb control cells, whereas it had little effect on TfR2-alpha-expressing CHO cells when measured by clonogenic and cell cycle analysis. In addition, CHO cells that express TfR2-alpha developed into tumors in nude mice whereas CHO control cells did not. In conclusion, TfR2 expression may be regulated by the cell cycle rather than cellular iron status and may support cell growth both in vitro and in vivo.     

The Cytoplasmic domain of transferrin receptor 2 dictates its stability and response to holo-transferrin in Hep3B cells

Transferrin receptor 2 (TfR2) is a homolog of transferrin receptor 1 (TfR1), the receptor responsible for the uptake of iron-loaded transferrin (holo-Tf) into cells. Unlike the ubiquitous TfR1, TfR2 is predominantly expressed in the liver. Mutations in TfR2 gene cause a rare autosomal recessive form of the iron overload disease, hereditary hemochromatosis. Previous studies demonstrated that holo-Tf increases TfR2 levels by stabilizing TfR2 at the protein level. In this study we constructed two chimeras, one of which had the cytoplasmic domain of TfR2 and the remaining portion of TfR1 and the other with the cytoplasmic and transmembrane domain of TfR1 joined to the ectodomain of TfR2. Similar to TfR2, the levels of the chimera containing only the cytoplasmic domain of TfR2 increased in a time- and dose-dependent manner after the addition of holo-Tf to the medium. The half-life of the chimera increased 2.7-fold in cells exposed to holo-Tf like the endogenous TfR2 in HepG2 cells. Like TfR2 and unlike TfR1, the levels of the chimera did not respond to intracellular iron content. These results suggest that although holo-Tf binding to the ectodomain is necessary, the cytoplasmic domain of TfR2 is largely responsible for its stabilization by holo-Tf.     

Structural and functional stability of the mature transferrin receptor from human placenta

The transferrin receptor (TfR) is a N- and O-glycosylated transmembrane protein mediating the cellular iron uptake by binding and internalization of diferric transferrin. In this study, rate constants and dissociation constants of 125I-ferri-transferrin binding to the human TfR were examined dependent on receptor glycan composition, pH, bivalent cations, and temperature. To do so, purified human placental TfR was noncovalently immobilized to polystyrene surfaces and subjected to alterations in various parameters. We found that transferrin binding was clearly dependent on a receptor pretreatment with buffers of various pH in that most of the TfR molecules irreversibly lost transferrin binding activity below pH 6.5. However, the dissociation constant of the remaining active binding sites was not affected. Similarly, we were able to define the thermal stability of the receptor as a function of transferrin binding ability. Binding of transferrin was completely lost provided that the receptor was pretreated at temperatures of at least 65 degrees C. Treatment with EDTA also caused an irreversible loss of transferrin binding activity, indicating that the functionally active conformation of the mature TfR depends on bivalent cations. In order to examine the role of the receptor glycans, we enzymatically removed the sialic acid residues, the hybrid and oligomannosidic N-glycans, or all types of N-glycans. In contrast to the parameters described above, all desialylated and N-deglycosylated TfR variants had exactly the same transferrin binding properties as the native TfR. To assess changes in the secondary structure of the receptor, circular dichroic spectra were recorded from TfR at pH 5.0, from heat pretreated receptor and from deglycosylated TfR. Since the receptor did not exhibit detectable changes in the CD spectrum of the deglycosylated receptor, it can be concluded that the N-linked carbohydrates of the mature, fully processed TfR are not essential for transferrin binding and conformational stability.