Inhibition of transferrin receptor expression by interferon-alpha in human lymphoblastoid cells and mitogen-induced lymphocytes

125I-Transferrin binding to lymphoblastoid K562 and Daudi cells markedly increased after exposure of the cells to culture conditions that stimulated proliferation. Treatment of these cells with interferon-alpha (IFN-alpha) resulted in concurrent inhibition of cell growth and of the rise in transferrin binding. Scatchard analyses revealed that IFN reduced the number of transferrin receptors without altering the binding constant. When 125I-transferrin binding was measured using permeabilized cells, the IFN-induced reduction of binding was comparable to that observed with intact cells, indicating that IFN diminished the total number of cellular transferrin receptors. We also found that addition of IFN-alpha to phytohemagglutinin-stimulated human lymphocytes inhibited the mitogen-induced enhancement of [3H]thymidine incorporation as well as surface binding of 125I-transferrin. Our findings suggest that the decrease in transferrin receptor expression on IFN-alpha-treated cells may be one of the mechanisms responsible for the antiproliferative action of IFN.     

Ligand-induced heterodimerization between the ligand binding domains of the Drosophila ecdysteroid receptor and ultraspiracle.

The insect ecdysteroid receptor consists of a heterodimer between EcR and the RXR-orthologue, USP. We addressed the question of whether this heterodimer, like all other RXR heterodimers, may be formed in the absence of ligand and whether ligand promotes dimerization. We found that C-terminal protein fragments that comprised the ligand binding, but not the DNA binding domain of EcR and USP and which were equipped with the activation or DNA binding region of GAL4, respectively, exhibit a weak ability to interact spontaneously with each other. Moreover, the heterodimer formation is greatly enhanced upon administration of active ecdysteroids in a dose-dependent manner. This was shown in vivo by a yeast two-hybrid system and in vitro by a modified electromobility shift assay. Furthermore, the EcR fragment expressed in yeast was functional and bound radioactively labelled ecdysteroid specifically. Ligand binding was greatly enhanced by the presence of a USP ligand binding domain. Therefore, ecdysteroids are capable of inducing heterodimer formation between EcR and USP, even when the binding of these receptor proteins to cognate DNA response elements does not occur. This capability may be a regulated aspect of ecdysteroid action during insect development.     

Iodination significantly influences the binding of human transferrin to the transferrin receptor

The human transferrin receptor (TfR) and its ligand, the serum iron carrier transferrin, serve as a model system for endocytic receptors. Although the complete structure of the receptor's ectodomain and a partial structure of the ligand have been published, conflicting results still exist about the magnitude of equilibrium binding constants, possibly due to different labeling techniques. In the present study, we determined the equilibrium binding constant of purified human TfR and transferrin. The results were compared to those obtained with either iodinated TfR or transferrin. Using an enzyme-linked assay for receptor-ligand interactions based on the published direct calibration ELISA technique, we determined an equilibrium constant of Kd=0.22 nM for the binding of unmodified human Tf to surface-immobilized human TfR. In a reciprocal experiment using soluble receptor and surface-bound transferrin, a similar constant of Kd=0.23 nM was measured. In contrast, covalent labeling of either TfR or transferrin with 125I reduced the affinity 3-5-fold to Kd=0.66 nM and Kd=1.01 nM, respectively. The decrease in affinity upon iodination of transferrin is contrasted by an only 1.9-fold decrease in the association rate constant, suggesting that the iodination affects rather the dissociation than the association kinetics. These results indicate that precautions should be taken when interpreting equilibrium and rate constants determined with covalently labeled components.     

Mechanism for multiple ligand recognition by the human transferrin receptor

Transferrin receptor 1 (TfR) plays a critical role in cellular iron import for most higher organisms. Cell surface TfR binds to circulating iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes, where low pH promotes iron to dissociate from transferrin (Tf) in a TfR-assisted process. The iron-free form of Tf (apo-Tf) remains bound to TfR and is recycled to the cell surface, where the complex dissociates upon exposure to the slightly basic pH of the blood. Fe-Tf competes for binding to TfR with HFE, the protein mutated in the iron-overload disease hereditary hemochromatosis. We used a quantitative surface plasmon resonance assay to determine the binding affinities of an extensive set of site-directed TfR mutants to HFE and Fe-Tf at pH 7.4 and to apo-Tf at pH 6.3. These results confirm the previous finding that Fe-Tf and HFE compete for the receptor by binding to an overlapping site on the TfR helical domain. Spatially distant mutations in the TfR protease-like domain affect binding of Fe-Tf, but not iron-loaded Tf C-lobe, apo-Tf, or HFE, and mutations at the edge of the TfR helical domain affect binding of apo-Tf, but not Fe-Tf or HFE. The binding data presented here reveal the binding footprints on TfR for Fe-Tf and apo-Tf. These data support a model in which the Tf C-lobe contacts the TfR helical domain and the Tf N-lobe contacts the base of the TfR protease-like domain. The differential effects of some TfR mutations on binding to Fe-Tf and apo-Tf suggest differences in the contact points between TfR and the two forms of Tf that could be caused by pH-dependent conformational changes in Tf, TfR, or both. From these data, we propose a structure-based model for the mechanism of TfR-assisted iron release from Fe-Tf.     

The control of transferrin receptor synthesis in mitogen-stimulated human lymphocytes

Human lymphocytes cultured in the presence of the plant mitogenic lectin phytohemagglutin in (PHA) become activated and leave the G0 phase of the cell cycle. In the presence of PHA and lymphokines produced in situ the cells will enter S phase and undergo cell division. We have determined the time course of appearance for the receptor for transferrin as an initial attempt to understand the molecular mechanisms regulating the onset of lymphocyte differentiation and proliferation in the 48 h following PHA addition. Using three different assay methods we have shown that the increase in the number of surface receptor molecules is due to the accumulation of newly synthesized receptor and not to the redistribution of a previously existing pool of receptor molecules. The total amount of transferrin receptor increased at least four-fold. In vitro translation of RNA from activated lymphocytes indicates that the new receptor synthesis is due, at least in part, to increased availability of mRNA encoding the transferrin receptor.     

Dynamic stabilization of nuclear receptor ligand binding domains by hormone or corepressor binding

We have developed a novel assembly assay to examine structural changes in the ligand binding domain (LBD) of the thyroid hormone receptor (TR). Fragments including the first helix of the TR LBD interact only weakly with the remainder of the LBD in the absence of hormone, but this interaction is strongly enhanced by the addition of either hormone or the corepressor NCoR. Since neither the ligand nor the corepressor shows direct interaction with this helix, we propose that both exert their effects by stabilizing the overall structure of the LBD. Current models of activation of nuclear hormone receptors focus on a ligand-induced allosteric shift in the position of the C-terminal helix 12 that generates the coactivator binding site. Our results suggest that ligand binding also has more global effects that dynamically alter the structure of the receptor LBD.     

TrkA immunoglobulin-like ligand binding domains inhibit spontaneous activation of the receptor

The extracellular region of the nerve growth factor (NGF) receptor, TrkA, contains two immunoglobulin (Ig)-like domains that are required for specific ligand binding. We have investigated the possible role of these two Ig-like domains in receptor dimerization and activation by using different mutants of the TrkA extracellular region. Deletions of each Ig-like domain, of both, and of the entire extracellular region were made. To probe the structural constraints on ligand-independent receptor dimerization, chimeric receptors were generated by swapping the Ig-like domains of the TrkA receptor for the third or fourth Ig-like domain of c-Kit. We also introduced single-amino-acid changes in conserved residues within the Ig-like domains of TrkA. Most of these TrkA variants did not bind NGF, and their expression in PC12nnr5 cells, which lack endogenous TrkA, promoted ligand-independent neurite outgrowth. Some TrkA mutant receptors induced malignant transformation of Rat-1 cells, as assessed by measuring proliferation in the absence of serum, anchorage-independent growth, and tumorigenesis in nude mice. These mutants exhibited constitutive phosphorylation and spontaneous dimerization consistent with their biological activities. Our data suggest that spontaneous dimerization of TrkA occurs when the structure of the Ig-like domains is altered, implying that the intact domains inhibit receptor dimerization in the absence of NGF.     

Structural domains of the insulin receptor and IGF receptor required for dimerisation and ligand binding

We investigated structural requirements for dimerisation and ligand binding of insulin/IGF receptors. Soluble receptor fragments consisting of N-terminal domains (L1/CYS/L2, L1/CYS/L2/F0) or fibronectin domains (F0/F1/F2, F1/F2) were expressed in CHO cells. Fragments containing F0 or F1 domains were secreted as disulphide-linked dimers, and those consisting of L1/CYS/L2 domains as monomers. None of these proteins bound ligand. However, when a peptide of 16 amino acids from the alpha-subunit C-terminus was fused to the C-terminus of L1/CYS/L2, the monomeric insulin and IGF receptor constructs bound their respective ligands with affinity only 10-fold lower than native receptors.     

Mapping the membrane topology and extracellular ligand binding domains of the retinol binding protein receptor

STRA6 is a multitransmembrane domain protein not homologous to any other proteins with known function. It functions as the high-affinity receptor for plasma retinol binding protein (RBP) and mediates cellular uptake of vitamin A from the vitamin A-RBP complex. Consistent with the diverse roles of vitamin A and the wide tissue expression pattern of STRA6, mutations in STRA6 are associated with severe pathological phenotypes in humans. The structural basis for STRA6's biochemical function is unknown. Although computer programs predict 11 transmembrane domains for STRA6, its topology has never been studied experimentally. Elucidating the transmembrane topology of STRA6 is critical for understanding its structure and function. By inserting an epitope tag into all possible extracellular and intracellular domains of STRA6, we systematically analyzed the accessibility of each tag on the surface of live cells, the accessibility of each tag in permeabilized cells, and the effect of each tag on RBP binding and STRA6-mediated vitamin A uptake from the vitamin A-RBP complex. In addition, we used a new lysine accessibility technique combining cell-surface biotinylation and tandem-affinity purification to study a region of the protein not revealed by the epitope tagging method. These studies not only revealed STRA6's extracellular, transmembrane, and intracellular domains but also implicated extracellular regions of STRA6 in RBP binding.     

Ionic residues of human serum transferrin affect binding to the transferrin receptor and iron release

Efficient delivery of iron is critically dependent on the binding of diferric human serum transferrin (hTF) to its specific receptor (TFR) on the surface of actively dividing cells. Internalization of the complex into an endosome precedes iron removal. The return of hTF to the blood to continue the iron delivery cycle relies on the maintenance of the interaction between apohTF and the TFR after exposure to endosomal pH (≤6.0). Identification of the specific residues accounting for the pH-sensitive nanomolar affinity with which hTF binds to TFR throughout the cycle is important to fully understand the iron delivery process. Alanine substitution of 11 charged hTF residues identified by available structures and modeling studies allowed evaluation of the role of each in (1) binding of hTF to the TFR and (2) TFR-mediated iron release. Six hTF mutants (R50A, R352A, D356A, E357A, E367A, and K511A) competed poorly with biotinylated diferric hTF for binding to TFR. In particular, we show that Asp356 in the C-lobe of hTF is essential to the formation of a stable hTF-TFR complex: mutation of Asp356 in the monoferric C-lobe hTF background prevented the formation of the stoichiometric 2:2 (hTF:TFR monomer) complex. Moreover, mutation of three residues (Asp356, Glu367, and Lys511), whether in the diferric or monoferric C-lobe hTF, significantly affected iron release when in complex with the TFR. Thus, mutagenesis of charged hTF residues has allowed identification of a number of residues that are critical to formation of and release of iron from the hTF-TFR complex.     

Mapping ligand binding domains in chimeric fibroblast growth factor receptor molecules. Multiple regions determine ligand binding specificity

Fibroblast growth factors (FGFs) mediate essential cellular functions by activating one of four alternatively spliced FGF receptors (FGFRs). To determine the mechanism regulating ligand binding affinity and specificity, soluble FGFR1 and FGFR3 binding domains were compared for activity. FGFR1 bound well to FGF2 but poorly to FGF8 and FGF9. In contrast, FGFR3 bound well to FGF8 and FGF9 but poorly to FGF2. The differential ligand binding specificity of these two receptors was exploited to map specific ligand binding regions in mutant and chimeric receptor molecules. Deletion of immunoglobulin-like (Ig) domain I did not effect ligand binding, thus localizing the binding region(s) to the distal two Ig domains. Mapping studies identified two regions that contribute to FGF binding. Additionally, FGF2 binding showed positive cooperativity, suggesting the presence of two binding sites on a single FGFR or two interacting sites on an FGFR dimer. Analysis of FGF8 and FGF9 binding to chimeric receptors showed that a broad region spanning Ig domain II and sequences further N-terminal determines binding specificity for these ligands. These data demonstrate that multiple regions of the FGFR regulate ligand binding specificity and that these regions are distinct with respect to different members of the FGF family.     

Similarities between the transferrin receptor proteins on human reticulocytes and human placentae

The transferrin receptor of the human reticulocyte was isolated by two different immunoaffinity procedures. These included indirect immunoprecipitation with a transferrin/anti-transferrin complex and direct immunoprecipitation with antiserum to purified transferrin receptor from placentae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the receptor isolated from reticulocytes reveals a polypeptide at Mr = 94,000 identical in molecular weight with that of the placenta. A radioimmunoassay using purified 125I-labeled transferrin receptor from placentae and antiserum to transferrin receptor fails to distinguish any immunological differences between the reticulocyte and placental forms of the protein. In addition, proteolytic digests of both of these polypeptides with Staphylococcus aureus protease show identical proteolytic patterns, indicating similar sequences.     

Combined radiolabel-binding and immunocytochemical evaluation of receptor–ligand interactions. Studies of transferrin receptors on activated lymphocytes

A protocol that involved both immunohistological and radiolabel-binding procedures was devised for the study of transferrin-receptor interactions. This composite approach yielded considerably more information than did either technique used alone, and also provided a simple means for exclusion of several common potential sources of error.     

Hemopexin-dependent down-regulation of expression of the human transferrin receptor

To investigate the regulation mechanism of the uptake of iron and heme iron by the cells and intracellular utilization of iron, we examined the interaction between iron uptake from transferrin and hemopexin-mediated uptake of heme by human leukemic U937 cells or HeLa cells. U937 cells exhibited about 40,000 hemopexin receptors/cell with a dissociation constant (Kd) of 1 nM. Heme bound in hemopexin was taken up by U937 cells or HeLa cells in a receptor-mediated manner. Treatment of both species of cells with hemopexin led to a rapid decrease in iron uptake from transferrin in a hemopexin dose-dependent manner, and the decrease seen in case of treatment with hemin was less than that seen with hemopexin. The decrease of iron uptake by hemopexin contributed to a decrease in cell surface transferrin receptors on hemopexin-treated cells. Immunoblot analysis of the transferrin receptors revealed that the cellular level of receptors in U937 cells did not vary during an 8-h incubation with hemopexin although the number of surface receptors as well as iron uptake decreased within the 2-h incubation. After 4 h of incubation of the cells with hemopexin, a decrease of the synthesis of the receptors occurred. Thus, the down-regulation of transferrin receptors by hemopexin can be attributed to at least two mechanisms. One is a rapid redistribution of the surface receptor into the interior of the cells, and the other is a decrease in the biosynthesis of the receptor. 59Fe from the internalized heme rapidly appeared in non-heme iron (ferritin) coincidently with the induction of heme oxygenase. The results suggest that iron released from heme down-regulates the expression of the transferrin receptors and iron uptake.     

Restricted expression of tumor necrosis factor-related apoptosis-inducing ligand receptor 4 in human peripheral blood lymphocytes

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumor but not normal cells, thus providing therapeutic possibilities for human cancers. However, it is not fully clear how widespread TRAIL receptors are, or how TRAIL signaling is modulated in normal cells. We characterized cell surface expression of TRAIL receptors in normal healthy donor peripheral blood and report that each of the TRAIL receptors are characteristically expressed on restricted cell populations. TRAIL-R1 is distinctively expressed on B-lymphocytes, TRAIL-R2 on monocytes, TRAIL-R3 on neutrophils and most impressively, CD8+ lymphocytes and NKT lymphocytes but not CD4+ lymphocytes express TRAIL-R4.     

Rosetting of human T lymphocytes with sheep and human erythrocytes. Comparison of human and sheep ligand binding using purified E receptor

Previous studies have shown that the purified T lymphocyte glycoprotein, cluster differentiation 2 (CD2) (also known as T11, lymphocyte function-associated antigen (LFA)-2, and the erythrocyte (E) rosette receptor) interacts with the LFA-3 molecule on human E. We have examined the interaction of the purified CD2 molecule with the T11 target structure (T11TS) molecule on sheep E, and compared the two interactions. Purified, 125I-labeled CD2 bound to sheep E and the binding was inhibited by anti-T11TS monoclonal antibody (mAb). Reciprocally, the binding of T11TS mAb to sheep E was inhibited by pretreatment of sheep E with purified CD2. High concentrations of purified CD2 aggregated sheep E, possibly by inserting into the membrane, and the aggregation was inhibited by T11TS mAb. The affinity and number of binding sites for purified CD2 on sheep and human E was found to be similar, with Ka of 9 X 10(7)/M and 6 X 10(7)/M and 9800 and 8300 CD2 binding sites/E, respectively. Thus, the human T lymphocyte CD2 molecule is a receptor that cross-reacts between LFA-3 on human E and T11TS on sheep E, suggesting that LFA-3 and T11TS are functionally homologous ligands. As measured by saturation mAb binding, there are 8100 and 3900 ligand molecules/sheep and human E, respectively. Human and sheep E have surface areas of 145 and 54 micron 2, respectively. The 3.2- to 5.6-fold higher ligand density on sheep E appears to account for the ability of sheep but not human E to rosette with certain types of human T lymphocytes.     

New insights into receptor ligand binding domains from a novel assembly assay

Previous studies have demonstrated that hormone binding stabilizes the ligand binding domain (LBD) of the nuclear hormone receptors against proteolysis. We have confirmed and extended this observation using a newly developed assembly assay. In this assay, the LBD is divided into two parts, of which one includes the first helix of this domain and the other corresponds to the remainder of the LBD. Several independent criteria demonstrate that these two fragments can assemble into a functional LBD in the presence of a ligand, but not in its absence, and that this is a reflection of the stabilizing effect of ligand. We have also used this assay to demonstrate that binding of the nuclear receptor corepressor NCoR can directly stabilize the LBD. Overall, these results highlight the dynamic nature of the LBD and suggest that current models for activation based solely on allosteric effects on the C-terminal helix may be too limited.     

Roles of specific extracellular domains of the glucagon receptor in ligand binding and signaling

To identify structural determinants of ligand binding in the glucagon receptor, eight receptor chimeras and additional receptor point mutants were prepared and studied. Amino acid residues 103-117 and 126-137 in the extracellular N-terminal tail and residues 206-219 and 220-231 in the first extracellular loop of the glucagon receptor were replaced with the corresponding segments of the glucagon-like peptide-1 receptor or the secretin receptor. Specific segments of both the N-terminal tail and the first extracellular loop of the glucagon receptor are required for hormone binding. The 206-219 segment of the first loop appears to be important for both glucagon binding and receptor activation. Functional studies with a synthetic chimeric peptide consisting of the N-terminal 14 residues of glucagon and the C-terminal 17 residues of glucagon-like peptide 1 suggest that hormone binding specificity may involve this segment of the first loop. The binding selectivity may arise in part from aspartic acid residues in this segment. Mutation of R-202 located at the junction between the second transmembrane helix and the first loop resulted in a mutant receptor that failed to bind glucagon or signal. We conclude that high-affinity glucagon binding requires multiple contacts with residues in the N-terminal tail and first extracellular loop domain of the glucagon receptor, with hormone specificity arising primarily from the amino acid 206-219 segment. The data suggest a model whereby glucagon first interacts with the N-terminal domain of the receptor followed by more specific interactions between the N-terminal half of the peptide and the first extracellular loop of the receptor, leading to activation.     

The role of the transferrin receptor for the activation of human lymphocytes

The proliferative response of peripheral blood mononuclear cells (PBMC) in synthetic serum-free media depends on the presence of sufficient amounts of transferrin (Tf). In the present communication we show that the reduction of Tf concentration in culture media results in a decreased proliferation, whereas lymphokine production and the expression of activation markers (IL-2 receptor; transferrin receptor, (TfR); HLA class II) remain unchanged. To examine whether this effect is due to iron depletion we added iron chelates (ferric citrate, FeCi; ferric nitrilotriacetic acid, FeNTA) which can be internalized by cells without the requirement for Tf. The iron chelates could fully restore the proliferative response even in complete absence of Tf, suggesting that the observed inhibitory effect was indeed caused by iron depletion. Addition of a monoclonal TfR antibody, J 64, also caused a marked inhibition of proliferation of PBMC in regular serum-containing medium as well as in Tf-free synthetic medium; this effect could not be overcome by any of the tested iron chelates. Therefore, growth inhibition caused by J 64 cannot simply be attributed to iron starvation. These data suggest that J 64 may interfere with processes others than iron uptake and that the TfR might confer a necessary promoting signal for lymphocyte proliferation.     

Influence of transferrin glycans on receptor binding and iron-donation

Human bi-bi-antennary transferrin (Tf) was partially deglycosylated by subsequently incubating with one or more of the following exoglycosidases: neuraminidase, β-galactosidase or N-Acetyl-β-D-glucosaminidase. Aglyco-Tf obtained from serum of a patient suffering from the Carbohydrate Deficient Glycoprotein syndrome was isolated. Receptor binding and the Tf and iron uptake capacities of the fully glycosylated-, partially deglycosylated- and aglyco-Tf were compared using the human hepatoma cell line PLC/PRF/5. No difference in binding capacity between the iso-Tf fractions could be demonstrated, however, the Tf and iron uptake capacity of aglyco-Tf was clearly reduced compared with the other Tf fractions. This revised version was published online in November 2006 with corrections to the Cover Date.