Transferrin receptors on human B and T lymphoblastoid cell lines.

Experiments demonstrating the existence of receptors for iron-saturated transferrin on both B and T lymphoblastoid cell lines of human origin are described. Binding of 125I-labeled transferrin is rapid, saturable and reversible. It can be specifically inhibited by unlabeled transferrin but not by other proteins. The number of receptors on T cell lines determined by Scatchard analysis is almost double the number on B cell lines but the binding affinities are equal. The putative transferrin receptor can be removed from the cell by the proteolytic enzymes papain and trypsin, and is re-expressed during overnight incubation at 37 degrees C. Resynthesis is inhibited by puromycin. The receptor can be solubilized by deoxycholate, and retains transferrin binding capacity when non-covalently attached to an amphipathic matrix consisting of deoxycholate-coupled poly(L-lysyl) Agarose.     

Transferrin binding to K562 cell line : Effect of heme and sodium butyrate induction

Experiments demonstrating the existence of receptors for iron-saturated transferrin on K562 cells are described. Binding of ¹²⁵I-labelled transferrin is rapid, saturable and reversible, and can be specifically inhibited by unlabelled transferrin, but not by other proteins. The number of receptors determined by Scatchard analysis significantly decreased when K562 cells moved from the exponential to the quiescent phase of growth. Induction by hemin or sodium butyrate resulted in a marked reduction of transferrin binding. This phenomenon was due entirely to reduction in the number of receptors and was without effect on the affinity of interaction. The effect of butyrate and hemin on the number of transferrin receptors in other hematopoietic cell lines was investigated. Butyrate on the various cell lines was variable in its effect, whereas hemin constantly elicited a significant reduction in the number of transferrin receptors.     

Transferrin receptor expression during exponential and plateau phase growth of human tumour cells in culture

Transferrin receptor expression by the human tumour cell lines CCRF-CEM leukaemia and PMC-22B melanoma was studied, measuring the specific binding of fluorescein isothiocyanate (FITC)-labelled transferrin using a fluorescence-activated cell sorter. By measuring the fluorescence of cells stained at subsaturating concentrations of conjugate it was possible to calculate the average numbers of receptors per cell and the binding affinity by Scatchard analysis. These values (1.9 × 10⁵ binding sites/cell, K^A 1.2 × 10⁹ M^?1 for CCRF-CEM during exponential growth and 6.9 × 10⁴ binding sites/cell, K_A 1.4 × 10^?9 M^?1 for PMC-22B) are in close agreement with previously published data obtained using radiolabelled transferrin. The present method, however, allowed the transferrin receptor expression of individual cells within a population to be measured and thus it has been possible to test the hypothesis that transferrin receptor is a marker for cycling cells. Frequency-distribution histograms of transferrin receptor showed a wide range of values for both cell lines during exponential growth. When the extreme ranges were sorted and the cells examined for cellular DNA content it was found that those with the highest transferrin receptor expression were enriched with cells in S, G₂, and M phases of the cell cycle, whereas those with low transferrin receptor expression were mainly in G₁. However, two-parameter-correlated dot plots of transferrin receptor expression versus DNA content showed there was considerable overlap between the ranges of receptor expression for the different cell cycle compartments. Using a stathmokinetic method we have measured the proportion of quiescent cells in fed plateau phase cultures. Transferrin receptor expression was downgraded under these growth conditions but, contrary to expectation, the decline affected the population uniformly, without the emergence of a distinct, transferrin receptor-negative subpopulation corresponding to the increasing proportion of quiescent cells. Thus, although transferrin receptor expression bears some relation to cell cycle phase and reflects the proliferative activity of populations of cells, it is incapable of identifying individual cells which are out of cycle.     

The role of the transferrin receptor in human B lymphocyte activation

Transferrin receptors are expressed on proliferating cells and are required for their growth. Transferrin receptors can be detected after, but not before, mitogenic stimulation of normal peripheral blood T and B cells. T cells demonstrate a functional requirement for transferrin receptors in the activation process. These receptors, in turn, are induced to appear by T cell growth factor (interleukin 2). In the experiments reported here, we examined the regulation of transferrin receptor expression on activated human B cells and whether these receptors are necessary for activation to occur. Activation was assessed by studying both proliferation and immunoglobulin secretion. We determined that transferrin receptor expression on B cells is regulated by a factor contained in supernatants of mitogen-stimulated T cells (probably B cell growth factor). This expression is required for proliferation to occur, because antibody to transferrin receptor (42/6) blocks B cell proliferation. Induction of immunoglobulin secretion, however, although dependent on phytohemagglutinin-treated T cell supernatant, is not dependent on transferrin receptor expression and can occur in mitogen-stimulated cells whose proliferation has been blocked by anti-transferrin receptor antibody. These findings support a model for B cell activation in which mitogen (or antigen) delivers two concurrent but distinct signals to B cells: one, dependent on B cell growth factor and transferrin receptor expression, for proliferation; and a second, dependent on T cell-derived factors and not requiring transferrin receptors, which leads to immunoglobulin secretion.     

Transferrin binding and iron uptake in mouse hepatocytes

Methods were developed for obtaining highly viable mouse hepatocytes in single cell suspension and for maintaining the hepatocytes in adherent static culture. The characteristics of transferrin binding and iron uptake into these hepatocytes was investigated. (1) After attachment to culture dishes for 18–24 h hepatocytes displayed an accelerating rate of iron uptake with time. Immediately after isolation mouse hepatocytes in suspension exhibited a linear iron uptake rate of 1.14·10⁵molecules/cell per min in 5 μM transferrin. Iron uptake also increased with increasing transferrin concentration both in suspension and adherent culture. Pinocytosis measured in isolated hepatocytes could account only for 10–20% of the total iron uptake. Iron uptake was completely inhibited at 4°C. (2) A transferrin binding component which saturated at 0.5 μM diferric transferrin was detected. The number of specific, saturable diferric transferrin binding sites on mouse hepatocytes was 4.4·10⁴±1.9·10⁴ for cells in suspension and 6.6·10⁴±2.3·10⁴ for adherent cultured cells. The apparent association constants were 1.23·10⁷ 1·mol^?1 and 3.4·10⁶ 1·mol^?1 for suspension and cultured cells respectively. (3) Mouse hepatocytes also displayed a large component of non-saturable transferrin binding sites. This binding increased linearly with transferrin concentration and appeared to contribute to iron uptake in mouse hepatocytes. Assuming that only saturable transferrin binding sites donate iron, the rate of iron uptake is about 2.5 molecules iron/receptor per min at 5 μM transferrin in both suspension and adherent cells and increases to 4 molecules iron/receptor per min at 10 μM transferrin in adherent cultured cells. These rates are considerably greater than the 0.5 molcules/receptor per min observed at 0.5 μM transferrin, the concentration at which the specific transferrin binding sites are fully occupied. The data suggest that either the non-saturable binding component donates some iron or that this component stimulates the saturable component to increase the rate of iron uptake. (4) During incubations at 4°C the majority of the transferrin bound to both saturable and nonsaturable binding sites lost one or more iron atoms. Incubations including 2 mM α,α′-dipyridyl (an Fe¹¹ chelator) decreased the cell associated ⁵⁹Fe at both 4 and 37°C while completely inhibiting iron uptake within 2–3 min of exposure at 37°C. These observations suggest that most if not all iron is loosened from transferrin upon interaction of transferrin with the hepatocyte membrane. There is also greater sensitivity of ⁵⁹Fe uptake compared to transferrin binding to pronase digestion, suggesting that an iron acceptor moiety on the cell surface is available to proteolysis.     

The effect of lead on iron uptake from transferrin in human erythroleukemia (K562) cells

The effect of lead on cellular iron metabolism has been investigated using human erythroleukemia (K562) cells. When the cells were cultured with 100 m Pb²⁺ for 48 h, the rate of cellular iron uptake from transferrin decreased to 46% of that in untreated cells. Scatchard analysis of the binding data revealed that this reduction was the result of a decrease in the number of transferrin receptors rather than an alteration in ligand-receptor affinity. The results of immunoprecipitation of transferrin receptors on the cell surface also confirmed the decreased expression of transferrin receptors by lead-treated cells. The down-regulation of transferrin receptors by treatment with lead did not result from a decrease in the total amount of the receptor, as determined by immunoblotting. Moreover, the biosynthesis of the receptor was unaffected by lead treatment. Thus, the down-regulation of surface transferrin receptors in lead-treated cells might be due to a redistribution of receptors rather than an actual loss of receptors from the cell. Using kinetic analysis, it was shown that redistribution of the receptor did not result from the alteration in the rates of transferrin receptor recycling. A comparison of the amounts of transferrin receptor on the cell surface and in the cycling pool revealed that the sequestration of the receptor from normal flow through the cycle might cause down-regulation of the surface receptor.     

A rapid redistribution of the transferrin receptor to the cell surface of HL-60 cells and K562 cells upon treatment with dimethyl sulfoxide due to slowing of endocytosis

Treatment of two human leukemia cell lines with 1.25% dimethyl sulfoxide at 37 degrees C results in a rapid increase in the number of transferrin receptors on the cell surface detected by fluorescein-labeled anti-transferrin receptor antibodies. Both HL-60 cells, a human myeloid cell line, and K562 cells, a human erythroid-myeloid cell line, showed a 25-65% increase in cell surface transferrin binding in parallel experiments. Scatchard plot analysis of the data indicates that the number of receptors increases while the affinity of transferrin for the receptor remains the same. This rapid increase in the number of receptors at the cell surface appears to be due to a slowing of endocytosis rather than an increase in externalization of the receptor.     

Murine cell surface transferrin receptor: Studies with an anti-receptor monoclonal antibody

A rat monoclonal antibody against the murine transferrin receptor has been identified. The receptor is a 95,000 molecular weight species that exists in the cell membrane as a disulphide-bonded dimer. Whereas 29 of 29 murine hematopoietic tumor cell lines express detectable numbers of transferrin receptors, less than 1% of adult thymocytes or spleen cells and only 5% of bone marrow cells are positive. However, fetal liver and neonatal spleen contain substantial numbers of transferrin receptor-positive cells. Induction of Friend cells in vitro with dimethyl-sulphoxide leads to an overall increase in the expression of transferrin receptors on the cell surface. The anti-transferin receptor antibody we have obtained partially blocks iron uptake from ⁵⁹Fe-transferrin by a variety of murine cell lines and inhibits the growth of a murine myeloma cell line in vitro.     

Demonstration of two distinct transferrin receptor recycling pathways and transferrin-independent receptor internalization in K562 cells

The endocytosis and recycling of the human transferrin receptor were evaluated by several experimental modalities in K562 cells perturbed with 10(-5) M monensin. The work presented is an extension of a previous study demonstrating both complete inhibition of release of internalized human transferrin and a 50% reduction in the number of cell surface transferrin binding sites in K562 cells treated with monensin (Stein, B. S., Bensch, K. G., and Sussman, H. H. (1984) J. Biol. Chem. 259, 14762-14772). The data directly reveal the existence of two distinct transferrin receptor recycling pathways. One pathway is monensin-sensitive and is felt to represent recycling of transferrin receptors through the Golgi apparatus, and the other pathway is monensin-resistant and most likely represents non-Golgi-mediated transferrin receptor recycling. A transferrin-free K562 cell culture system was developed and used to demonstrate that cell surface transferrin receptors can be endocytosed without antecedent ligand binding, indicating that there are factors other than transferrin binding which regulate receptor internalization. Evidence is presented suggesting that two transferrin receptor recycling pathways are also operant in K562 cells under ligand-free conditions, signifying that trafficking of receptor into either recycling pathway is not highly ligand-dependent.     

Cell surface ceramide controls translocation of transferrin receptor to clathrin-coated pits

Transferrin receptor mediates internalization of transferrin with bound ferric ions through the clathrin-dependent pathway. We found that binding of transferrin to the receptor induced rapid generation of cell surface ceramide which correlated with activation of acid, but not neutral, sphingomyelinase. At the onset of transferrin internalization both ceramide level and acid sphingomyelinase activity returned to their basic levels. Down-regulation of acid sphingomyelinase in cells with imipramine or silencing of the enzyme expression with siRNA stimulated transferrin internalization and inhibited its recycling. In these conditions colocalization of transferrin with clathrin was markedly reduced. Simultaneously, K⁺ depletion of cells which interfered with the assembly of clathrin-coated pits inhibited the uptake of transferrin much less efficiently than it did in control conditions. The down-regulation of acid sphingomyelinase activity led to the translocation of transferrin receptor to the raft fraction of the plasma membrane upon transferrin binding. The data suggest that lack of cell surface ceramide, generated in physiological conditions by acid sphingomyelinase during transferrin binding, enables internalization of transferrin/transferrin receptor complex by clathrin-independent pathway.     

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.     

Transferrin receptor induction is required for human B-lymphocyte activation but not for immunoglobulin secretion

Transferrin receptors are expressed on proliferating cells and are required for their growth. Transferrin receptors can be detected after, but not before, mitogenic stimulation of normal peripheral blood T and B cells. In the experiments reported here we have examined the regulation of transferrin receptor expression on activated human B cells and whether or not these receptors are necessary for activation to occur. Activation was assessed by studying both proliferation and immunoglobulin secretion. We have determined that transferrin receptor expression on B cells is regulated by a factor contained in supernatants of mitogen-stimulated T cells (probably B-cell growth factor). This expression is required for proliferation to occur, since antibody to transferrin receptor (42/6) blocks B-cell proliferation. Induction of immunoglobulin secretion, however, although dependent on PHA-treated T-cell supernatant, is not dependent on transferrin receptor expression and can occur in mitogen-stimulated cells whose proliferation has been blocked by antitransferrin receptor antibody. In addition, we have demonstrated that IgM messenger RNA induction following mitogen stimulation is unaffected by antitransferrin receptor antibody. These findings support a model for B-cell activation in which mitogen (or antigen) delivers two concurrent but distinct signals to B cells: one, dependent on B-cell growth factor and transferrin receptor expression, for proliferation, and a second, dependent on T cell-derived factors and not requiring transferrin receptors, which leads to immunoglobulin secretion.     

The bacterial receptor protein, transferrin-binding protein B, does not independently facilitate the release of metal ion from human transferrin.

Pathogenic Gram-negative bacteria of the Pasteurellaceae and Neisseriaceae acquire iron for growth from host transferrin through the action of specific surface receptors. Iron is removed from transferrin by the receptor at the cell surface and is transported across the outer membrane to the periplasm. A periplasmic binding protein-dependent pathway subsequently transports iron into the cell. The transferrin receptor is composed of a largely surface-exposed lipoprotein, transferrin binding protein B, and a TonB-dependent integral outer membrane protein, transferrin binding protein A. To examine the role of transferrin binding protein B in the iron removal process, complexes of recombinant transferrin binding protein B and transferrin were prepared and compared with transferrin in metal-binding and -removal experiments. A polyhistidine-tagged form of recombinant transferrin binding protein B was able to purify a complex with transferrin that was largely monodisperse by dynamic light scattering analysis. Gallium was used instead of iron in the metal-binding studies, since it resulted in increased stability of recombinant transferrin binding protein B in the complex. Difference absorption spectra were used to monitor removal of gallium by nitrilotriacetic acid. Kinetic and equilibrium binding studies indicated that transferrin binds gallium more tightly in the presence of transferrin binding protein B. Thus, transferrin binding protein B does not facilitate metal ion removal and additional components are required for this process.     

An evaluation of lysosomal enzyme leakage as a factor influencing the behaviour of transformed cells.

The effect of concanavalin A on transferrin and iron uptake by reticulocytes was determined using rabbit reticulocytes and rabbit transferrin labelled with ⁵⁹Fe and ¹²⁵I and concanavalin A (ConA) labelled with ¹³¹I. In concentrations of 50–200 μg/ml ConA markedly inhibited iron uptake but did not inhibit transferrin uptake or release from the cells. ConA was itself taken up by rabbit blood cells in a manner similar to that of transferrin except that the uptake was not specific for reticulocytes but occurred also with mature erythrocytes. The inhibition of iron uptake by concanavalin and the uptake of concanavalin by the cells were both inhibited by α-methyl-d-mannoside. It is concluded that the effects observed were due to the binding of concanavalin to glycoproteins of the cell membrane, either by a direct interaction with transferrin receptors or by the production of a non-specific change in the structure of the membrane.     

Modulation of cell surface iron transferrin receptors by cellular density and state of activation

This report describes investigations of plasma membrane transferrin receptors on a variety of lymphoid cell lines and normal peripheral blood lymphocytes during activation and cell growth cycles. Transformed lymphoid cell lines have as many as 1,000 times the number of receptors found on normal resting lymphocytes. The number of iron transferrin receptors on continuous cell lines as well as normal human fibroblasts is down-regulated during the transition from log-phase growth to stationary plateau growth. When normal lymphocytes are transformed by mixed lymphocyte culture or mitogens, they rapidly express a 50-fold increase in the number of transferrin binding sites. This appearance of iron transferrin receptors anticipates nuclear changes during cell activation and subsequent mitosis of normal cells.     

Differential effect of cyclosporin A on the expression of T and B lymphocyte activation antigens

We have used a monoclonal antibody (Mab) to the interleukin 2 (IL 2) receptor as well as a Mab to the transferrin receptor to analyze the effects of cyclosporin A (CsA) on the induction and expression of these activation antigens on mitogen-stimulated murine T and B lymphocytes. The same concentration (0.25 microgram/ml) of CsA that produced optimal inhibition of the T cell proliferative response to concanavalin A (Con A) was also very effective at inhibiting IL 2 production and the induction of IL 2 responsiveness, as well as the expression of the IL 2 and transferrin receptors when measured 72 hr after mitogen activation. Surprisingly, CsA only minimally inhibited expression of these receptors 24 hr after the addition of mitogen; however, T cell blasts recovered from these cultures failed to respond to IL 2 even though IL 2 receptor expression was only modestly decreased. These results suggest that inhibition of the maturation of receptor expression is secondary to an early effect of CsA in blocking the induction of IL 2 responsiveness or to an arrest in the sequence of events required for maturation of T cells that bear high densities of these receptors. In contrast to the results observed with T lymphocytes, CsA had no effect on the B cell proliferative response to lipopolysaccharide (LPS) or on the induction of the IL 2 and transferrin receptors on activated B lymphocytes.     

Selection of cell lines resistant to anti-transferrin receptor antibody: evidence for a mutation in transferrin receptor.

Some anti-murine transferrin receptor monoclonal antibodies block iron uptake in mouse cell lines and inhibit cell growth. We report here the selection and characterization of mutant murine lymphoma cell lines which escape this growth inhibition by anti-transferrin receptor antibody. Growth assays and immunoprecipitation of transferrin receptor in hybrids between independently derived mutants or between mutants and antibody-susceptible parental cell lines indicate that all of the selected lines have a similar genetic alteration that is codominantly expressed in hybrids. Anti-transferrin receptor antibodies and transferrin itself still bind to the mutant lines with saturating levels and Kd values very similar to those of the parental lines. However, reciprocal clearing experiments by immunoprecipitation and reciprocal blocking of binding to the cell surface with two anti-transferrin receptor antibodies indicate that the mutant lines have altered a fraction of their transferrin receptors such that the growth-inhibiting antibody no longer binds, whereas another portion of their transferrin receptors is similar to those of the parental lines and binds both antibodies. These results argue that the antibody-selected mutant cell lines are heterozygous in transferrin receptor expression, probably with a mutation in one of the transferrin receptor structural genes.     

Changes in glycosylation alter the affinity of the human transferrin receptor for its ligand

When transferrin receptors of human erythroleukemic cells were pulse-labeled with [35S]methionine and then chased in the absence of radioactive precursor, the first detectable immunoprecipitable form of the receptor had a molecular mass of 85 kDa. This form of the receptor was converted to the mature form of 93 kDa with a half-time of about 40-60 min. Both the immature (85 kDa) and mature (93 kDa) receptors associated as dimers, the native form of the receptor. The 85-kDa, as well as the 93-kDa, receptors bound to a monoclonal antibody raised against the transferrin receptor or to transferrin-Sepharose. In order to determine whether glycosylation was necessary for ligand binding, purified receptors were isolated from cells grown in the presence of tunicamycin. When K562 cells were grown in the presence of tunicamycin, an 80-kDa nonglycosylated form of the receptor was synthesized. This nonglycosylated receptor was also capable of dimer formation; however, much less of it reached the cell surface than the fully glycosylated form, although both untreated and tunicamycin-grown cells appeared to synthesize transferrin receptors at similar rates. Although the number of receptor molecules/cell was similar in control and tunicamycin-treated cells, the nonglycosylated receptors exhibited a much lower affinity for transferrin than those of untreated cells; in contrast, when receptors were purified by immunoprecipitation and digested with bacterial alkaline phosphatase, no difference was observed between the affinity of these receptors and undigested immunoprecipitated receptors. These results suggest that glycosylation is not necessary for specific binding of transferrin to its receptor, but the affinity of this binding can be influenced greatly by the presence or absence of carbohydrate residues.     

Mitogenic agents induce redistribution of transferrin receptors from internal pools to the cell surface.

Incubation of serum-growth HeLa cells in serum-free medium causes a rapid (t1/2 3 min) 30-60% decrease in the binding of 125I-diferric transferrin to the cell surface. Addition of fetal bovine serum to cells in serum-free medium results in a rapid (t1/2 3 min) and concentration-dependent increase in binding activity. The loss or gain in ligand binding is a result of changes in surface receptor number rather than an alteration in ligand-receptor affinity. A variety of hormones (insulin, insulin-like growth factor, interleukin 1 and platelet-derived factor) were found to mimic the effect of serum on receptor number. The alteration in surface receptor number was found to be calcium-dependent. Changes in surface receptor number were independent of either receptor biosynthetic rate or the absolute cellular content of receptors. The effect of insulin or serum on Hela cell transferrin receptor distribution was unaffected by the presence of transferrin, demonstrating that receptor distribution in this cell type is ligand-independent. The ability of serum or insulin to modify surface transferrin receptor number was also observed in mouse L-cells, human skin fibroblasts, and J774 macrophage tumour cells. However, transferrin receptors on K562 and Epstein-Barr virus-transformed human lymphoblasts were unaltered by these agents. The quantities of receptors whose distribution is predominantly on the surface (i.e. epidermal growth factor or low density lipoprotein receptor) were unaltered by addition of the mitogenic agents. These results extend our previous studies [H.S. Wiley & J. Kaplan (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7456-7460] demonstrating that mitogenic agents can induce redistribution of receptor pools in selected cell types.     

Externalization of transferrin receptor in established human cell lines

The externalization of transferrin receptors was found in established human tumor cell lines at the rate of 10-35 ng/hour/10(6) cells, when they were incubated with transferrin at 37 degrees C. This externalization is inhibited by lowering the incubation temperature to 4 degrees C or eliminating the ligand from the culture medium. Metabolic inhibitors such as sodium azide, colchicine, cytochalasin B and chloroquine also decreased the rate of externalization. Almost 95% of released transferrin receptors were precipitated by centrifugation at 100,000 x g for 30 min, suggesting that transferrin receptor is externalized into the medium as a vesicular form.