Rapid endocytosis of the transferrin receptor in the absence of bound transferrin

The rate of endocytosis of transferrin receptors, occupied or unoccupied with transferrin, was measured on the cell line K562. At 37 degrees C, receptors, radioiodinated on the cell surface at 4 degrees C, were internalized equally rapidly in the presence or absence of transferrin. In both cases, 50% of the labeled receptors became resistant to externally added trypsin in 5 min. An antitransferrin antibody was used to show directly that the receptors had entered the cells without bound transferrin. The distribution of the receptors on the cell surface was revealed by antibody and protein A-gold staining after prolonged incubation in the presence or absence of transferrin. The receptors were concentrated in coated pits under both conditions. The data suggest that endocytosis of transferrin receptors is not "triggered" by ligand binding and raise the possibility that ligand-induced down-regulation of surface receptors may not occur by this mechanism. Instead receptors may be recognized as being ligand-occupied, not at the cell surface, but at some other site in the recycling pathway such as the endosome.     

Targeting of tumor necrosis factor to tumor cells: secretion by myeloma cells of a genetically engineered antibody-tumor necrosis factor hybrid molecule

The construction, synthesis and secretion of a genetically engineered antibody-cytokine fusion molecule is described. To target tumor necrosis factor (TNF) to tumor cells, recombinant antibody techniques were used to produce a Fab-like antibody-TNF conjugate. At the gene level, the heavy chain gene of an antitransferrin receptor antibody was linked to a synthetic TNF gene encoding human TNF. Transfection of the heavy chain-TNF gene into a myeloma derived cell line which was producing the light chain of the same antibody, allowed the isolation of a cell line secreting a fusion protein of the expected molecular weight and composition. The culture supernatant of the cell line contained TNF cytotoxic activity towards murine L929 cells and human MCF-7 cells. Cytotoxicity towards the human cancer cells was inhibited by an excess of the original antitransferrin receptor antibody, indicating that the antibody-TNF molecules are targeted to the transferrin receptor rich tumor cells. Since the antibody genes used are chimeric (i.e. composed of mouse variable and human constant regions) and since DNA encoding human TNF was used, the hybrid protein is an example of a humanized immunotoxin-like molecule. These results illustrate the possibilities of antibody engineering technology to create and produce improved agents for cancer therapy. Furthermore, they demonstrate for the first time the ability of myeloma cells to secrete an antibody-cytokine chimeric molecule.     

The appearance and internalization of transferrin receptors at the margins of spreading human tumor cells

Using gold complexes stabilized with a monoclonal antibody specific for the human transferrin receptor, the distribution of transferrin receptors on the surfaces of human epidermoid carcinoma A431 cells has been mapped at high resolution. On prefixed cells and cells incubated at 5 degrees C, the receptors are predominantly within and around clathrin-coated microdomains near the free cell margin. By preincubating the cells with saturating concentrations of free antibody at 5 degrees C and warming them to 37 degrees C in the presence of the gold complexes, the appearance of new receptors in the membrane has been followed. The majority first appear near the free cell margin and then move centripetally. At first, they are monodisperse, but as they move toward the site of internalization they form loose aggregates. Within the immediate vicinity of the clathrin-coated microdomains the migrating receptors form closely packed, ordered aggregates. These observations indicate recycling transferrin receptors move to their site of internalization without cross-linking.     

Isolation by fluorescence-activated cell sorting of Chinese hamster ovary cell lines with pleiotropic, temperature-conditional defects in receptor recycling.

We have isolated several Chinese hamster ovary cell lines with temperature-sensitive defects in the recycling of receptors after endocytosis. These cell lines were selected using fluorescence-activated cell sorting for retention of a pulse of labeled transferrin after a chase in the presence of unlabeled transferrin. One of these cell lines, TfT1.11, was selected for further characterization. In TfT1.11 the trapping of transferrin within the cells is paralleled by a loss of cell surface transferrin receptors. Within 4 h after the shift from 33 to 41 degrees C the surface binding of transferrin is reduced to 18% of parental cells at 41 degrees C. The trapping of transferrin and the loss of transferrin receptor from the cell surface are caused by a temperature-conditional 5.5-fold decrease in the initial rate of transferrin recycling. TfT1.11 cells also rapidly lose 89% of their ability to take up alpha 2-macroglobulin after the temperature shift to 41 degrees C. These data indicate that the TfT1.11 cell line has a pleiotropic defect in receptor recycling.     

Synthesis of the transferrin receptor by cultures of embryonic chicken spinal neurons

We have purified a glycoprotein from chicken sciatic nerves, sciatin, which has pronounced trophic effects on avian skeletal muscle cells in culture. Recent studies have shown that sciatin is identical to the iron-transport protein, transferrin, in terms of its physicochemical structure, immunological reactivity, and biological activity. To determine whether transferrin is synthesized and released by neuronal tissue, we incubated cultures of dissociated chicken spinal neurons in a medium free of L-leucine containing either L-3H-amino acids or L-[14C]leucine and immunoprecipitated transferrin with highly specific antibodies. The radiolabeled protein precipitated by rabbit heteroclonal, goat heteroclonal, or mouse monoclonal antitransferrin antibodies increased in specific activity in a linear manner for at least 30 min. Synthesis of this protein was abolished by the presence of puromycin (20 micrograms/ml) or cycloheximide (10(-5) M). The disappearance of the radiolabeled protein from cells was linear with a half-life (t 1/2) of 8-10 h. When immunoprecipitates were separated by SDS gel electrophoresis, a prominent band corresponding to transferrin (Mr 84,000) was visualized by staining with Coomassie Blue. However, when such gels were fluorographed, no radioactivity was apparent in the transferrin region of the gel although a prominent radioactive band was visualized at an Mr of 56,000. The protein of Mr 56,000 was not simply a degradation product of transferrin because this particular protein band was not generated by incubating radiolabeled transferrin with unlabeled neuronal homogenates. The protein of Mr 56,000 was purified from embryonic chicken brain and spinal cord by immunoabsorption chromatography on mouse monoclonal antitransferrin IgG conjugated to Sepharose 4B followed by affinity chromatography on immobilized transferrin. The purified protein bound radioiodinated transferrin and was precipitated by rabbit anti-chicken transferrin-receptor antibodies. Furthermore, this receptor protein was found to be localized on the plasma membrane of dorsal root ganglion neurons by immunocytochemistry using the peroxidase-antiperoxidase technique, and by blocking experiments, which showed that antitransferrin receptor IgG could inhibit the binding of fluorescein-conjugated transferrin at 4 degrees C to cultured neurons in vitro. From these data, we conclude that transferrin is not synthesized by cultures of chicken spinal cord neurons, but that the receptor for transferrin is synthesized by these cultures and is precipitated by antitransferrin antibodies as an antigen-receptor complex.     

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.     

Receptor-mediated endocytosis of transferrin in K562 cells

Human diferric transferrin binds to the surface of K562 cells, a human leukemic cell line. There are about 1.6 X 10(5) binding sites per cell surface, exhibiting a KD of about 10(-9) M. Upon warming cells to 37 degrees C there is a rapid increase in uptake to a steady state level of twice that obtained at 0 degree C. This is accounted for by internalization of the ligand as shown by the development of resistance to either acid wash or protease treatment of the ligand-cell association. After a minimum residency time of 4-5 min, undegraded transferrin is released from the cell. Internalization is rapid but is dependent upon cell surface occupancy; at occupancies of 20% or greater the rate coefficient is maximal at about 0.1-0.2 min-1. In the absence of externally added ligand only 50% of the internalized transferrin completes the cycle and is released to the medium with a rate coefficient of 0.05 min-1. The remaining transferrin can be released from the cell only by the addition of ligand, suggesting a tight coupling between cell surface binding, internalization, and release of internalized ligand. There is a loss of cell surface-binding capacity that accompanies transferrin internalization. At low (less than 50%) occupancy this loss is monotonic with the extent of internalization. Even at saturating levels of transferrin, the loss of surface receptors upon internalization never exceeds 60-70% of the initial binding capacity. This suggests that receptors enter the cell with ligand but are replaced so as to maintain a constant, albeit reduced, receptor number on the cell surface. In the absence of ligand, the cell surface receptor number returns at 37 degrees C. Neither sodium azide nor NH4Cl blocks internalization of ligand. However, they both prevent the release of transferrin from the cell thus halting the transferrin cycle. Excess ligand can overcome the block due to NH4Cl but not azide although the cycle is markedly slower. Iron is delivered to these cells by transferrin at 37 degrees C with a rate coefficient of 0.15 to 0.2 min-1. The iron is released from the transferrin and the majority is found in intracellular ferritin. There is a large internal receptor pool comprising 70 to 80% of the total cell receptors and this may be involved in maintaining the steady state iron uptake.     

Transferrin receptor number, synthesis, and endocytosis during erythropoietin-induced maturation of Friend virus-infected erythroid cells

Erythropoietin (EP) responsive Friend virus-infected erythroid cells had 200,000 steady-state binding sites for transferrin at 37 degrees C when isolated from the spleens of Friend virus-infected mice. Upon culture of these cells with EP, the synthesis of transferrin receptors increased 4- to 7-fold and the number of transferrin-binding sites per cell doubled after 24 h. However, the rate of uptake of 59Fe from transferrin remained constant at approximately 35,000 atoms of 59Fe per minute per cell during this period in culture. The amount of 125I-transferrin internalized during the steady-state binding did not change during this culture period while the transferrin bound to the surface increased 3-fold. At all stages of erythroid maturation, the maximum rate of endocytosis was determined to be 18,000 molecules of transferrin per minute per cell, and the interval that 125I-transferrin remains in the interior of the cell was calculated to be 6.9 min. After 48 h of culture with EP, the number of steady-state transferrin-binding sites was reduced in part due to the sequestration of surface receptors within the cell. The uptake of iron from transferrin was limited by the level of endocytosis of transferrin during the initial phase of culture and the number of transferrin receptors at the cell surface during the latter stages of erythroid maturation of these 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.     

Transferrin receptor activity in rat mammary epithelial cells

The binding of 125I-transferrin to rat mammary cells isolated by collagenase and hyaluronidase digestion has been investigated. Surface binding was determined at 4 degrees C and total binding also at 4 degrees C but in the presence of 0.1% w/v saponin. KD values between 20 and 25 nM were obtained. Binding assays at 37 degrees C showed the internalisation of the receptor and the bound transferrin was occurring but also provided evidence for an impaired recycling of the receptors to the cell surface in the freshly isolated cells. No differences in total binding were observed in cells prepared at different stages of lactation with a mean value of 29 fmol transferrin bound/micrograms cellular DNA, equivalent to 180,000 receptors per cell.     

Coated pits and asialoglycoprotein receptors redistribute to the substratum during hepatocyte adhesion to galactoside surfaces

Rat hepatocytes bind in a sugar-specific and concentration-dependent manner to flat polyacrylamide matrices containing covalently attached galactosyl (Gal) groups. Previous studies (Weigel, P.H., J. Cell Biol. 87, 855, 1980) concluded that binding was likely mediated by the asialoglycoprotein receptor. Here we confirm that adhesion is mediated by this receptor, since cell binding is inhibited by antireceptor antibody and a threshold binding response is also observed when hepatocytes adhere to surfaces coated with asialoorosomucoid, a ligand for this receptor. Cells that had bound to a Gal surface and were then sheared from the surface left a membrane patch behind on the substratum. The cytoplasmic side of these plasma membrane patches was visualized on the substratum by indirect immunofluorescence using antireceptor antibody or anticlathrin antibody. The density of punctate coated pits, visualized with the latter antibody, was enriched in a circular membrane region of about 4 microns 2 area that mediated cell binding. This zone also contained concentrated receptors, although the staining pattern with antireceptor antibody was more uniform and less punctate. The results show that both asialoglycoprotein receptors and coated pits are redistributed at the substratum interface on hepatocytes bound to Gal surfaces.     

125I-thrombin binds to clustered receptors on noncoated regions of mouse embryo cell surfaces

We used electron microscope autoradiography (EMAR) to visualize the interaction of 125I-thrombin with its surface receptors on mouse embryo (ME) cells. Autoradiographic grains were spaced over the surface of cells in a periodic nonrandom pattern, indicating 125I-thrombin association with clusters of thrombin receptors. The grain spacing varied slightly from cell to cell, indicating subpopulations of cells with different numbers of thrombin receptors. The average distance between grains on ME cells after binding 125I-thrombin (125 ng/ml) at 37 degrees C was 1.65 +/- 0.49 microns. The average distance between grains on prefixed cells and cells incubated with 125I-thrombin at 4 degrees C was not significantly different from that observed at 37 degrees C. This indicates that thrombin receptors are clustered before thrombin binding and that the thrombin receptor aggregates do not redistribute into large aggregates on the surface of cells subsequent to thrombin binding. The number of grains per cluster also does not change under these three binding conditions. Thus, the number of occupied receptors in each cluster appears to be constant. On the basis of the average grain number and spacing, we estimate that each cluster is approximately 400 nm in diameter containing approximately 550 thrombin-binding sites. These receptor-clusters are not associated with specialized structures or coated regions of the membrane. Additionally, grains observed within cells were not found associated with coated vesicles. Therefore, neither the clustering patterns nor internalization of 125I-thrombin are characteristic of molecules which bind to receptors and are internalized by receptor-mediated endocytosis.     

Receptor-mediated endocytosis and exocytosis of transferrin in Concanavalin A-stimulated rat lymphoblasts

Iron-loaded transferrin has been shown to be necessary for the support of cell proliferation in culture. This function depends upon interaction of transferrin with a specific high-affinity cell surface receptor. The present report is directed toward determining the consequences of the interaction of transferrin with this receptor on Concanavalin A-stimulated rat lymphocytes. Three specific questions have been posed: (a) Is transferrin endocytosed following binding to its specific receptor in a temperature-dependent fashion? (b) Following endocytosis, is the carrier protein released from the cell in a structurally and functionally intact form? and (c) Is the cell surface transferrin receptor also endocytosed following ligand binding? The results provide affirmative answers to all questions. Using two independent probes of the cell surface versus intracellular location of transferrin we observed that cell-bound transferrin moved from the cell surface to the inside of the cell and subsequently back to the medium. This process occurred in a temperature-dependent fashion. When cells containing only intracellular transferrin were further incubated at 37°C approximately 80% of cell-bound transferrin was released to the medium. Nearly all of this material retained reactivity with antibody to transferrin. In addition, exocytosed transferrin exhibited qualitatively and quantitatively equivalent binding reactivity with the transferrin receptor and showed identical electophoretic mobility on SDS gel electrophoresis. Finally, using similar methodology to that employed with transferrin itself, we provide evidence that the specific receptor is also endocytosed.     

A monoclonal antibody modulates the interaction of nerve growth factor with PC12 cells

A nerve growth factor (NGF) receptor interactive monoclonal antibody (192-IgG) which enhances beta-NGF binding to PC12 cells has been produced. The hybridoma clone was obtained by fusing Sp2/0- Ag14 myeloma cells with splenocytes from Balb/C mice which had been immunized with n-octyl glucoside solubilized proteins from isolated PC12 cell plasma membranes. The antibody is an IgG, which does not bind beta-NGF. It binds to the same number of sites on PC12 cells at low temperature as does beta-NGF. The 192-IgG increases the apparent affinity of beta-NGF binding to fast receptors on PC12 cells at low temperature by a factor of 2.5- to 4-fold and enhances the photoactivatable cross-linking of beta-NGF to the same receptor while decreasing the cross-linking of beta-NGF to the slow NGF receptor. At 37 degrees C 192-IgG partially inhibits the regeneration of neurites from primed PC12 cells. The 192-IgG also reduces the rate of appearance of binding to slow NGF receptors and increases the proportion of beta-NGF bound to fast receptors at 37 degrees C. These results implicate the slow receptor as the mediator of the biological response. This antibody provides a tool for examining steps in the mechanism of action of beta-NGF after binding to the receptor.     

Role of low density lipoprotein receptor-dependent and -independent sites in binding and uptake of chylomicron remnants in rat liver

The role of the low density lipoprotein (LDL) receptor in the binding of chylomicron remnants to liver membranes and in their uptake by hepatocytes was assessed using a monospecific polyclonal antibody to the LDL receptor of the rat liver. The anti-LDL receptor antibody inhibited the binding and uptake of chylomicron remnants and LDL by the poorly differentiated rat hepatoma cell HTC 7288C as completely as did unlabeled lipoproteins. The antireceptor antibody, however, decreased binding of chylomicron remnants to liver membranes from normal rats by only about 10%. This was true for intact membranes and for solubilized reconstituted membranes and with both a crude membrane fraction as well as with purified sinusoidal membranes. Further, complete removal of the LDL receptor from solubilized membranes by immunoprecipitation with antireceptor antibody only decreased remnant binding to the reconstituted supernatant by 10% compared to solubilized, nonimmunoprecipitated membranes. Treatment of rats with ethinyl estradiol induced an increase in remnant binding by liver membranes. All of the increased binding could be inhibited by the antireceptor antibody. The LDL receptor-independent remnant binding site was not EDTA sensitive and was not affected by ethinyl estradiol treatment. LDL receptor-independent remnant binding was competed for by beta-VLDL = HDLc greater than rat LDL greater than human LDL (where VLDL is very low density lipoprotein, and HDL is high density lipoprotein). There was weak and incomplete competition by apoE-free HDL, probably due to removal of apoE from the remnant. The LDL receptor-independent remnant-binding site was also present in membranes prepared from isolated hepatocytes and had the same characteristics as the site on membranes prepared from whole liver. In contrast, when chylomicron remnants were incubated with a primary culture of rat hepatocytes, the anti-LDL receptor antibody prevented specific cell association by 84% and degradation of chylomicron remnants completely. Based on these studies, we conclude that although binding of chylomicron remnants to liver cell membranes is not dependent on the LDL receptor, their intact uptake by hepatocytes is.     

A variant of human transferrin with abnormal properties.

Normal human skin fibroblasts cultured in vitro exhibit specific binding sites for 125I-labelled transferrin. Kinetic studies revealed a rate constant for association (Kon) at 37 degrees C of 1.03 X 10(7) M-1 X min-1. The rate constant for dissociation (Koff) at 37 degrees C was 7.9 X 10(-2) X min-1. The dissociation constant (KD) was 5.1 X 10(-9) M as determined by Scatchard analysis of binding and analysis of rate constants. Fibroblasts were capable of binding 3.9 X 10(5) molecules of transferrin per cell. Binding of 125I-labelled diferric transferrin to cells was inhibited equally by either apo-transferrin or diferric transferrin, but no inhibition was evident with apo-lactoferrin, iron-saturated lactoferrin, or albumin. Preincubation of cells with saturating levels of diferric transferrin or apo-transferrin produced no significant change in receptor number or affinity. Preincubation of cells with ferric ammonium citrate caused a time- and dose-dependent decrease in transferrin binding. After preincubation with ferric ammonium citrate for 72 h, diferric transferrin binding was 37.7% of control, but no change in receptor affinity was apparent by Scatchard analysis. These results suggest that fibroblast transferrin receptor number is modulated by intracellular iron content and not by ligand-receptor binding.     

Recycling of the asialoglycoprotein receptor and the effect of lysosomotropic amines in hepatoma cells

Receptor-mediated uptake and degradation of 125I-asialoorosomucoid (ASOR) in human hepatoma HepG2 cells is inhibited by the lysosomotropic amines chloroquine and primaquine. In the absence of added ligand at 37 degrees C, these amines induce a rapid (t1/2 5.5-6 min) and reversible loss of cell surface 125I-ASOR binding sites as well as a rapid decrease in 125I-ASOR uptake and degradation. There is no effect of these amines on the binding of 125I-ASOR to the cell surface at 4 degrees C or on the rate of internalization of prebound 125I-ASOR. The loss of 125I-ASOR surface binding at 37 degrees C is not attributable to altered affinity of ligand-receptor binding. In the presence of added ligand at 37 degrees C, there is a more rapid (t1/2 2.5-3 min) loss of hepatoma cell surface receptors. In addition, the amines inhibit the rapid return of the internalized receptor to the cell surface. We examined the nature of this loss of 125I-ASOR surface binding sites by following the fate of receptor molecules after biosynthetic labeling and after cell surface iodination. At 37 degrees C, chloroquine and primaquine induce a loss of asialoglycoprotein receptor molecules from the hepatoma cell surface to an internal pool.     

LDL receptor binds newly synthesized apoE in macrophages. A precursor pool for apoe secretion.

There appear to be multiple post-translational sites for regulation of macrophage apolipoprotein (apo)E secretion, including the presence of a distinct cell surface pool of apoE. Cell surface proteoglycans have been shown to be involved in forming this pool. The current studies were designed to investigate the role of an additional cell surface site, i.e., the low density lipoprotein (LDL) receptor. Antiserum to the LDL receptor displaced apoE from the macrophage cell surface and into the medium during a 4 degrees C incubation from apoE-expressing J774 cells, from proteoglycan-depleted apoE-expressing J774 cells, and from human monocyte-derived macrophages. Similar results were obtained when purified monoclonal antibody to the LDL receptor was added to human monocyte-derived macrophages. J774 cells transfected to express an LDL receptor binding-defective mutant of apoE did not show a similar response to addition of LDL receptor antibody. Studies were conducted in which cells were pulse labeled for 30 min, followed by various periods of chase at 4 degrees C or 37 degrees C in the presence or absence of LDL receptor antibody. The results of these studies indicated that nascent macrophage-derived apoE binds to the LDL receptor, and that this apoE served as a precursor pool for apoE released into the medium.These studies establish a role for the LDL receptor in forming the cell surface pool of apoE and, along with data regarding the importance of proteoglycans, indicate that cell surface binding sites for nascent macrophage-derived apoE are heterogeneous. The heterogeneity of such sites could have implication for the size and turnover of this cell surface pool.     

Demonstration of the specific binding of bovine transferrin to the human transferrin receptor in K562 cells: evidence for interspecies transferrin internalization

Specific binding of ferric bovine transferrin to the human transferrin receptor was investigated using K562 cells propagated in serum-free medium without transferrin supplemented with 10(-5) elemental iron. Affinity chromatography of solubilized extracts of K562 cells surface-labeled with 125I was performed using bovine transferrin- and human transferrin-Sepharose 4B resins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of resin eluates reveal that bovine transferrin specifically binds a Mr = 188,000 protein which dissociates into a Mr = 94,000 protein under reducing conditions, a finding identical to what is seen with human transferrin. The Mr = 94,000 reduced protein isolated by bovine transferrin resin shows an identical one-dimensional partial proteolytic digestion map with that of the human transferrin receptor. Unlabeled bovine transferrin was shown to specifically compete 125I-labeled human transferrin from the human transferrin receptor on the surface of K562 cells at 4 degrees C in a similar manner as unlabeled human transferrin; however, approximately a 2,000-fold higher concentration of bovine ligand was required to achieve comparable competition (50% inhibition of binding). Indirect immunofluorescence cytolocalization of bovine transferrin in K562 cells grown in serum-free medium supplemented with ferric bovine transferrin reveal patterns similar to those seen for human transferrin (both focal perinuclear and diffuse cytoplasmic fluorescence). Monensin treatment results in a dramatic accumulation of bovine ligand in perinuclear aggregates, suggesting that it is recycled through the Golgi, as is human transferrin. K562 cells grown in serum-free medium supplemented with either 300 micrograms/ml of ferric human or ferric bovine transferrin were found to demonstrate superimposable growth curves.     

Expression of the transferrin receptor in murine peritoneal macrophages is modulated in the different stages of activation

A specific high affinity binding site for the serum glycoprotein transferrin was identified on murine peritoneal macrophages. The binding reached equilibrium within 60 min and was reversible, saturable, and specific for transferrin. Although the presence of this receptor was detected on the cell surface by studies carried out using intact cells, the majority (70 to 90%) of the binding activity was detectable only in detergent extracts of such cells. This suggests that a substantial portion of the binding activity is localized within the macrophage. The association constant (Ka) for binding to intact cells (6 to 9 X 10(8) M-1) was comparable to values reported for transferrin receptors described on other cell types. The expression of transferrin binding activity was examined in macrophages exhibiting qualitatively and quantitatively different degrees of functional activation. Resident peritoneal macrophages, exudate macrophages primed by elicitation with pyran copolymer, and activated macrophages induced by chronic infection of mice with bacillus Calmette-Guerin (BCG) or elicitation with heat killed Propionibacterium acnes (P. acnes) had low numbers of binding sites (1000 to 5000 total sites/cell). Macrophages elicited by sterile inflammatory agents (thioglycollate broth, fetal bovine serum, or casein) all exhibited a greater number of transferrin receptors (15,000 to 20,000 total sites/cell). This modulation did not appear to result from differential shifts between surface and internal loci. Our results suggest that the expression of the transferrin receptor may be a useful marker of the responsive stage of macrophage functional activation and the membrane changes that accompany activation.