Selection and characterization of transferrin receptor mutants using receptor-specific antibodies

Lymphoma cell lines were selected by growth in transferrin receptor-specific antibodies and in transferrin receptor-specific antibody coupled to ricin toxin. Sequential selections were used to isolate lines with multiple mutations affecting the transferrin receptor molecule. Mutant cell lines were characterized by their growth in antibody and their antibody-binding properties. Two basic types of mutations were found. One type resulted in the loss of a binding determinant for the antibody used for selection on one of the two transferrin receptor allelic products. The other type of mutation resulted in the loss of cell-surface expression of the entire gene product of one of the transferrin receptor alleles.     

Co-migration and internalization of transferrin and its receptor on K562 cells

The incorporation of iron into human cells involves the binding of diferric transferrin to a specific cell surface receptor. We studied the process of endocytosis in K562, a human erythroid cell line, by using tetramethylrhodamine isothiocyanate-labeled transferrin (TRITC- transferrin) and fluorescein isothiocyanate-labeled Fab fragments of goat antireceptor IgG preparation (FITC-Fab-antitransferrin receptor antibody). Because the antireceptor antibody and transferrin bind to different sites on the transferrin receptor molecule it was possible to simultaneously and independently follow ligand and receptor. At 4 degrees C, the binding of TRITC-transferrin or FITC-Fab antitransferrin receptor antibody exhibited diffuse membrane fluorescence. At 20 degrees C, the binding of TRITC-transferrin was followed by the rapid formation of aggregates. However, the FITC-Fab antitransferrin receptor did not show similar aggregation at 20 degrees C unless transferrin was present. In the presence of transferrin, the FITC-Fab antitransferrin receptor antibody formed aggregates at the same sites and within the same time period as TRITC transferrin, indicating co-migration. Although the diffuse surface staining of either label was removed by proteolysis, the larger aggregates were not susceptible to enzyme degradation, indicating that they were intracellular. The internal location of the aggregates was also demonstrated using permeabilized cells that had been preincubated with transferrin and fixed with 4% paraformaldehyde. These cells showed aggregated receptor in the interior of the cell when reacted with fluorescein-labeled antibody to the receptor. This indicated that the transferrin and the transferrin receptor co-internalize and migrate to the same structures within the cell.     

Cloning, characterization, and modeling of a monoclonal anti-human transferrin antibody that competes with the transferrin receptor.

In this report we describe the isolation and characterization of a monoclonal antibody against human serum transferrin (Tf) and the cloning and sequencing of its cDNA. The antibody competes with the transferrin receptor (TR) for binding to human Tf and is therefore expected to bind at or very close to a region of interaction between Tf and its receptor. From the deduced amino acid sequence, we constructed a 3-dimensional model of the variable domains of the antibody based on the canonical structure model for the hypervariable loops. The proposed structure of the antibody is a first step toward a more detailed characterization of the antibody-Tf complex and possibly toward a better understanding of the Tf interaction with its receptor. The model might prove useful in guiding site-directed mutagenesis studies, simplifying the experimental elucidation of the antibody structure, and in the use of automatic procedures to dock the interacting molecules as soon as structural information about the structure of the human Tf molecule will be available.     

Characterization of transferrin receptor in an immortalized cell line of rat brain endothelial cells, RBE4

The content and distribution of transferrin receptors in an immortalized cell line, RBE4, derived from rat cerebral capillary endothelial cells was investigated using the monoclonal antibody MRC OX-26 (OX-26 mAb) specific for the rat transferrin receptor. An ELISA assay was developed with which the OX-26 mAb can be determined quantiatively. The detection limit of the assay was 10 pg or 0.07 fmol of murine antibody. With this technique accurate measurement of native antibody is now possible without the need for isotope labeling (iodination). Immunostaining of confluent monolayers of RBE4 cells using an antibody directed against the tight junction associated protein ZO-1 was indicative for structural intactness of RBE4 cell monolayers. OX-26 immunostaining demonstrated localization of the transferrin receptor at the plasma membrane and/or in the cytosol. Binding studies showed saturation of OX-26 mAb binding. The antibody binding analysis gave a dissociation constant (KD) of 17.1 +/- 1.2 nmol/l. The total amount of transferrin receptors present per cell was 70,800 +/- 17,000. Our results indicate that receptor binding of OX-26 mAb can be studied using an in vitro cell culture model of rat brain mircrovessel endothelium in conjunction with an ELISA technique for detection of native antibody. This approach will be used to investigate mechanisms of transendothelial transport of OX-26 in vitro.     

Cellular internalization of insulin-like growth factor binding protein-3: distinct endocytic pathways facilitate re-uptake and nuclear localization

Insulin-like growth factor binding protein-3 (IGFBP-3) is well established as a growth-inhibitory, apoptosis-inducing secreted molecule that acts via insulin-like growth factor (IGF)-independent as well as IGF-dependent pathways. Nuclear localization of IGFBP-3 has been observed and nuclear binding partners for IGFBP-3 demonstrated. However, little is known about the mechanism of IGFBP-3 internalization. We hypothesized that IGFBP-3 is first secreted then taken up again into cells and that its internalization could occur via binding to transferrin or caveolin. Incubation of cells with an IGFBP-3-neutralizing antibody demonstrated that nuclear translocation of endogenous IGFBP-3 requires IGFBP-3 secretion and re-uptake. Nuclear localization of exogenously added IGFBP-3 was rapid, occurring within 15 min, inhibited by co-incubation and extracellular sequestration with IGF-I, and dependent on the transferrin-binding C-terminal peptide region of IGFBP-3. Co-immunoprecipitation assays confirmed that IGFBP-3 binds transferrin but not directly to the transferrin receptor (TfR1); however, transferrin binds TfR1 and a ternary complex is formed. Specific binding to caveolin scaffolding docking sequence was confirmed utilizing radiolabeled IGFBP-3. Blocking TfR1-mediated endocytosis prevents both endogenous and exogenous IGFBP-3 re-uptake and inhibitors of caveolae formation also retard IGFBP-3 nuclear entry. Co-treatment with anti-transferrin receptor antibody and cholesterol depletion agents completely abolished endogenous and exogenous IGFBP-3 uptake. Suppression of IGFBP-3 internalization by TfR1 blockade inhibited IGFBP-3-induced apoptosis. Together, these data indicate that the actions of IGFBP-3 are mediated by internalization via distinct endocytic pathways.     

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.     

Modulation of transferrin receptor expression and function by anti-transferrin receptor antibodies and antibody fragments

It has been suggested that effects of anti-transferrin receptor antibodies on cell growth and receptor expression are the result of varying degrees of receptor crosslinking by bi- and multivalet binding agents. In order to study this question directly, we have cultured murine lymphoma cells in mono- and divalent fragments from IgG and IgM monoclonal anti-transferrin receptor antibodies and in intact antibodies. The studies presented here demonstrate that effects of antibody binding on transferrin receptor distribution, metabolism, and function depend, at least in part, on antibody valence, and therefore on the degree of crosslinking of receptors by antibody. We found that monovalent antibody fragments did not significantly alter cell growth, receptor surface expression, intracellular localization, or degradation. Diavalent antibody caused a uniform down-regulation of cell-surface receptor expression, which was accompanied by increased degradation only when antibody Fc was present. Normal receptor cycling apparently continued, despite the reduction in surface expression. Culture in multivalent IgM antibody, however, resulted in accumulation of antibody-complexed receptor on the cell surface without internalization and caused profound inhibition of cell growth. Thus, we show two mechanisms by which different degrees of antibody crosslinking can influence transferrin receptor function: by receptor down-regulation and blocking internalization.     

Characterization of Transferrin Receptor in an Immortalized Cell Line of Rat Brain Endothelial Cells,RBE4

Abstract

The content and distribution of transferrin receptors in an immortalized cell line, RBE4, derived from rat cerebral capillary endothelial cells was investigated using the monoclonal antibody MRC OX-26 (OX-26 mAb) specific for the rat transferrin receptor. An ELISA assay was developed with which the OX-26 mAb can be determined quantiatively. The detection limit of the assay was 10 pg or 0.07 fmol of murine antibody. With this technique accurate measurement of native antibody is now possible without the need for isotope labeling (iodination). Immunostaining of confluent monolayers of RBE4 cells using an antibody directed against the tight junction associated protein ZO-1 was indicative for structural intactness of RBE4 cell mono-layers. OX-26 immunostaining demonstrated localization of the transferrin receptor at the plasma membrane and/or in the cytosol. Binding studies showed saturation of OX-26 mAb binding. The antibody binding analysis gave a dissociation constant (K_D) of 17.1 ± 1.2 nmol/l. The total amount of transferrin receptors present per cell was 70,800 ± 17,000. Our results indicate that receptor binding of OX-26 mAb can be studied using an in vitro cell culture model of rat brain mircrovessel endothelium in conjunction with an ELISA technique for detection of native antibody. This approach will be used to investigate mechanisms of transendothelial transport of OX-26 in vitro.     

Inhibition of cell growth by monoclonal anti-transferrin receptor antibodies.

Five anti-murine transferrin receptor monoclonal antibodies have been characterized with respect to immunoglobulin class, effects on binding of transferrin, and effects on AKR1 lymphoma cell growth in vitro. The immunoglobulin M (IgM) antibodies, but not the IgG antibodies, prevent cell growth. We suggest that the profound effects of the IgM antibodies on cell growth are probably due to extensive cross-linking of cell surface receptors. In support of this, we are able to mimic the growth-inhibiting effects of the IgM antibodies by adding antiimmunoglobulin to an IgG antibody. By flow microfluorimetry, we show that an IgG antibody by itself induces up to a 10-fold downward regulation in the cell surface transferrin receptor, which is accompanied by accelerated receptor degradation. A similar downward regulation is seen in mutant cells resistant to growth inhibition by an IgM antibody, when grown in the selecting antibody. Wild-type cells grown in the presence of IgM antibody do not show receptor downward regulation. Inhibitory effects of antibody plus antiimmuoglobulin on mutant cells are also consistent with extensive cross-linking causing inhibition of growth.     

The role of transferrin receptors and iron delivery in mouse embryonic morphogenesis

The iron-carrying serum protein transferrin is required for the proliferation and differentiation of embryonic tissues in culture. We studied the expression and role of transferrin receptors in two model systems using a monoclonal antibody against the transferrin receptor of mice. The addition of 20-100 micrograms/ml antibody to a chemically defined culture medium containing transferrin (10 micrograms/ml) inhibited morphogenesis and cell proliferation in kidneys and teeth. However, the antibody did not inhibit development when iron was delivered to the cells by a lipophilic iron chelator i.e., by-passing the receptor-mediated pathway. Hence, the binding of the receptor antibody to the receptor apparently did not affect cell proliferation, and the antibody was not toxic to the tissues. Our results suggest that the antibody to the transferrin receptor inhibits development by blocking the normal endocytotic route of iron delivery. Cells derived from embryonic kidneys and teeth expressed the transferrin receptor when cultured as monolayers. However, using immunofluorescent techniques, we were unable to detect the receptor in frozen tissue sections. It is possible that the seeding of cells in monolayer cultures affects the expression of the transferrin receptor, since it is known that all types of cells require transferrin for continued proliferation in culture. Organ-cultured kidney mesenchymal cells are not initially responsive to transferrin, but they acquire responsiveness as a consequence of an inductive tissue interaction. Although it remains unknown as to whether the acquisition of transferrin responsiveness is directly related to the expression of transferrin receptors, our results suggest that transferrin and its receptors play a role in embryonic morphogenesis.     

3'-Azido-3'-deoxythymidine reduces the rate of transferrin receptor endocytosis in K562 cells.

K562 cells, exposed for at least 24 h to 5 microM 3'-azido-3'-deoxythymidine (AZT), gave rise to an overall increase in the number of cell surface transferrin binding receptors (18-20%). This effect was ascertained either with binding experiments by using 125I-transferrin and with immunoprecipitation by using a specific monoclonal antibody against the transferrin receptor. At higher AZT concentrations (20 and 40 microM), a further increase was found, that is, up to 23% by binding experiments and up to 110% by immunoprecipitation. However, Scatchard analysis of the binding data indicated that although the number of cell surface transferrin receptors increased, the affinity of transferrin for its receptor did not change (Ka=4.0x108 M). Surprisingly, immunoprecipitation of total receptor molecules showed that the synthesis of receptor was not enhanced by the drug treatment. The effect of AZT on transferrin internalization and receptor recycling was also investigated. In this case, data indicated that the increase in the number of receptors at the cell surface was probably due to a slowing down of endocytosis rate rather than to an increased recycling rate of the receptor to cell surface. In fact, the time during which half the saturated amount of transferrin had been endocytosed (t1/2) was 2.15 min for control cells and 3.41, 3.04, and 3.74 min for 5, 20, and 40 microM AZT-treated cells, respectively. Conversely, recycling experiments did not show any significant differences between control and treated cells. A likely mechanism through which AZT could interfere with the transferrin receptor trafficking, together with the relevance of our findings, is extensively discussed.     

人转铁蛋白受体及其特异性抗体的制备

目的:从胎盘中提取转铁蛋白受体并获得抗转铁蛋白受体的抗体。方法:人新鲜胎盘组织被破碎后,用去污剂TritonX-100裂解细胞膜,释放膜蛋白。利用膜蛋白中的转铁蛋白受体能与铁-转铁蛋白复合物特异性结合的特性对其进行亲和纯化。对纯化得到的目的蛋白,经脱盐后进行ELISA及肽质量图谱分析,证明为所需的转铁蛋白受体后,以其包被免疫管,从全合成人源噬菌体抗体库中筛选抗体。结果:从人源噬菌体抗体库中筛选到5个能够与转铁蛋白受体特异性结合的噬菌体单链抗体。结论:以人源转铁蛋白受体为抗体,可从全人源噬菌体抗体库中筛选到其特异性的抗体。     

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.     

Plasma membrane and intracellular pools of transferrin receptors decline during in vitro cultivation of U937 cells

Transferrin receptor expression in the monocyte-like cell line U937 was investigated during in vitro cultivation. U937 cells expressed a single class of high affinity surface transferrin receptors (KD approximately 4 nM), with apparent subunit Mr of 90-95,000 Da as determined by SDS-reducing PAGE. [125I]-transferrin binding studies on detergent-solubilized cells revealed that half to two-thirds of the total functional binding sites were located intracellularly. Radioligand binding, immunofluorescence and flow cytometry studies were performed on intact, detergent-solubilized, or saponin-permeabilized cells, using either transferrin or the anti-transferrin receptor monoclonal antibody OKT9 IgG. These studies demonstrated that functional and antigenic transferrin receptor levels were maximal on cells 24 h after subculture at low density and declined during the culture period. Scatchard analysis of radioligand binding data suggested that the decline in functional transferrin binding sites resulted from a decline in the number of available receptors. These results demonstrate that in U937 cells there is a density-dependent regulation of transferrin receptor expression, resulting in a loss of functional and antigenic receptors from both plasma membrane and intracellular locations.     

A dynamic model of the meningococcal transferrin receptor.

Iron is an essential nutrient for all organisms and consequently, the ability to bind transferrin and sequester iron from his source constitutes a distinct advantage to a blood-borne bacterial pathogen. Levels of free iron are strictly limited in human serum, largely through the action of the iron-binding protein transferrin. The acquisition of trasferrin-iron is coincident with pathogenicity among Neisseria species and a limited number of other pathogens of human and veterinary significance. In Neisseria meningitidis, transferrin binding relies on two co-expressed, outer membrane proteins distinct in aspects of both structure and function. These proteins are independently and simultaneously capable of binding human transferrin and both are required for the optimal uptake of iron from this source. It has been established that transferrin-binding proteins (designated TbpA and TbpB) form a discrete, specific complex which may be composed of a transmembrane species (composed of the TbpA dimer) associated with a single surface-exposed lipoprotein (TbpB). This more exposed protein is capable of selectively binding iron-saturated transferrin and the receptor complex has ligand-binding properties which are distinct from either of its components. Previous in vivo analyses of N. gonorrhoeae, which utilizes a closely related transferrin-iron uptake system, indicated that this receptor exists in several conformations influenced in part by the presence (or absence) of transferrin.Here we propose a dynamic model of the meningococcal transferrin receptor which is fully consistent with the current data concerning this subject. We suggest that TbpB serves as the initial binding site for iron-saturated transferrin and brings this ligand close to the associated transmembrane dimer, enabling additional binding events and orientating transferrin over the dual TbpA pores. The antagonistic association of these receptor proteins with a single ligand molecule may also induce conformational change in transferrin, thereby favouring the release of iron. As, in vivo, transferrin may have iron in one or both lobes, this dynamic molecular arrangement would enable iron uptake from either iron-binding site. In addition, the predicted molecular dimensions of the putative TbpA dimer and hTf are fully consistent with these proposals. Given the diverse data used in the formulation of this model and the consistent characteristics of transferrin binding among several significant Gram-negative pathogens, we speculate that such receptor-ligand interactions may be, at least in part, conserved between species. Consequently, this model may be applicable to bacteria other than N. meningitidis.     

The p97 antigen is mapped to the q24-qter region of chromosome 3; the same region as the transferrin receptor.

Since the p97 antigen, a membrane-associated iron-binding protein, has extensive amino acid sequence with homology with transferrin, is functionally related to the transferrin receptor, and has been previously mapped to chromosome 3, we have performed additional studies for regional mapping of the gene expressing p97 antigen. In these experiments, Chinese hamster-human cell lines were chosen that contained a large spectrum of autosomal human chromosomes, but mainly consisted of clones expressing all or a part of chromosome 3. These cell lines included a clone that previously allowed for mapping of human transferrin receptor to q22-qter region. Human p97 expression was assessed by specific binding of [125I]monoclonal antibody 96.5, and human transferrin receptor expression was tested by specific [125I]human transferrin binding and [125I]monoclonal antibody OKT-9 specific for human transferrin receptor. Based on these analyses, both human p97 antigenic expression and human transferrin receptor are mapped concordantly to the q24-qter region. These data and previous reports, therefore, suggest that the related iron-transport proteins are closely linked and may be under coordinate regulation. However, studies of several cell lines that exhibit up-regulation of human transferrin receptor expression with cellular proliferation, and down-regulation of receptor with increased transferrin-iron in the media, showed no change in expression of p97 antigen. p97 antigenic expression increased when melanocyte-stimulating hormone was added to a human melanoma cell line in tissue culture. These latter studies suggest that in mammalian cells the two proteins do not show coordinate regulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Entamoeba histolytica: transferrin binding proteins

Entamoeba histolytica trophozoites depend on iron for their growth; thus, they must use some host iron-containing molecules to fulfill this requirement. In this work we report that amoebas are able to utilize human holo-Tf as iron source and to recognize it through transferrin binding proteins. By use of an anti-human transferrin antiserum in an immunoblotting assay, two main polypeptides with apparent molecular masses of 70 and 140 kDa were found in total extract of trophozoites cultured in vitro. However, when a monoclonal anti-human transferrin receptor antibody was used, only one band with molecular mass of 140 kDa was observed. Both the human transferrin and the monoclonal antibody recognized a protein on the amoebic surface, demonstrated by confocal microscopy. Furthermore, the complex transferrin-transferrin binding protein was internalized by an endocytic process and probably dissociated inside the cell. This mechanism could be one manner in which E. histolytica acquires iron from the human host transferrin.     

NK recognition of target structures: is the transferrin receptor the NK target structure?

That the transferrin receptor acts as a target antigen for human NK cells has previously been suggested. In this study we used two models to examine the hypothesis that the transferrin receptor is recognized by NK cells. In the first model, we employed mouse cloned NK cells in conjunction with the species-specific monoclonal antibody R17 217, which binds to the murine transferrin receptor. We show that there is no correlation between the amount of transferrin receptor expressed on targets and the susceptibility of these targets to NK lysis or NK binding in cold target competition assays. In the second model, we used human NK cells and transferrin receptor-positive transformants as targets. These transformants were derived from mouse L cells transfected with human DNA and selected for the presence of human transferrin receptor. Results show that, in contrast to the mouse system, there is a correlation between the expression of the human transferrin receptor on targets and the ability of these targets to competitively inhibit the lysis of K562 by NK cells. However, because inhibition is not complete, other cell surface antigens probably play a role in human NK-target interactions.     

Expression of the transferrin receptor gene during the process of mononuclear phagocyte maturation

The expression of transferrin receptors by blood monocytes, human alveolar macrophages, and in vitro matured macrophages was evaluated by immunofluorescence, radioligand binding, and Northern analysis, using the monoclonal anti-human transferrin receptor antibody OKT9, [125I]-labeled human transferrin and a [32P]-labeled human transferrin receptor cDNA probe, respectively. By immunofluorescence, the majority of alveolar macrophages expressed transferrin receptors (86 +/- 3%). The radioligand binding assay demonstrated the affinity constant (Ka) of the alveolar macrophage transferrin receptor was 4.4 +/- 0.7 X 10(8) M-1, and the number of receptors per cell was 4.4 +/- 1.2 X 10(4). In marked contrast, transferrin receptors were not present on the surface or in the cytoplasm of blood monocytes, the precursors of the alveolar macrophages. However, when monocytes were cultured in vitro and allowed to mature, greater than 80% expressed transferrin receptors by day 6, and the receptors could be detected by day 3. Consistent with these observations, a transferrin receptor mRNA with a molecular size of 4.9 kb was demonstrated in alveolar macrophages and in vitro matured macrophages but not in blood monocytes. Thus, although blood monocytes do not express the transferrin receptor gene, it is expressed by mature macrophages, an event that probably occurs relatively early in the process of monocyte differentiation to macrophages.     

Monoclonal antibodies against a glycoprotein localized in coated pits and endocytic vesicles inhibit alpha 2-macroglobulin binding and uptake

Eight monoclonal antibodies, all IgG2a, which recognize a 180/90-kDa glycoprotein similar in properties to the receptor for alpha 2-macroglobulin of mouse embryo 3T3 cell plasma membranes, have been tested for their effect on the binding and uptake of alpha 2-macroglobulin by live cells. One antibody directly inhibited binding of 125I-alpha 2-macroglobulin under conditions in which 125I-transferrin binding to the transferrin receptor was unaffected. Another monoclonal antibody decreased alpha 2-macroglobulin binding when preincubated with cells at 37 degrees C. This antibody was also capable of specifically binding to ligand-receptor complexes formed by preincubating 125I-alpha 2-macroglobulin with detergent extracts of Swiss 3T3 cells. Immunoelectron microscopy showed that the 180/90-kDa glycoprotein was localized in coated pits of the cell surface and in intracellular endocytic vesicles (receptosomes/endosomes). The data suggest that the 180/90-kDa glycoprotein is a component of the receptor for alpha 2-macroglobulin.