The binding of apotransferrin to the transferrin receptor on the surface of human leukemic K562 cells was found to be significantly less tight than that of the holoprotein, diferric transferrin. The finding that both ligands displayed linear Scatchard plots with similar receptor number ("'150,000 per cell) and mutually inhibit each other's binding suggested that they bind to the same receptor. Both the dissociation and association rate of apotransferrin were markedly increased (28-fold and 15-fold, respectively) at pH 7.2 compared to pH 4.8. Using the values of these binding parameters, we propose a mechanism to account for the recycling of transferrin subsequent to internalization and residence within an acidic nonlysosomal organelle where iron is removed.A wide variety of molecules gain entry into cells by receptormediated endocytosis (1,2). Diverse ligands, including low density lipoproteins, asialoglycoproteins, epidermal growth factor, a2 macroglobulin, lysosomal enzymes, and certain hormones, toxins, and viruses traverse strikingly similar, if not identical, pathways. Binding to specific cell-surface receptors is followed by internalization involving specialized regions of the plasma membrane, the coated pits. Between their internalization and their ultimate fate, which is often lysosomal degradation, appropriately tagged ligands are visible by electron microscopy within a number of morphologically varied intracellular vesicles. We have examined this pathway for the receptor-mediated endocytosis of asialoglycoproteins in hepatocytes and have shown that the receptor enters the cell as a complex with its ligand, whereupon the latter subsequently dissociates and is degraded (3, 4). In contrast to the ligand, the receptor is reutilized (5, 6). We have presented evidence that the intracellular dissociation of asialoglycoproteins is facilitated by encounter with an acidic environment prior to delivery to lysosomes (4, 7), a step that we believe to be critical in receptor reutilization. There is growing evidence that a number of other ligands are similarly exposed to nonlysosomal environments of low pH in their movement through the cell (8-11). This suggests that an acidic endocytic vesicle may participate in the intracellular transit of many ligands. As with asialoglycoproteins, a number of ligands interact with their respective receptors in a highly pH-dependent fashion, and many of these receptors have been shown to be reutilized (1).A notable exception to this generalized picture of receptormediated endocytosis involves the iron-binding protein transferrin (reviewed in ref. 12). Diferric transferrin enters the cell bound to its specific receptor, but internalization of the receptor-bound transferrin does not result in transferrin degradation (13)(14)(15). Instead, both the receptor and the apoprotein ligand are returned to the cell's exterior with retention of iron within the cell. Although coated pits (16) and an acidic, nonlysosomal microenvironment (9) have been shown to be involved in the transfe...

At physiological temperature, the Fe-carrier transferrin is taken up by K562 human erythroleukemia cells through receptor-mediated endocytosis. Both ligand (now minus Fe) and receptor recycle back to the cell surface where the receptor is rapidly reutilized. After endocytosis, transferrin becomes transiently lodged within an acidic compartment inside the cell, as judged by the changed spectral characteristics and quantum yield of fluorescein isothiocyanate-labeled transferrin that is cell-associated at 37C. Upon binding to transferrin, anti-fluorescein antibody strongly quenches the emission of the fluoresceinlabeled residues on the protein and is used to assess whether the transferrin is at the cell surface (incubation at 0C) or mainly internalized into the cell (incubation at 37C). Using Percoll gradient fractionation of postnuclear supernatants, we show that the acidic compartment is not the lysosomal compartment.All cells require Fe, and the physiologic apparatus for Fe delivery utilizes a serum glycoprotein, transferrin (1). Despite some controversy concerning the pathway of Fe uptake, there is accumulating evidence pointing to receptor-mediated endocytosis of transferrin, with the subsequent transfer of Fe to the cell (2-5). There has been a great deal of interest in this general cellular process, and certain patterns are emerging from a number of endocytic systems (6). Such endocytosis is an energy-dependent process mediated by specific cell surface receptors, which are generally preferentially internalized through coated pits (7). Although the exact cellular path taken by the ligand is yet to be elucidated, most end up in lysosomes in which they are degraded (8, 9). The pathway taken by the receptor is even less clear. However, in several systems, the receptors clearly are not degraded and are reutilized (10, 11). We have studied certain details of the pathway taken by the asialoglycoprotein receptor (12). We showed that the ligand enters the cell attached to the receptor and that both receptor and ligand are internalized. Soon after internalization, the ligand is released from the receptor, and, after an obligate processing time, the ligand is degraded in the lysosomes. The receptor recycles back to the cell surface. The initial replacement for internalized receptor is derived from a cryptic spare receptor pool.In striking contrast to many other endocytosed ligands, transferrin is not degraded (3,13). Rather, mono-or diferric transferrin is taken up, the Fe removed, and the apotransferrin is released into the medium. Data from our laboratory (unpublished observations) concerning the transferrin uptake by K562 cells have led to the following conclusions. The K562 cells contain about 2 x 105 high-affinity (Kd = 1 x 10-9 M) transferrin surface-receptors per cell. Transferrin containing one or two ferric ions binds to these receptors and, when the cells are warmed, is rapidly internalized (t412 = 3 min). Upon internalization of the receptor-transferrin complex, the Fe is removed from the transferri...

Treatment of human K562 cells with 4f3 phorbol 12-myristate 13-acetate (PMA) resulted in an approximately 50% reduction in cell surface transferrin receptors within 30-45 min as judged by binding of both ligand and antireceptor antibody. The affinity of the remaining surface receptors for diferric transferrin appeared to be unaltered. The time-dependent loss in transferrin receptors was also dependent upon PMA concentration, with a half-maximal effect observed at approximately 1 nM. The kinetic parameters for the binding, internalization, intracellular residency, and recycling of 2'I-labeled transferrin were unchanged by PMA treatment, as were the rate and extent of internalization of anti-receptor antibody. Moreover, despite the decrease in surface receptors, uptake of 59Fe from transferrin proceeded at a rate comparable to that seen in untreated cells. Accounting for this observation was the fact that ligand induced a reduction in surface receptors in untreated but not PMA-treated cells. Quantitative immunoprecipitation of transferrin receptors from surface-iodinated K562 cells revealed that little receptor internalization occurred in untreated cells in the absence of ligand, but internalization of ligand-occupied receptors in these cells was readily detected. In contrast, PMA treatment resulted in the rapid internalization of surface receptors irrespective of occupancy. Thus, binding of ligand appeared to trigger the internalization of receptors that were relatively static in their unoccupied state, and a signal for receptor internalization was also provided by PMA treatment. The possibility that this signal involves phosphorylation of the transferrin receptor is discussed.Phorbol esters have been studied for several decades, largely because of their action as tumor promoters (reviewed in ref. 1). Cells undergo a wide variety of changes after treatment with phorbol esters, including alterations in the synthesis and turnover of phosphatidylcholine, prostaglandins, polyamines, proteins, DNA, and RNA. Recently, interest has been heightened by the identification of specific phorbol ester receptors (2), the discovery that phorbol esters activate protein kinase C (3), and the likely role of this kinase as the receptor (4-6). How activation of this kinase leads to the many cellular events triggered by phorbol esters is unknown. Whereas multiple changes in the cell surface upon treatment with phorbol esters have been described (1), it is not clear that all such changes are attributable to phosphorylation events. Phorbol esters may alter, directly or indirectly, a wide variety of cell surface receptors. Exposure to phorbol esters leads to a loss of high-affinity binding sites for epidermal growth factor (EGF) (7) and somatomedin (8) and has been shown to induce phosphorylation of the receptor for EGF (9), insulin, and somatomedin C (10). In this report, we present evidence for a specific alteration in the dynamics of the human transferrin (Tf) receptor that accompanies treatment of K562 cells with 43-phorbol 1...

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Hen ovalbumin, the major secretory product of oviduct cells, is a 43 000-dalton glycoprotein. Many studies have led to controversy over the question of whether ovalbumin (OA) can be fully renatured after chemical denaturation. We have studied the renaturation of OA after denaturation with guanidinium chloride, urea or alkaline pH. Denatured OA displays an intrinsic viscosity consistent with nearly complete unfolding of the protein. Removal of the denaturant results in a complete reversal of the changes in intrinsic viscosity. However, closer examination of the renatured protein reveals major differences from the native form. Renatured OA (OAR) can be completely separated from the native form (OAN) by affinity chromatography on phenyl-Sepharose. OAR displays altered tryptophan fluorescence, u.v.-absorption and c.d. spectra. Only OAR binds anilinonaphthalenesulphonate (as measured by fluorescence enhancement). OAR, but not OAN, binds about 2 mol of the covalent hydrophobic affinity probe phenyl isothiocyanate/mol. Renaturation, and the production of OAR, occurs regardless of the oxidation state of the disulphide bonds, of phosphorylation of the protein, and of the presence or the absence of the single carbohydrate chain. OAR may be either monomeric or an irreversible aggregate. Which of these two states is formed depends on the protein concentration during renaturation. Monomeric and aggregated OAR can be distinguished on the basis of some spectroscopic characteristics, but they share the essential hydrophobic characteristics that distinguish them from OAN. OAN and OAR do not spontaneously interconvert. Antibodies raised to each can be made monospecific by immunoabsorption. Thus two stable forms of OA can be obtained, one of which, OAR, displays hydrophobic characteristics. OAN, but not OAR, is formed when OA is synthesized in vitro in a translation system.

A Chinese hamster ovary cell mutant defective in the receptor-mediated endocytosis of several unrelated ligands (Robbins, A. R., S. S. Peng, and J. L. Marshall, 1983, J. Cell Biol., 96:1064-1071) failed to accumulate iron provided in the form of diferric transferrin. Analysis of the steps of the transferrin cycle indicated that binding and internalization of transferrin proceeded normally in mutant cells. However, the mutant appeared unable to dissociate iron from transferrin, as evidenced by release of diferric transferrin from the mutant versus apotransferrin from the parent. Uptake of ferric ions from the growth medium was enhanced in the mutant.

The three-dimensional structures of the transforming region of the product of the EJ/T24 human bladder oncogene and of the c-Ha ras8-gene product have been calculated by using conformational energy calculations. These two genes, representing a transforming oncogene and its normal cellular homologue, encode 21,000-dalton peptides that differ by one amino acid at position 12. We therefore examined the energetically allowed conformations of the hydrophobic decapeptide surrounding this substitution site. The calculations show that the most favorable form of the c-Ha ras-l gene product exists when glycine-12 is in a left-handed bend conformation. No other amino acid can adopt this conformation and thus the bladder oncogene peptide containing valine at position 12 has a markedly different three-dimensional structure. A simple model is proposed to account for the consequences of a position 12 mutation.by restriction mapping (12)(13)(14). In our routine DNA transfection assays, however, the cloned oncogene has a transforming potency that is several orders of magnitude higher than that of cloned c-Ha ras-1. By chimeric reconstruction, this difference in transforming potency was localized to a 350-nucleotide sequence in the 5' end of the p21 coding sequences. The sequences of these regions of DNA have been determined (15)(16)(17), and the difference in transforming potential was pinpointed to a single-base change-from guanosine to thymidine. This mutation results in substitution of valine for glycine at residue 12 of the predicted amino acid sequence of p21 and occurs in the middle of a hydrophobic sequence. The sequence for the first 20 amino acid residues for the normal (nontransforming) protein is Harvey murine sarcoma virus (Ha-MuSV) was originally isolated from a tumor-bearing rat that had been inoculated with Moloney murine leukemia virus (1). The resultant retrovirus induces solid tumors and erythroleukemia in susceptible mice (2), and the naked viral DNA is capable of transforming NIH 3T3 cells after transfection (3). The region of the viral DNA responsible for the transforming activity has been localized to a one-kilobase segment of nucleic acid derived from the original host rat. A 21,000-dalton protein (p21) is encoded by this region of the viral DNA (4-7). Sequences homologous to the Ha-MuSV transforming region have been molecularly cloned from normal rat DNA (8). Moreover, homology between these sequences and DNA from other species has allowed the identification and molecular cloning from normal human DNA of genes related to the oncogene of Ha-MuSV (9). These cellular homologues (conc or more specifically in this case c-Ha ras) encode a 21,000-dalton protein immunologically and biochemically related to viral p21 (10).Through DNA transfer techniques, oncogenic sequences have been identified in several human tumors (11). The tumor oncogenes found in T24 and EJ human bladder carcinoma cell lines have been shown to be related to the c-Ha ras gene (12)(13)(14). The normal cellular counterpart of a tumor o...

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