Binding of apotransferrin to K562 cells: explanation of the transferrin cycle.

Abstract

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...

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

“…We first analyzed the interaction by a cellular enzyme-linked immunosorbent assay (CELISA), an assay that detects the binding of specific ligands to cell-surface proteins (Arunachalam et al, 1990). We carried out an inhibition binding assay in which different dilutions of anti-Tf MAbs and peroxidase-labeled human Tf (HPR-Tf) were added to wells of a microtiter plate coated with K562 cells, which present TRs in large numbers (about 150,OOO per cell) on their plasma membranes (Klausner et al, 1983). This assay allows a direct study of human Tf/TR interaction in a physiological context, thus circumventing the problem of extraction and purification of TR.…”

Low affinity of Trypanosoma brucei transferrin receptor to apotransferrin at pH 5 explains the fate of the ligand during endocytosis

“…In mammalian cells, the receptor-ligand complex is delivered to endosomes where the low pH triggers the release of iron from Tf. Apo-Tf remains tightly bound to ~ts receptor and is recycled to the cell surface to mediate further cycles of iron uptake [6,8]• Presumably also in trypanosomes the receptor-ligand complex is delivered to an endosomal system like other internalised macromolecules [15][16][17]. The acidic environment of this compartment certainly leads to liberation of iron from Tf and, because of the low affinity of the resulting apo-Tf for its receptor (see Figs.…”

“…Binding of one molecule of transferrin Tf) [2] requires association of both pESAG6 and pESAG7 as ~hown by coexpression in heterologous systems [3][4][5]. Despite :he profound difference in receptor structure, the apparent tissociation constant (Kd) for iron-loaded Tf (holo-Tf) is of ::he same order of magnitude for both the trypanosome TfR 3.6-108 nM [2]) and the mammalian TfR (2-110 nM [6,7]). Fhe fate of Tf differs, however, in mammalian cells and try3anosomes.…”

“…In mammalian cells, the Tf-TfR complex is transported to endosomes where Tf releases its iron. The iron-free l'f (apo-Tf) remains bound to its receptor and is recycled back ~.o the cell surface where it dissociates from the receptor [6,8]. In contrast, Tf is delivered in trypanosomes to lysosomes ~vhere it is proteolytically degraded [2,9].…”

Analysis of Receptor Tyrosine Kinase Internalization Using Flow Cytometry

“…Acid stripping cells by washing with low pH solutions readily dissociates most ligands from their receptors (4,13,16). However the optimal acid stripping conditions for removing cell surface associated ligand without damaging the cells must be empirically determined for each cell line and for each receptor/ligand combination.…”