Exposure of K562 cells to anti-receptor monoclonal antibody OKT9 results in rapid redistribution and enhanced degradation of the transferrin receptor.

Abstract

Abstract. When the human erythroleukemia cell line K562 is treated with OKT9, a monoclonal antibody against the transferrin receptor, effects on receptor dynamics and degradation ensue. The apparent half-life of the receptor is decreased by >50% as a result of OKT9 treatment. The transferrin receptor is also rapidly redistributed in response to OKT9 such that a lower percentage of the cellular receptors are displayed on the cell surface. OKT9 treatment also leads to a decrease in the total number of receptors participating in the transferrin cycle for cellular iron uptake. The reduction in iron uptake that results from the loss of receptors from the cycle leads to enhanced biosynthesis of the receptor. Receptors with bound OKT9 continue to participate in multiple cycles of iron uptake. However, OKT9 treatment appears to result in a relatively small increase per cycle in the departure of receptors from participation in iron uptake to a pathway leading to receptor degradation. Radiolabeled OKT9 is itself degraded by K562 cells and this degradation is inhibitable by leupeptin or chloroquine. In the presence of leupeptin, OKT9 treatment results in the enhanced intracellular accumulation of transferfin. Because the time involved in the transferrin cycle is shorter 02.5 min) than the normal half-life of the receptor (8 h), a small change in recycling efficiency caused by OKT9 treatment could account for the marked decrease in receptor half-life. In this paper the implications of these findings are discussed as they relate to systems in which receptor number is regulated by ligand. p ROLIFERATING cells require exogenous iron and acquire it via receptor-mediated endocytosis of the ironcarrier protein transferrin (Tf) 1 (for review see reference 6). Iron uptake via the Tf cycle involves binding of diferric Tf to a specific, high-affinity surface receptor followed by internalization of the receptor ligand complex. Within the cell, the complex encounters an acidic (~pH 5) environment that is instrumental in iron unloading (22, 31). Apo-Tf is then released intact from the cell, and the transferrin receptors (TtR) are reused (2, 9). The Tf cycle shares certain aspects with other systems of receptor-mediated endocytosis but also exhibits distinctive features, including the return of undegraded ligand and receptor to the cell exterior.In the present study, we describe perturbations of the pathway traversed by the TfR that result from exposure of K562 cells to a monoclonal anti-receptor antibody (OKT9). The monoclonal antibody OKT9 recognizes the human TfR at a site distinct from the ligand binding site and has been used to examine the structural features of the receptor as well as in the chromosomal localization and cloning of the TfR gene (3,15,25 Materials and Methods Cells and ChemicalsK562, a human erythroleukemia cell line, was grown in RPMI 1640 with 25 mM Hepes containing 10% fetal bovine serum, penicillin, and streptomycin (growth medium). Ceils were maintained at densities of 2-6 × 105/ml at 37"C in 5% CO2 incubator....

Down-Regulation of Cell Surface Receptors Is Modulated by Polar Residues within the Transmembrane Domain

“…Further support for this idea came from cross-linking the wild-type receptor with dimeric Tf, which also resulted in effective down-regulation of the complexes. This last result is consistent with previous studies that showed that the TR was rapidly degraded after monoclonal antibody cross-linking (Weissman et al, 1986).…”

“…It is interesting that one of the proteins identified in the search, P-selectin, was also a protein in which a cytoplasmic tail lysosomal targeting signal was difficult to localize (Straley et al, 1998). And finally in support of our proposed model, it is now clear that receptor down-regulation requires multiple rounds of recycling before lysosomal delivery occurs (Lai et al, 1989;Felder et al, 1992), suggesting that down-regulation, rather than being a distinctive all-or-nothing event, may simply reflect a loss of recycling fidelity (Weissman et al, 1986).…”

“…A critical unanswered question, however, is what is the mechanism that distinguishes recycling receptors from down-regulated receptors in the sorting endosome? Clearly, one mechanism for down-regulation is by receptor cross-linking with multivalent ligands (Mellman and Plutner, 1984;Weissman et al, 1986), but how this relates to the normal clearance process is unclear.…”

Structural Requirements for Major Histocompatibility Complex Class II Invariant Chain Endocytosis and Lysosomal Targeting

“…As expected, apo-Tf released into the medium from cells expressing the wild-type TR was undegraded (Fig. 5), because TR-apo-Tf complexes are efficiently recycled back to the cell surface through sorting and recycling compartments (35,37,43,44). Only about 2% of the counts were in the TCA soluble fraction after 2 h (TCA soluble counts).…”

Modulation of doxorubicin resistance in a doxorubicin-resistant human leukaemia cell by an immunoliposome targeting transferring receptor

“…Thus CIL-targeting cells with multidrug-resistant phenotype could lead to a distinct intracellular DOX distribution that may result in the decreased excretion of DOX. A membrane transferrin receptor (TFR) is associated with cell growth in malignant cells and some normal cells (Hamilton et al, 1979;Trowbridge and Omary, 1981) and is internalized into cells by endocytosis through the binding of transferrin or anti-TFR antibodies (Weissman et al, 1986;Esserman et al, 1989;Girones and Davis, 1989). Thus TFR possess properties suitable for a target antigen for endocytosis of CILs.…”