Functional Expression of the Human Transferrin Receptor cDNA in Chinese Hamster Ovary Cells Deficient in Endogenous Transferrin Receptor

Transferrin (Tf) receptor-variant Chinese hamster ovary cells have been isolated by selection for resistance to two Tf-toxin conjugates. The hybrid toxins contain Tf covalently linked to ricin A chain or a genetically engineered diphtheria toxin fragment. The Tf-receptor-variant (TRV) cells do not have detectable cell-surface Tf receptor; they do not bind fluorescein-Tf or ~25I-Tf. TRV cells are at least 100-fold more resistant to the Tf-diphtheria toxin conjugate than are the parent cells. The TRV cells have retained sensitivity to native diphtheria toxin, indicating that the increased resistance to the conjugate is correlated with the loss of Tf binding. The endocytosis of fluoresceinlabeled t1:-macroglobulin is normal in TRV cells, demonstrating that the defect does not pleiotropically affect endocytosis. Since these cells lack endogenous Tf receptor activity, they are ideally suited for studies of the functional expression of normal or altered Tf receptors introduced into the cells by cDNA transfection. One advantage of this system is that Tf binding and uptake can be used to monitor the behavior of the transfected receptor. A cDNA clone of the human Tf receptor has been transfected into TRV cells. In the stably expressing transfectants, the behavior of the human receptor is very similar to that of the endogenous Chinese hamster ovary cell "If receptor. Tf binds to cell surface receptors, and is internalized into the para-Golgi region of the cell. Iron is released from Tf, and the apo-Tf and its receptor are recycled back to the cell surface. Thus, the TRV cells can be used to study the behavior of genetically altered Tf receptors in the absence of interfering effects from endogenous receptors. T RANSVERRIN (Tf) ~ is an iron-carrying serum glycoprotein responsible for Fe +3 delivery to vertebrate cells (1). Diferric Tf uptake is mediated by binding to a specific cell surface receptor (20). Once bound, the Tfreceptor complex is internalized, via receptor-mediated endocytosis, into an acidic intracellular compartment (for review see reference 8) where the acidic pH facilitates Fe ÷3 release from transferrin. The qY receptor (Tf-R) and apo-Tf are recycled back to the cell surface. At physiological extracellular pH apo-Tf is released from the receptor, and the recycled receptor participates in multiple rounds of internalization (4, 6). The general process of receptor-mediated endocytosis has been morphologically well characterized for a number of ligands in a variety of cell types (for review see reference 7). Little is known about the features of the receptors that serve to signal internalization and intracellular routing. The avail1. Abbreviations used in this paper: DT, diphtheria toxin; f-TE fluorescein transferrin; med 2, medium consisting of 150 mM NaC1, 20 mM Hepes pH 7.4, 1 mM CaCl2, 5 mM KCI, 1 mM MgCl2; Tf, transferrin; Tf-R, transferfin receptor; Tf-RA, transferrin-ricin A chain; TRV, transferrin receptor variant. ability of cDNA clone of the human Tf-R (17, 26) provides a direct approach to these questions since the cloned gene can be specifically mutated in vitro and transfected into cells. The effect of specific mutations on receptor function can then be characterized. An ideal cell line for such studies would be one that is deficient in Tf-R expression. Tf-R is expressed on most cultured cell lines, presumably reflecting the nutritional requirement for iron (10). Thus, studies of the transfected receptor would necessarily be performed in a background of endogenous Tf-R. The presence of the endogenous receptor would prohibit the use of "If binding and uptake in the characterization of the transfected receptor, although the expression of the receptor could be monitored using anti-human Tf-R monoclonal antibodies. Mouse L cell transformants expressing the human Tf-R have been isolated using human specific monoclonal antibodies (15, 19). Tf uptake in Chinese hamster ovary (CHO) cells has been characterized, and the properties are similar to those found in other cell types studied (30). Mutant CHO cells that cannot release iron from "IT are able to transport sufficient iron to support cell growth, apparently by increased uptake of iron salts (14). Since CHO cells have an alternative to qT-mediated iron uptake, it should be possible to select for ceils that © The Rockefeller University Press, 0021-9525/87/07/207/8 $2.00 The Journal of Cell Biology, Volume 105, July 1987 207-214 207 on O cber 9, 2017 jcb.rress.org D ow nladed fom are deficient in Tf-R expression. Such cells could then be used for studies of the functioning of the transfected human Tf-R. A further advantage of using CHO cells for these studies is that they have been used for many studies of receptormediated endocytosis, including the isolation of mutant cells defective in receptor-mediated endocytosis (12, 24). A strategy for isolating Tf-R-variant CHO cells is to select for resistance to a Tf-toxin conjugate. In the conjugate, Tf replaces the cell surface-binding domain of the toxin. The specificity of the cytotoxicity of the conjugate is conferred by Tf binding to cells. The rationale behind this approach is that "If-R-variant cells would not specifically bind the conjugate and would thereby display greater resistance than cells expressing high affinity Tf-R. The efficacy of isolating cells deficient in the expression of cell surface markers by selection with toxin conjugates has been previously established (18, 23). Such an approach, using a Tf-ricin A chain (Tf-RA) conjugate, has been used to isolate a Tf-R-modified human leukemia cell line (22). In this study we report the isolation of Tf-R-variant CHO cells by selection for resistance to two Tf conjugate toxins: a ricin A chainand a modified diphtheria toxin (DT)-Tf conjugate. The Tf-R-variant cells do not bind detectable amounts of Tf. A human Tf-R cDNA has been transfected into these cells. The behavior of the human receptor in these cells is very similar to that of the endogenous receptor in wild type CHO cells. 59Fe is efficiently internalized by the transfectants when presented to the cells as diferric Tf, demonstrating that the human receptor is biologically active. These results demonstrate the utility of using the receptorvariant CHO cells for structure-function studies of the human Tf-R. Materials and Methods Cells and Cell Culture WTB Chinese hamster ovary cells were used as the parental cell line (29). Ceils were maintained in Ham's nutrient F12 medium (Gibco, Grand Island, NY) supplemented with 5 % FCS (referred to as complete medium), or in Ham's F12 containing 500 ng/ml Se203, 5 llg/m] insulin, and 1 mg/ml BSA fraction V (referred to as serum-free medium) at 3"/°C in a humidified atmosphere of 5 % CO2 in air. Single cell colony lines were isolated by two rounds of cloning ring colony purification.