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

• Published in: The Journal of cell biology Vol. 105; no. 1; pp. 207 - 214
• Main Authors: McGraw, Timothy E., Greenfield, Lawrence, Maxfield, Frederick R.
• Format: Journal Article
• Language: English
• Published: New York, NY Rockefeller University Press 01.07.1987; The Rockefeller University Press
• Subjects: Animals; Biological and medical sciences; Cell Line; Cell lines; Cell receptors; Cell structures and functions; Cells; CHO cells; Cricetinae; Diphtheria Toxin - administration & dosage; Diphtheria Toxin - pharmacology; DNA - genetics; Drug Resistance; Female; Fibroblasts - drug effects; Fibroblasts - metabolism; Fluorescence; Fundamental and applied biological sciences. Psychology; Humans; Iron - metabolism; L cells; Molecular and cellular biology; Ovary; Protein synthesis; Receptors; Receptors, Transferrin - biosynthesis; Receptors, Transferrin - genetics; Receptors, Transferrin - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Ricin - administration & dosage; Ricin - pharmacology; Toxins; Transfection; Transferrin - administration & dosage; Transferrin - metabolism; Transferrin receptors; Transferrins; Human; Cell culture; Transferrin; Rodentia; Receiver; Variant; Ovary; Toxin; Vertebrata; Mammalia; Transfection; Complementary DNA; Investigation method; Immunofluorescence; Hamster
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.105.1.207

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 or125I- 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 fluorescein-labeled α2-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 Tf 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.