Generation of the soluble transferrin receptor requires cycling through an endosomal compartment.
The transmembrane protein, transferrin receptor (TfR), is found in a soluble form in human serum and in the medium of cell lines grown in tissue culture. The soluble form is generated by proteolytic cleavage between Arg-100 and Leu-101. We used two mutant human TfRs expressed in Chinese hamster ovary (CHO) cells lacking endogenous transferrin receptor to characterize the protease that cleaves the TfR and determine its location in the cell. The T104D mutant TfR lacks the O-linked carbohydrate at position 104, and is more susceptible to proteolytic cleavage at Arg-100 than the wildtype human TfR in these cells. We find that the protease is not a component of the serum in the growth medium, and it is not secreted by the cells. Cleavage does not occur during biosynthesis of the TfR, and occurs after the TfR has reached the cell surface. Expression of the T104D TfR in a temperature-sensitive acidification defective CHO cell line, G.7.1, shows that cleavage of the TfR is not dependent on acidification of endosomes. The C20A23 TfR is an endocytosis deficient mutant lacking an internalization signal. This mutant TfR, which is mainly localized to the cell surface, is cleaved less efficiently than the wild-type TfR, indicating that the protease is localized to an intracellular compartment.
Rutledge
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EA
Department of Cell Biology and Anatomy, Oregon Health Sciences University, Portland 97201.
Green
F A
FA
Enns
C A
CA
D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002451:Cell Compartmentation; D006224:Cricetinae; D004705:Endocytosis; D010450:Endopeptidases / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011990:Receptors, Transferrin / Q000378:metabolism*; D011994:Recombinant Proteins; D012995:Solubility; D013329:Structure-Activity Relationship
D011990:Receptors, Transferrin; D011994:Recombinant Proteins; D010450:Endopeptidases