A region of the C-terminal portion of the human transferrin receptor contains an asparagine-linked glycosylation site critical for receptor structure and function.

The transferrin receptor is a cell surface protein and is responsible for the uptake of iron into many eukaryotic cells. In its mature form, the receptor possesses three asparagine-linked oligosaccharides. The effect of asparagine-linked glycosylation on the processing and cell surface localization of the human transferrin receptor is examined here by site-directed mutagenesis. Each of the extracellular consensus sequences (Asn-X-Ser/Thr) for asparagine-linked glycosylation was mutated individually and in all possible combinations. The constructs were transfected stably into NIH-3T3 cells and a Chinese hamster ovary cell line lacking endogenous transferrin receptors. Of the seven possible combinations of glycosylation sites, single mutations eliminating glycosylation at either Asn251 or Asn317 do not affect the processing and surface localization of the receptor. Eliminating both of these sites together has a small effect on the behavior of the receptor. However, mutation of the C-terminal glycosylation site (Asn727) has the most profound negative effect on the appearance of the receptor at the cell surface. The mutants lacking glycosylation at Asn727 appear to be retained in the endoplasmic reticulum as an increased association with binding immunoglobulin protein (BiP) is observed. Addition of a new glycosylation site in the C-terminal region of the unglycosylated mutated transferrin receptor restores the cell surface localization and the transferrin binding of the transferrin receptor, indicating that glycosylation in this region is critical for the correct transport of this receptor to the cell surface.