A REGION OF THE C-TERMINAL PORTION OF THE HUMAN TRANSFERRIN RECEPTOR CONTAINS AN ASPARAGINE-LINKED GLYCOSYLATION SITE CRITICAL FOR RECEPTORSTRUCTURE 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 s urface localization of the human transferrin receptor is examined here by site-directed mutagenesis. Each of the extracellular consensus seq uences (Asn-X-Ser/Thr) for asparagine-linked glycosylation was mutated individually and in all possible combinations. The constructs were tr ansfected stably into NIH-3T3 cells and a Chinese hamster ovary cell l ine lacking endogenous transferrin receptors. Of the seven possible co mbinations of glycosylation sites, single mutations eliminating glycos ylation at either Asn251 or Asn317 do not affect the processing and su rface localization of the receptor. Eliminating both of these sites to gether has a small effect on the behavior of the receptor. However, mu tation of the C-terminal glycosylation site (Asn727) has the most prof ound negative effect on the appearance of the receptor at the cell sur face. The mutants lacking glycosylation at Asn727 appear to be retaine d in the endoplasmic reticulum as an increased association with bindin g immunoglobulin protein (BiP) is observed. Addition of a new glycosyl ation site in the C-terminal region of the unglycosylated mutated tran sferrin receptor restores the cell surface localization and the transf errin binding of the transferrin receptor, indicating that glycosylati on in this region is critical for the correct transport of this recept or to the cell surface.