Elimination of the O-linked glycosylation site at Thr 104 results in the
generation of a soluble human-transferrin receptor
EA Rutledge, BJ Root, JJ Lucas and CA Enns
Department of Cell Biology and Anatomy, Oregon Health Sciences University,
Portland 97201.
The transferrin receptor (TfR) is the plasma membrane protein responsible
for the binding and internalization of the major iron- transport protein,
transferrin. The function of the single O-linked oligosaccharide near the
transmembrane domain of the TfR at amino acid Thr 104 is unknown. To
elucidate the effect of the O-linked carbohydrate on TfR function, the
oligosaccharide was eliminated by replacing Thr 104 with Asp and the
mutated cDNA was expressed in a cell line lacking endogenous TfR.
Elimination of the oligosaccharide at Thr 104 results in a form of the
receptor that is susceptible to cleavage. A 78-kD soluble TfR that can bind
transferrin is released into the growth medium. The intact mutant TfR is
not grossly altered in its structure and does not differ significantly from
the wild-type human receptor in many respects: (1) It shows the same
distribution between the plasma membrane and intracellular compartments;
(2) the binding constant for transferrin is similar to that of the
wild-type TfR; and (3) it is not rapidly degraded. Protein-sequence
analysis of the soluble form indicates that the sequence begins at amino
acid 101 of the intact receptor. This is the same cleavage site reported
for a soluble form of normal receptor found in human serum. Substitution of
Gly, Glu, or Met at position 104 also results in increased cleavage of the
TfR and suggests that elimination of the O-linked carbohydrate at position
104 enhances the susceptibility of TfR to cleavage and may mimic a
naturally occurring process previously described as being related to
erythropoiesis.
Volume 83,
Issue 2,
pp. 580-586,
01/15/1994
Copyright © 1994 by The American Society of Hematology
