The Transferrin Receptor Cytoplasmic Domain Determines Its Rate of Transport through the Biosynthetic Pathway and Its Susceptibility to Cleavage Early in the Pathway

Elizabeth A Rutledge,Isabelle Gaston,Barbara J Root,Timothy E Mcgraw,Caroline A Enns

doi:10.1074/jbc.273.20.12169

Abstract

The soluble human transferrin receptor (TfR) found in blood is the result of a proteolytic cleavage occurring in the ectodomain of the receptor close to the transmembrane domain at Arg-100. We have discovered another cleavage site between Gly-91 and Val-92 even closer to the transmembrane domain. Cleavage at Gly-91 differs markedly from the normal cleavage site. It occurs when the entire cytoplasmic portion or the proximal 31 amino acids of the transmembrane domain are deleted. A soluble disulfide-bonded dimer of the TfR is released into the medium in contrast to the cleavage at Arg-100 where a dimer lacking intersubunit disulfide bonds is released. Whereas the cleavage at Arg-100 is generated by cycling through the endosomal system, pulse-chase experiments indicate that cleavage at Gly-91 occurs predominantly during the biosynthesis of the receptor. Pulse-chase analysis of the biosynthesis of mutant TfRs that lack the membrane-proximal cytoplasmic domain show that they exit the endoglycosidase H-sensitive compartment at a slower rate than the wild type TfR. These results suggest that the cytoplasmic domain influences the trafficking of the TfR either by influencing the folding of the ectodomain or by providing a positive signal for its transport through the biosynthetic pathway.

Highlights

The soluble human transferrin receptor (TfR) found in blood is the result of a proteolytic cleavage occurring in the ectodomain of the receptor close to the transmembrane domain at Arg-100
Pulse-chase analysis of the biosynthesis of mutant TfRs that lack the membrane-proximal cytoplasmic domain show that they exit the endoglycosidase H-sensitive compartment at a slower rate than the wild type TfR. These results suggest that the cytoplasmic domain influences the trafficking of the TfR either by influencing the folding of the ectodomain or by providing a positive signal for its transport through the biosynthetic pathway
While examining the trafficking of various mutated TfRs, we discovered a different cleavage site in the ectodomain of the TfR which results in the release of a soluble disulfide-bonded dimer of the TfR

Summary

Introduction

The soluble human transferrin receptor (TfR) found in blood is the result of a proteolytic cleavage occurring in the ectodomain of the receptor close to the transmembrane domain at Arg-100. Pulse-chase analysis of the biosynthesis of mutant TfRs that lack the membrane-proximal cytoplasmic domain show that they exit the endoglycosidase H-sensitive compartment at a slower rate than the wild type TfR. These results suggest that the cytoplasmic domain influences the trafficking of the TfR either by influencing the folding of the ectodomain or by providing a positive signal for its transport through the biosynthetic pathway. The longer the TfR spends in the endo H-sensitive compartment (e.g. ER/cis-Golgi), the greater the extent of cleavage at Gly-91 These results imply that the region of the cytoplasmic domain of the TfR proximal to the membrane is required for rapid transit through the early biosynthetic compartments. The deletion of amino acids 29 –59 increases cleavage of the TfR by slowing its transit through the early compartments, thereby increasing the time the TfR is in the compartment containing the protease

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Results

Conclusion