Cell Surface Relocalization of the Endoplasmic Reticulum Chaperone and Unfolded Protein Response Regulator GRP78/BiP

Yi Zhang,Ren Liu,Min Ni,Parkash Gill,Amy S Lee

doi:10.1074/jbc.m109.087445

Yi Zhang, Ren Liu + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m109.087445

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Abstract

The recent discovery that GRP78/BiP, a typical endoplasmic reticulum (ER) lumenal chaperone, can be expressed on the cell surface, interacting with an increasing repertoire of surface proteins and acting as receptor in signaling pathways, represents a paradigm shift in its biological function. However, the mechanism of GRP78 trafficking from the ER to the cell surface is not well understood. Using a combination of cellular, biochemical, and mutational approaches, we tested multiple hypotheses. Here we report that ER stress actively promotes GRP78 localization on the cell surface, whereas ectopic expression of GRP78 is also able to cause cell surface relocation in the absence of ER stress. Moreover, deletion of the C-terminal ER retention motif in GRP78 alters its cell surface presentation in a dose-dependent manner; however, mutation of the putative O-linked glycosylation site Thr(648) of human GRP78 is without effect. We also identified the exposure of multiple domains of GRP78 on the cell surface and determined that binding of extracellular GRP78 to the cell surface is unlikely. A new topology model for cell surface GRP78 is presented.

Highlights

The physiological function of cell surface GRP78 is still emerging, evidence is accumulating that GRP78 can form cell surface complexes with specific proteins that in turn play an important role in signal transduction [19, 20]
We report on the characterization of cell surface relocalization of GRP78 through a combination of cellular, biochemical, and mutational approaches and test various hypotheses that could help explain how GRP78 is expressed at the cell surface
We demonstrate that endoplasmic reticulum (ER) stress can actively promote GRP78 surface expression, and overexpression of GRP78 can lead to cell surface localization independent of ER stress

Summary

Introduction

Construction of Expression Plasmids for Epitope-tagged GRP78 Bearing Specific Mutations—Human GRP78 is a highly conserved protein containing 654 amino acids, which include ER signal peptide (aa 1–18) at the N terminus, an ATPase domain (aa 125–280), a peptide-binding domain (aa 400 –500), and a KDEL motif (aa 651– 654) at the C terminus (Fig. 1). These plasmids, following transfection into 293T cells, produced FLAG-tagged wild type and mutant GRP78 proteins readily detectable in the cell lysates (Fig. 2A).

Results

Conclusion