COPI-mediated retrograde trafficking from the Golgi to the ER regulates EGFR nuclear transport

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Abstract

Emerging evidence indicates that cell surface receptors, such as the entire epidermal growth factor receptor (EGFR) family, have been shown to localize in the nucleus. A retrograde route from the Golgi to the endoplasmic reticulum (ER) is postulated to be involved in the EGFR trafficking to the nucleus; however, the molecular mechanism in this proposed model remains unexplored. Here, we demonstrate that membrane-embedded vesicular trafficking is involved in the nuclear transport of EGFR. Confocal immunofluorescence reveals that in response to EGF, a portion of EGFR redistributes to the Golgi and the ER, where its NH2-terminus resides within the lumen of Golgi/ER and COOH-terminus is exposed to the cytoplasm. Blockage of the Golgi-to-ER retrograde trafficking by brefeldin A or dominant mutants of the small GTPase ADP-ribosylation factor, which both resulted in the disassembly of the coat protein complex I (COPI) coat to the Golgi, inhibit EGFR transport to the ER and the nucleus. We further find that EGF-dependent nuclear transport of EGFR is regulated by retrograde trafficking from the Golgi to the ER involving an association of EGFR with γ-COP, one of the subunits of the COPI coatomer. Our findings experimentally provide a comprehensive pathway that nuclear transport of EGFR is regulated by COPI-mediated vesicular trafficking from the Golgi to the ER, and may serve as a general mechanism in regulating the nuclear transport of other cell surface receptors.

Research highlights

ARF1 activation is involved in the EGFR transport to the ER and the nucleus. ► Assembly of γ-COP coatomer mediates EGFR transport to the ER and the nucleus. ► Golgi-to-ER retrograde trafficking regulates nuclear transport of EGFR.

Introduction

Endocytosis is characterized by membrane and vesicular trafficking along the secretory pathway, which transports budding vesicles from a donor membrane and then fuses them with an acceptor organelle. Luminal and membrane cargo proteins are carried by budding vesicles and sorted into destinations regulated by distinct assemblies of coat proteins [1]. Endosomal trafficking after endocytosis to the biosynthetic/secretory compartments, such as the endoplasmic reticulum (ER) and the Golgi apparatus, known as retrograde transport, is important for diverse cellular functions [2]. Several mammalian cargo proteins and exogenous viruses/toxins are routed from the early endosomes to the Golgi apparatus and the ER, respectively, by retrograde transport [2], [3], [4].

In eukaryotes, vesicular transport from the Golgi to the ER via a retrograde route is mediated by coat protein complex I (COPI) vesicles, which consist of the small GTPase ADP-ribosylation factor (ARF) and coatomer composed of seven subunits (α, β, β′, γ, δ, ε, and ζ) [5]. COPI coat assembly is initiated by the membrane recruitment and activation of ARF1, with GDP-GTP exchange mediated by ARF-guanine exchange factors [6]. Membrane-bound ARF1 recruits a preassembled coatomer complex, and cargo proteins are then recruited through multiple recognition sites on separate subunits [7]. Ultimately, disassembly from the membrane occurs when ARF-GTPase-activating protein hydrolyzes the GTP on ARF1 [7], [8].

Multiple cell surface receptor tyrosine kinases (RTKs), such as insulin-like growth factor 1 receptor (IGF-1R), cMet, fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), and the four members of the epidermal growth factor receptor (EGFR) family, have been reported to localize in the nucleus [9], [10], [11], [12], [13], [14]. Among these, nuclear EGFR/ErbB-1 has been shown to be involved in transcriptional regulation, cell proliferation, DNA repair, DNA replication, and chemo- and radio-resistance [15], [16], [17], [18], [19], [20], [21]. It has been reported that endocytosis is required for the nuclear translocation of EGFR [16], [22], [23]. However, after endocytosis, it is unclear which pathway routes EGFR proteins to the nucleus. In this study, we found that EGF-dependent nuclear transport of EGFR is regulated by a retrograde trafficking from the Golgi to the ER involving an association of EGFR with γ-COP.

Section snippets

Experimental reagents

The following antibodies and chemicals were purchased for our study: anti-EGFR antibodies (Santa Cruz; NeoMarker); anti-calregulin, anti-calnexin, and anti-γ-COP antibodies (Santa Cruz); anti-tubulin antibodies, mouse IgG, brefeldin A, and recombinant human EGF (Sigma); anti-lamin B antibodies (Calbiochem); and Texas red-labeled EGF (Molecular Probes). siRNA oligonucleotides targeting γ-COP and nonspecific siRNA control were purchased from Drarmacon. The GalNAc-T2-GFP plasmid was a gift from

Distribution and orientation of EGFR in the Golgi and ER membrane for EGF response in vivo

Previous studies were postulated that the nuclear transport of EGFR is via a retrograde route from the Golgi to the ER [25], [26]. If the nuclear transport of EGFR requires a membrane-bound environment involving the intracellular organelles such as the Golgi/ER, we would expect the COOH– and NH2-terminal domains of EGFR to reside on opposite sides of the membrane of these organelles and the nucleus. To address this issue, we sought to determine the orientation of EGFR in human breast carcinoma

Acknowledgments

We thank Dr. B. Storrie (University of Arkansas for Medical Sciences) for providing the GalNAc-T2-GFP plasmid and Dr. J.S. Bonifacino (National Institutes of Health) for the ARF1/wt, ARF1/T31N, and ARF1/Q71L expression vectors. This study was partially funded by the National Institutes of Health Grants RO1 109311 and PO1 099031; the National Breast Cancer Foundation, Inc.; the Sister Institutional fund from China Medical University Hospital and M.D. Anderson Cancer Center (to M.-C.H.); and

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    1

    Present address: Texas Children’s Cancer Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA.

    2

    These authors contributed equally.

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