Elsevier

Methods

Volume 35, Issue 4, April 2005, Pages 373-381
Methods

The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress

https://doi.org/10.1016/j.ymeth.2004.10.010Get rights and content

Abstract

The multiple implications of ER stress and the unfolded protein response in health and disease highlight the importance of identifying convenient monitoring systems for its onset under various experimental or physiological settings. A large volume of studies establish that induction of GRP78 is a marker for ER stress. GRP78, also referred to as BiP, is a central regulator for ER stress due to its role as a major ER chaperone with anti-apoptotic properties as well as its ability to control the activation of transmembrane ER stress sensors (IRE1, PERK, and ATF6) through a binding-release mechanism. In the following report, we present several methods to measure GRP78 induction. This can be achieved by measuring the Grp78 promoter activity or by measuring the level of Grp78 transcripts or GRP78 protein. These techniques can be applied to tissue culture cells as well as tissues and organs.

Introduction

The endoplasmic reticulum (ER) is a perinuclear, cytoplasmic compartment where proteins and lipids are synthesized. Within the ER reside molecular chaperones that facilitate proper protein folding, maintain proteins in a folded state, and prevent protein folding intermediates from aggregating. One of the best characterized ER chaperone proteins is the glucose regulated protein (GRP78). GRP78 was first discovered as a 78,000 Da protein whose synthesis was enhanced in tissue culture cells grown in medium deprived of glucose [1]. Subsequently, GRP78 was determined to be an ER resident protein and its synthesis can be stimulated by a variety of environmental and physiological stress conditions that perturb ER function and homeostasis [2], [3]. GRP78 is also commonly referred to as BiP, the immunoglobulin heavy chain-binding protein. BiP was originally found to bind to the immunoglobulin heavy chains of pre-B cells [4]. Through analysis of proteins related to the heat shock protein family (HSP70) and using an antibody against BiP, it was discovered that BiP is identical to the previously reported GRP78; furthermore, it was determined that BiP is not restricted to B cells. In fact, the level of BiP in pre-B and B cells is comparable to that found in fibroblasts and its level is greatly elevated in antibody secreting plasma cells [5].

Another confusion that exists in literature concerning GRP78 is whether or not it is a heat shock protein. From amino acid sequence comparisons, GRP78 shares about 60% homology with HSP70, including the ATP-binding domain required for their shared chaperone function in facilitating protein folding [6]. However, GRP78 differs from HSP70 in two important aspects. First, GRP78 has a signal peptide sequence that targets it to the ER, whereas HSP70 does not contain such a sequence and is cytosolic, relocalizing to the nucleus upon stress. Second, the synthesis of GRP78 is not significantly affected by heat shock conditions that greatly elevate the level of HSP70 and other members of the heat shock protein family. In contrast, the most potent chemical inducers for GRP78 are thapsigargin (Tg), which inhibits the ER calcium ATPase pump, the calcium ionophore A23187, and tunicamycin, which blocks N-linked protein glycosylation [7], [8]. As expected, treatment of cells with azetidine, a proline analog that leads to the formation of malfolded proteins both in the cytoplasm and ER, transcriptionally activates both HSP70 and GRP78 [9], [10].

A large amount of work has established that specific induction of GRP78 is indicative of ER stress. In addition to the chemical inducers mentioned above, ER stress can occur under various physiological settings that have significant implications in health and disease [3], [11]. For example, in highly specialized secretory cells such as plasma cells and pancreatic β-cells, the ER compartment is expanded considerably and because of the high volume of protein traffic, the ER can experience accumulation of partially folded proteins that require chaperone assistance. Malfolded protein accumulation has also been associated with neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases, as well as prion protein diseases. Further, natural induction of GRP78 in multiple types of solid tumors can be attributed to glucose starvation resulting from poor perfusion within tumors as well as metabolic characteristics of cancer cells, which have much higher glucose utilization rates [12], [13].

Why is GRP78 such an integral part of ER stress and the unfolded protein response (UPR)? The answer may lie in the role of GRP78 as an ER stress signaling regulator as well as its ability to block the apoptotic process [14], [15]. The role of GRP78 in modifying UPR signaling and cell survival is summarized in Fig. 1. In mammalian cells, several ER-resident transmembrane proteins have been identified that act as transducers of ER stress signaling: the serine/threonine kinase and endoribonuclease IRE1, the PERK serine/threonine kinase (also referred to as PEK), and the basic leucine-zipper transcription factor ATF6 [11], [16]. In non-stressed cells, GRP78 binds to all three transducers which are maintained in an inactive state. Upon ER stress, all three sensors are released from GRP78. In the case of IRE1 and PERK, they homodimerize through their luminal domains, autophosphorylate their respective cytoplasmic domains, and become activated [17]. For ATF6, a fraction of it is translocated from the ER to the Golgi complex, where it is cleaved by the proteases S1P and S2P. The cleaved form of ATF6 enters the nucleus and acts as an active transcription factor for the UPR target genes, including Grp78 [18], [19], [20]. Therefore, GRP78 is a key regulator of ER stress transducers since their activation upon ER stress is dependent on their release from GRP78.

While it is well established that the ER plays an essential role in cellular homeostasis, recent discovery points to the ER as a site of convergence of both pro- and anti-apoptotic molecules and represents a novel focal point for the regulation of apoptosis [21], [22]. For survival under ER stress, the UPR shuts down general protein translation while selectively activating expression of chaperone proteins such as GRP78, which exhibits anti-apoptotic properties through interference with caspase activation and probably other yet undefined mechanisms [15], [23]. Thus, GRP78 induction under pathological conditions may represent a major cellular protective mechanism for cells to survive ER stress and could have implications in organ preservation as well as cancer progression [3].

Taken together, GRP78 induction can serve as a general indicator that the ER is undergoing stress and that the UPR is being triggered. In the following report, we present several methods to measure GRP78 induction. This can be achieved by measuring the Grp78 promoter activity, or by measuring the level of Grp78 transcripts or GRP78 protein. These techniques can be applied to tissue culture cells as well as tissues and organs.

Section snippets

Activation of the Grp78 promoter activity

GRP78 is activated by ER stress at the transcriptional level. While it is feasible to measure directly the rate of transcription of Grp78 by nuclear run-on experiments as previously described [8], a more convenient method is to measure the activity of the reporter gene linked to the Grp78 promoter. The rat Grp78 promoter is a well characterized promoter system where the localization of various cis-acting regulatory elements and trans-acting factors contributing to both the basal and ER

Assay of Grp78 transcript level

The level of Grp78 transcripts in tissue culture cells after ER stress is easily detectable by Northern blot assays, which are generally more reliable than RT-PCR measurements unless they are performed in a quantitative manner. Since the nucleic acid sequence of Grp78 cDNA is highly conserved among rat, mouse, hamster, and human, the cDNA probe from any one of these species can easily cross-hybridize with the other species [30], [31]. The size of the Grp78 transcript is about 2.7 kb and for

Measurement of GRP78 protein level

In tissue culture cells, GRP78 is generally detectable in whole cell lysates by Western blot assays and its level is elevated after prolonged stress treatment (Fig. 3). However, due to the stability of the GRP78 protein (half-life of about 72 h), the fold increase in GRP78 protein level several hours after ER stress treatment is usually less than that observed in Northern blots or promoter–reporter gene assays. Nonetheless, for study of GRP78 regulation and function, it is important to measure

Discussion

While the majority of the studies on GRP78 induction thus far has been performed in tissue culture cells, progress has been made in studying the induction of GRP78 in whole organisms during growth, differentiation, and pathological conditions. For example, through immunohistochemical staining, GRP78 was found to be highly elevated in the mouse embryonic heart, somites, and other embryonic tissues but much less in adult tissues such as the brain, heart, and lung ([36], and our unpublished

Acknowledgments

This work was supported in part by the National Institutes of Health Grant CA27607 to A.S.L. I thank members of the Lee laboratory, in particular, Min Hong, Peter Baumeister, Vince Tai, Dezheng Dong, Shengzhu Luo, and Brenda Lee for assistance.

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