GRP78: Mechanisms & Interactions
Immunofluorescent detection of GRP78 in heat shocked HeLa cells using Anti-GRP78 polyclonal antibody at 1:100
As noted, ER stress elicits numerous intracellular pathways collectively termed the unfolded protein response (UPR). This corrective pathway reconstitutes the proteostasis equilibrium by increasing the ER folding capacity as well as decreasing the incoming protein client load (for a review see 48, 170-172). The UPR covers three basic signaling pathways triggered by three ER sensors located to the ER membrane (Figure 4): (i) protein kinase RNA (PKR)-like ER kinase (PERK); (ii) activating transcription factor 6 (ATF-6); and (iii) inositol-requiring enzyme 1 (IRE-1). In their inactive state, these sensors constitute stable complexes with Grp78/BiP at the luminal site of the ER membrane thus blocking the UPR under normal conditions. In case of an ER luminal overload of unfolded proteins, Grp78/BiP is released from the sensors and binds to hydrophobic patches on nascent polypeptides followed by homodimerization and autophosphorylation of PERK and IRE-1 173, culminating in the activation of these signaling pathways. In contrast, activation of ATF-6 obviously occurs via alternative glycosylation of the sensor leading to UPR initiation 174. In any case, activated sensors ultimately act as transcription factors triggering the transcription of several effector genes, including, e.g., those encoding ER chaperones, proteins involved in lipid biosynthesis, amino acid regulation, redox homeostasis, and apoptosis 175-178.
In detail, ATF-6 is a type II ER transmembrane protein exposing a stress-sensing domain to the ER lumen. In response to ER stress, ATF-6 is released from Grp78/BiP and translocates to the Golgi where it is subjected to enzymatic cleavage by site-1 and site-2 proteases (S1P and S2P) 179, 180. An N-terminal fragment of ATF-6 is then released and translocates to the nucleus, subsequently acting as a transcription factor in order to up-regulate the expression of various proteins such as Grp78/BiP itself and those implicated in lipid biosynthesis (see above). Grp78/BiP dissociation also leads to dimerization and trans-autophosphorylation of PERK followed by the phosphorylation and thus activation of the eukaryotic initiation factor 2α (eIF-2α). Phosphorylated eIF2α (eIF-2αP) is known to block the guanine nucleotide exchange capacity of the guanine nucleotide exchange factor eIF-2B 48, 170-172, 181 thereby blocking ribosomal function and protein synthesis, a crucial constituent of stress adaption. However, eIF-2αP selectively up-regulates the expression of other key molecules involved in ER stress pathways such as the transcription factor ATF-4. The corresponding mRNAs harbor an internal ribosome entry site (IRES) located to the 5ʹ-untranslated region 182. ATF-4 was noted to increase the expression of a broad spectrum of molecules such as the CCAAT-enhancer-binding protein homologous protein (CHOP) 183, growth arrest and DNA damage inducible protein 34 (GADD34) 184 as well as proteins engaged in apoptosis, macroautophagy, amino acid regulation, and redox homeostasis 183, 185. It is interesting to note that GADD34 and others are able to dephosphorylate eIF-2αP, thus providing a negative feedback mechanism in the phosphorylation of eIF-2α 186.
Inositol-requiring enzyme 1 (IRE-1) represents a highly conserved ER stress sensor with two isoforms in mammals. While IRE-1α is ubiquitiously expressed, IRE-1β is preferentially expressed in the epithelial lining of the lungs and gut 187, 188. Like PERK, IRE-1 dimerizes and autophosphorylates upon sensing unfolded/misfolded polypeptides after dissociation from Grp78/BiP. Both IRE-1 isoforms show an intrinsic endonuclease activity able to cleave numerous mRNA species. However, IRE-1 specifically cleaves Xbp-1 in higher eukaryotes. Whereas the spliced XBP1 mRNA is translated into an active transcription factor, the unspliced XBP1 mRNA is translated into a strong UPR inhibitor 189. Xbp-1 up-regulates the expression of folding chaperones (e.g., Grp78/BiP, ERdj4) as well as ERAD constituents and stimulates phospholipid synthesis and ER size 190-192. From these observations one can conclude that the IRE-1 pathway serves as a common reply to ER stress by enhancing ER volume and folding capacity as well as blocking the up-take of further polypeptides into the ER.
Evidence increases to demonstrate that IRE-1α is also connected to the innate immune system. IRE-1αP has been identified to associate with TNF receptor associated factor 2 (TRAF-2) and -6 (TRAF-6), thus coupling the UPR to several inflammatory processes including the c-Jun N-terminal kinase (JNK) pathway 193. As summarized by Judith Smith, ER stress and the UPR impact innate immune signaling and cytokine production at different levels (Figure 4) 170. Briefly, (i) ER stress and the UPR activate multiple pattern recognition receptors (PRRs), e.g., stimulator of interferon gene (STING), NLRP3, and other inflammasomes through up-regulation of thioredoxin-interacting protein (TXNIP) and reactive oxygen species (ROS) as well as the NOD-1/2 receptors. (ii) The UPR triggers inflammatory signaling cascades resulting in activation of mitogen-activated protein kinases (p38MAPK, ERK) as well as phosphorylation and degradation of inhibitory factor κB (IκB), the negative regulator of NF-κB. (iii) The UPR activates pro-inflammatory and IFN-regulatory transcription factors such as activator protein 1 (AP-1), NF-κB, and IRF-3. ‘Core’ UPR-generated transcription factors including CHOP and Xbp-1 are also able to directly activate cytokine production by interacting with cytokine promoter and enhancer elements 170. Apart from the Grp78/BiP-dependent activation of the UPR, Grp78/BiP-independent mechanisms of UPR activation can be observed. As summarized by Gong et al., ATF-6 and PERK can also be activated by the induction of the vascular endothelial growth factor (VEGF) receptor (VEGFR)/phospholipase C-γ (PLC-γ)/mTOR complex 1 (mTORC-1) signaling in tumors 171. Solid tumors are known to exist under hypoxic conditions and depend on adaptive signal transduction pathways such as HIF-1α, UPR, and macroautophagy to maintain protein homeostasis and energy balance. VEGF is a key pro-angiogenic factor which is induced by HIF-1α, Xbp-1, ATF-6, and ATF-4, and secreted in an autocrine/paracrine manner. Liganded VEGFR has been shown to induce PLC-γ activation followed by phosphorylation of mTORC-1 which in turn activates ATF-6 and PERK. Consequently, further activating of UPR and mTORC-2 occurs 171. The dissociation of Grp78/BiP from UPR sensors is not a prerequisite for the activation of the VEGF/VEGFR/PLC-γ/mTORC-1 pathway in tumors, as demonstrated by Urra and Hetz 194. VEGF/VEGFR interactions also activate the phosphoinositide-dependent kinase 1 (PDK1)-dependent as well as the mTORC-2-dependent phosphorylation of AKT, which modulates endothelial cell survival and angiogenesis 194. Nevertheless, ER stress induces phosphorylation of Rictor, the core component of mTORC-2, through glycogen synthase kinase 3β (GSK-3β), thereby affecting AKT/mTORC-2 binding and AKT phosphorylation 171, 195. Studies by the group of Theodore Fotsis clearly defined that glucagon enhanced protein kinase A (PKA)-mediated phosphorylation and activation of IRE-1α 196. From these findings one can hypothesize that the ER and the UPR machinery constitute components of the VEGF signaling circuit regulating endothelial cell survival and angiogenesis, thereby extending their role beyond adaptation to ER stress 195, 196.