Glucose‐Regulated Protein (GRP78) Is an Important Cell Surface Receptor

Received: 3 March 2021 Accepted: 1 May 2021 DOI: 10.1002/iub.2502

CRITICAL REVIEW

Glucose-regulated protein (GRP78) is an important cell surface receptor for viral invasion, cancers, and neurological disorders

Mario Gonzalez-Gronow1,2 | Udhayakumar Gopal2 | Richard C. Austin3 | Salvatore V. Pizzo2

1Department of Biological Sciences, Laboratory of Environmental Abstract Neurotoxicology, Faculty of Medicine, The 78 kDa glucose-regulated protein (GRP78) is an endoplasmic reticulum Universidad Catolica del Norte, (ER)-resident molecular chaperone. GRP78 is a member of the 70 kDa heat Coquimbo, Chile shock family of proteins involved in correcting and clearing misfolded proteins 2Department of Pathology, Duke University Medical Center, Durham, in the ER. In response to cellular stress, GRP78 escapes from the ER and North Carolina moves to the plasma membrane where it (a) functions as a receptor for many 3 Department of Medicine, Division of ligands, and (b) behaves as an autoantigen for autoantibodies that contribute Nephrology, McMaster University and The Research Institute of St. Joseph's to human disease and cancer. Cell surface GRP78 (csGRP78) associates with Hamilton, Hamilton, Ontario, Canada the major histocompatibility complex class I (MHC-I), and is the port of entry for several viruses, including the predictive binding of the novel SARS-CoV-2. Correspondence Mario Gonzalez-Gronow, Department of Furthermore, csGRP78 is found in association with partners as diverse as the Biological Sciences, Laboratory of teratocarcinoma-derived growth factor 1 (Cripto), the melanocortin-4 receptor Environmental Neurotoxicology, Faculty (MC4R) and the DnaJ-like protein MTJ-1. CsGRP78 also serves as a receptor of Medicine, Universidad Catolica del α α Norte, Coquimbo, Chile. for a large variety of ligands including activated 2-macroglobulin ( 2M*), plas- Email: [email protected] minogen kringle 5 (K5), microplasminogen, the voltage-dependent anion channel (VDAC), tissue factor (TF), and the prostate apoptosis response-4 pro- Funding information Fondo Nacional de Desarrollo Científico y tein (Par-4). In this review, we discuss the mechanisms involved in the translo- Tecnologico de Chile, FONDECYT, Grant/ cation of GRP78 from the ER to the cell surface, and the role of secreted Award Number: 1130451 GRP78 and its autoantibodies in cancer and neurological disorders.

Abbreviations: 1-LN, human prostate cells; Akt, protein kinase B; ALS, amyotrophic lateral sclerosis; ATF6, activating transcription factor 6; B16F1, murine melanoma cells; Bcl-2, B-cell lymphoma 2 regulator of apoptosis; C-MYC, protooncogene transcription factor; Cripto, teratocarcinoma- derived growth factor 1; csGRP78, cell surface 78 kDa glucose-regulated protein; eIF2α, eukaryotic translation initiation factor 2; ER, endoplasmic reticulum; ERK 1/2, extracellular signal-regulated protein kinases 1 and 2; EV71, enterovirus 71; FOSL1, Fos-related antigen 1 encoded by the FOSL1 gene; GADD45, growth arrest and DNA-damage-inducible protein; Gαq11, guanine nucleotide-binding protein 11; HAT, histone acetyltransferase; HDAC, histone deacetylases; Hep3B, human hepatoma cells; HepG2, hepatoma cells; ID2, protein inhibitor of DNA binding; IKK, IκB kinase; IM, ionomycin; IRE1, inositol-requiring enzyme 1; K5, plasminogen kringle 5; LIMK, Lim-kinase; MC4R, melanocortin-4 receptor; MERs, middle east respiratory syndrome coronaviruses; MHC-1, major histocompatibility complex class 1; MTJ-1, DnaJ-like protein; NBD, nucleotide binding domain; NF-κB, nuclear factor kappa b; p58, DnaJ protein p58; PAK-2, serine/threonine-proteinkinase PAK 2; Par-4, prostate apoptosis response-4 protein; PDI, protein disulfide isomerase; PERK, protein kinase RNA-like endoplasmic reticulum kinase; Phospho-Ask1, apoptosis signal-regulating kinase1; PI3-K, phosphoinositide 3 kinase; Rac-1, Ras-related C3 botulinum toxin substrate 1; SBD, substrate binding domain; SIL 1, nucleotide exchange factor SIL 1; SOD1, superoxide dismutase 1; SubA, Escherichia coli subtilase cytotoxin A subunit; TF, tissue factor; TG, thapsigargin; tPA, tissue-type plasminogen activator; TRAF2, tumor necrosis receptor-associated factor 2; VDAC, voltage-dependent anion channel; Wnt/β-catenin, canonical

Wnt/β-catenin pathway; XBP-1, transcriptional factor X-box binding protein; α2M*, activated α2-macroglobulin.

KEYWORDS cancer, GRP78, GRP78 autoantibodies, GRP78 signaling, heat shock protein, neurological disorders

1 | INTRODUCTION autoantibodies, which may act as ligands that facilitate either pro-proliferative or apoptotic signaling depending GRP78, also known as heavy chain binding protein (Bip) on whether they bind to either its N-terminal or C- or heat-shock 70-kDa protein 5 (Hspa5), belongs to the terminal domains, confirming that csGRP78 functions as heat-shock protein (HSP) 70 kDa family of molecular a compartmentalized receptor with independent signal- chaperones.1 GRP78 is primarily referred to as an endo- ing pathway. Finally, we discuss how csGRP78 or ER plasmic reticulum (ER)-resident molecular chaperone GRP78 may function as neuroprotectors via finely regu- where it participates in the quality control of proteins lated mechanisms that respond expeditiously and accu- processed in the ER as well as the regulation of ER sig- rately in neurodegenerative diseases. naling induced by protein misfolding that activates the unfolded protein response (UPR).1,2 GRP78 normally functions in the ER; however, in response to cellular ER 2 | MOLECULAR MECHANISMS stress it escapes from ER retention mechanisms and AND STRESS CONDITIONS translocates to the plasma membrane,3 where it activates DIRECTING GRP78 TO THE CELL multiple signal transduction pathways.4 Expression of SURFACE csGRP78 was first reported in hamster fibroblasts in 1984,5 and confirmed later in cancer cells, where its Expression of csGRP78 is associated with increased expression is associated with increased malignant behav- malignancy in many types of cancer,14 and is potentiated ior by endowing various cancer cells with increased pro- under conditions that increase ER stress such as liferative ability, improved survival, and augmented hypoxia,9 accumulation of misfolded proteins,1 and radia- invasive and metastatic potential.6 CsGRP78 is also tion chemotherapy.15 These conditions facilitate translo- expressed in the central nervous system, where it can cation of GRP78 from the ER to the cell surface and, elicit neuroprotection by attenuating ER stress via activa- from a physiological perspective, it is important to under- tion of pro-survival signaling pathways.7 CsGRP78 serves stand the mechanisms involved. There are at least two as a receptor for a large variety of ligands as diverse different pathways mediating this process: ER Ca2 8 + as activated α2-macroglobulin (α2M*), several -depletion and accumulation of unglycosylated proteins. classes of viruses,3 plasminogen kringle 5 (K5),9 In the first pathway, a fraction of the GRP78 synthesized microplasminogen,9 major histocompatibility complex during ER stress is translocated to the cell surface under class I (MHC-I),10 the voltage-dependent anion channel conditions analogous to those induced by agents that pro- + (VDAC),9 tissue factor (TF),11 the prostate apoptosis mote Ca2 depletion such as ionomycin (IM),16 or response-4 protein (Par-4),12 and the teratocarcinoma- thapsigargin (TG).17 The second pathway influences the derived growth factor 1 (Cripto).13 accumulation of unglycosylated proteins resulting from The biological roles of csGRP78 have been briefly ER stress induced by exposure to tunicamycin-stimulated analyzed in previous reviews by other laboratories.3,7 A csGRP78 translocation.16 major aim of this review is to show most of the new evi- GRP78 is structurally composed of two major dences on the multiple roles played by csGRP78. First, domains; a nucleotide binding domain (NBD) at the we report the mechanisms involving ER stress conditions amino-terminal region and a substrate binding domain that direct GRP78 to the cell surface, followed by a (SBD) at the carboxyl-terminal region.18 GRP78 binds description on how csGRP78, as a receptor for several unfolded peptides to the SBD which requires energy to viruses, facilitates not only the virus entry into the cells, prevent aggregation by ATP hydrolysis in the NBD.18 but also their association to ER GRP78, necessary for pro- Under normal conditions, GRP78 is inactively bound to liferation and propagation of their associated diseases. three transmembrane UPR stress sensors: activating tran- Then, we discuss how csGRP78 facilitates its secretion scription factor 6 (ATF6), protein kinase RNA-like endo- via exosomes in the tumor microenvironment, promoting plasmic reticulum kinase (PERK), and inositol-requiring tumor progression in an autocrine/paracrine manner. enzyme 1 (IRE1).19 When unfolded proteins accumulate Furthermore, we also analyze how csGRP78 may in the ER lumen, GRP78 translocates from these UPR act as an autoantigen, inducing the production of sensors embedded in the ER membrane to capture the GONZALEZ-GRONOW ET AL. 845 nascent polypeptides, thereby liberating and activating site upstream from its C-terminal KDEL sequence,28 these sensors.20 The activated UPR relieves ER stress by facilitating export via vesicular transport from the ER to decreasing protein translation and increasing the folding the cell surface. Studies from our laboratory show that capacity of the ER resulting from the upregulation of csGRP78 localized in lipid raft/caveolae pits forms a ter- GRP78 and other ER-resident chaperones.20 Once acti- nary complex with MTJ-1 and the guanine nucleotide- vated, PERK phosphorylates the α-subunit of the eukary- binding protein 11 (Gαq11) that initiates signaling 30 otic translation initiation factor 2 (eIF2α) which in turn induced by α2M*. inhibits protein synthesis and reduces the influx of A small subpopulation of csGRP78 is bound to GPI- nascent proteins into the lumen of the ER.21 Once anchored proteins.4 GRP78 substrate binding activity is released from GRP78, ATF6 is shuttled from the ER to required for cell surface translocation, whereas the ATP Golgi where it is cleaved before migration to the nucleus, binding activity is not necessary.4 Based on the structural where it acts as an active transcription factor similarity between Hsp70 and GRP78, it was hypothe- upregulating expression of proteins that increase the ER sized that both proteins could be anchored on the cell folding capacity of several chaperones, including GRP78, surface as peripheral proteins by inserting the tryptophan GRP94, and protein disulfide-isomerase (PDI).22 Acti- residue contained within their SBD into the lipid vated IRE1 has endoribonuclease activity and splices a bilayer31; however, mutation of this conserved trypto- 26-base intron from the mRNA encoding the transcrip- phan has no effect on GRP78 translocation to the cell sur- tional factor X-box binding protein 1 (XBP-1),23 which face.4 We summarize all these findings in a model targets genes involved in protein folding and ER- depicting the movement of GRP78 to the cell surface dur- associated degradations.15 ing conditions of ER stress (Figure 1). GRP78 contains a C-terminal KDEL sequence which is recognized by the KDEL receptor, a seven-transmembrane-domain protein involved in retrograde transport of 3 | csGRP78 AS A MULTIFACETED protein chaperones from the Golgi back to the ER.24 RECEPTOR When GRP78 is overexpressed in the ER, a fraction of it can escape the motif retention and translocate to the cell csGRP78 exists predominantly as a peripheral protein on surface mainly because the KDEL receptors are fully sat- the plasma membrane where its interaction with other urated and allow GRP78 to escape from this retention.25 cell surface-anchored proteins affect multiple functions The KDEL receptor system plays a major role in this pro- essential to maintain cell viability. csGRP78 exhibits the cess because it controls the traffic of chaperones from the properties of a transmembrane protein with a pattern Golgi complex to the ER, acting not as a retainer of chap- suggesting that both N-terminal and C-terminal regions erones, but more as a retrieval system shepherding these are localized outside the membrane.32 Both the NBD and proteins between the Golgi and ER via retrograde path- the SBD appear to be localized on the extracellular ways, while permitting them to move toward the plasma domain; however, csGRP78 may function as a receptor membrane via anterograde transport mechanisms.26 independently from its chaperone activity where signal- The Dna-J-like transmembrane protein (MTJ-1) is ing pathways triggered by different agonists depend on necessary for csGRP78 localization.27 The mechanisms whether they bind to the N-terminal or C-terminal involved in this process were inferred from studies using regions.32 + TG, which depletes Ca2 ER stores, abolishes the reten- The csGRP78 N-terminal region comprising amino tion of GRP78, and promotes its translocation to the cell acid residues 1–50 is a receptor for the 12 amino acid surface without inducing any new synthesis.28 In the ER, angiogenic peptides ROY and ADAM15,33 and the GRP78 is normally associated with the DnaJ protein plasminogen-derived kringle 5 (K5), a potent angiogenic p58.29 However, in TG-treated cells, GRP78 is transported inhibitor, which causes apoptosis of proliferating human from the Golgi to p58-containing pre-Golgi intermediate microvascular endothelial cells.34 The csGRP78 N- compartments (IC), arresting any further transportation terminal region Leu98–Leu115 is the binding site of 35 36 to the ER. At the same time, p58 that normally cycles α2M*, the GPI-anchored T-cadherin, the GPI- between the ER, IC, and cis-Golgi, is largely depleted anchored oncogene Cripto,13 and the prostate apoptosis from the cell periphery and accumulates in large-sized IC response-4 protein (Par-4).12 The center region of GRP78 elements and vesicles in the Golgi region, demonstrating is also an independent receptor for several additional pro- that TG selectively blocks p58 recycling from the IC back teins including MHC-I,10 and the cell surface voltage- + to the ER.28 As a result, depletion of ER-Ca2 causes the dependent anion channel (VDAC).29 Furthermore, dissociation of GRP78 from p58. At this point, GRP78 csGRP78 C-terminal region is a docking region possibly forms a complex with MTJ-1 which binds to a for the coagulation protein tissue factor (TF),11 846 GONZALEZ-GRONOW ET AL.

FIGURE 1 Mechanisms and stress conditions involved in the translocation of GRP78 from the ER to the cell surface. In stressed cells the ER- transmembrane signaling molecules ATF6, IRE1, and PERK are dissociated from GRP78 initiating a cycle that induces overexpression of GRP78 that returns to the ER both overcoming the capacity of the KDEL receptor to retain GRP78 and dissociating it from the DnaJ protein p58 at the same time. At this point, free GRP78 in the ER binds MTJ-1 and Gαq11, migrating to the cell membrane in vesicles via anterograde transport mechanisms. Details are described in the text

microplasminogen,9 and the melanocortin-4 receptor viruses including the Japanese encephalitis virus,42 (MC4R) that belongs to the G-protein-coupled receptor.37 human papillomavirus 16,43 Tembusu virus,44 and Mea- sles virus.45 Similarly, the receptor for the hepatitis B virus on the cell surface has not been identified; however, 3.1 | csGRP78 as a receptor for entry of in the ER GRP78 interacts with the large surface protein viruses of this virus in vitro and in vivo,46 and regulates viral replication and survival in human hepatocytes.46 A similar One of the partners of csGRP78 is the fully assembled case is observed with the enterovirus 71 (EV71), where MHC-I molecule and both molecules in combination ER GRP78 is necessary for EV71 replication and infectiv- serve as the internalization pathway of the Coxsack- ity in human brain microvascular endothelial cells, but it ievirus A9 to mammalian cells.38 CsGRP78 is also a is not an attachment receptor for EV71 on the cell sur- receptor for dengue virus serotype 2,39 and the binding face.47 Recent studies have now demonstrated that mid- region appears to be located in the C-terminal region dle east respiratory syndrome coronaviruses (MERs) and between amino acids 467 and 530.39 CsGRP78 is also a bat coronaviruses also utilize csGRP78 for attachment cell receptor for entry of the Borna disease virus, via onto host cells.48 Using combined molecular docking and a region located in the N-terminal region,40 and the hepa- structural bioinformation, Ibrahim et al. have predicted a titis E virus, docking to a domain shared by the co- SARS-CoV-2 spike binding site on csGRP78,49 a binding chaperone MTJ1 in the csGRP78 SBD.41 Furthermore, that seems more favorable between region IV of the csGRP78 also acts as a port of entry for several additional SARS-CoV-2 spike protein and the GRP78 SBD50; GONZALEZ-GRONOW ET AL. 847 however, this prediction requires a further experimental compartmentalized receptor with independent functions validation. In summary, viruses utilize both csGRP78 for on its N-terminal and C-terminal regions. their entry into cells and ER GRP78 for proliferation and propagation of their associated diseases. 5 | MODULATION OF SIGNALING PATHWAYS TRANSDUCED BY THE 4 | csGRP78 AS AN AUTOANTIGEN csGRP78 N-TERMINAL REGION COMPARTMENT Exposure of the immune system to proteins that normally reside intracellularly induces the generation of autoanti- We identified the linear N-terminal primary amino acid bodies against a number of chaperones in patients with sequence LIGRTWNPSVQQDIFKL (Leu98–Leu115) as the 25 autoimmune diseases or malignancies. GRP78 is one of binding sequence of several agonists including α2M* and 35 the chaperones with high expression frequency affected in autoantibodies to csGRP78. Binding of α2M* to this site these pathologies. Autoantibodies to GRP78 have been induces multiple downstream signaling targets including 2+ identified in serum from patients with autoimmune pathol- Ca elevation and the IP3 cascade, which lead to activa- ogies such as rheumatoid arthritis,51 neuromyelitis tion of Serine/threonine-proteinkinase PAK 2 (PAK-2), optica,52 Lambert–Eaton myasthenic syndrome,53 Ras-related C3 botulinum toxin substrate 1 (Rac-1), Lim- atherosclerosis,54 and type 1 diabetes.55 Neurological kinase (LIMK), and cofilin in a tyrosine kinase- disorders such as multiple sclerosis, neuromyelitis optica, dependent and PI3-kinase-dependent manner.60 The neuropsychiatric systemic lupus erythematosus, and neuro- stimulatory effect of α2M* on cell proliferation after bind- Behçet diseases show the presence of GRP78 autoanti- ing to csGRP78 functions via activation of PI3-kinase, bodies, which move across the blood–brain barrier (BBB) Akt, ERK 1/2, and p38 mitogen-activated protein which to the brain.56 GRP78 autoantibodies are also present in induces an up-regulation of Bcl-2, phospho-Bad, the serum of patients with prostate, ovarian, gastric, malig- phospho-FOXO1, phospho-GSK3β, XIAP, and the nant melanoma, and colorectal cancers.32,57 GRP78 autoan- nuclear factor kappa B (NF-κB) and its upstream activa- tibodies from prostate or malignant melanoma cancer tors IKK and IκB, cyclin D1, GADD45, phospho-Ask1, patients are specific against an epitope in the N-terminal and TRAF2.61 The complex between csGRP78, the ER region and induce tumor cell proliferation.32 Furthermore, membrane Dna J-like protein MTJ-1, and the G-protein- GRP78 N-terminal region autoantibodies also accelerate alpha-11 (Gαq11),30 facilitates signaling through the development of atherosclerotic lesions.54 PI3-kinase/Akt as well as NF-κB-dependent pathways.62 Studies from our laboratory show that the humoral The association between GRP78 and Gα proteins was response to GRP78 N-terminal region increases the pro- recently confirmed by a report describing that csGRP78 is gression of cancer in a murine malignant melanoma translocated from the ER to the plasma membrane via model,58 whereas antibodies against GRP78 C-terminal interaction with a Gα- interacting vesicle-associated pro- region significantly slow tumor growth.58 The specificity tein Girdin, a member of the family of guanine nucleo- of the humoral response to different regions in csGRP78 tide exchange modulators known for its role in the was confirmed on cells using the Escherichia coli sub- activation of heterotrimeric G protein signaling down- tilase cytotoxin A subunit (SubA), a serine proteinase, stream of multiple cell surface receptors in cancer cells.62 that displays extreme substrate specificity for GRP78. Recent findings from our laboratory show that bind-

SubA cleaves GRP78 between the amino acid residues ing of α2M*to csGRP78 activates PDK1 to induce PLK1 Leu416 and Leu417, located within the hinge region phosphorylation required for c-MYC activity in proliferat- between the ATPase and C-terminal protein binding ing cancer cells.63 PLK1 could directly bind to c-MYC domains, releasing a 28-kDa GRP78 C-terminal frag- and promote transcriptional activation of FOSL1 and ID2 ment.59 Cells expressing high levels of csGR78 were incu- genes by phosphorylating histone H3S10.64 Binding of bated with SubA.59 SubA specifically cleaves csGRP78 on α2M* to csGRP78 also promotes the Warburg effect in HepG2, 1-LN, and B16F1 cells without affecting intracel- cancer cells, enhancing acetyl-CoA production and his- lular GRP78, and has no effect on Hep3B, PC3, or B16F0 tone acetylation required for tumor growth.65 cells with low levels of expression of csGRP78. The T-cadherin associates with csGRP78 and activates sig- predicted 28-kDa GRP78 C-terminal fragment is released naling pathways similar to those initiated by α2M*, pro- into the culture medium and C-terminal domain moting cell survival and proliferation, thereby suggesting signal transduction is abrogated, whereas pro- that these proteins bind to the same receptor site on the 36 proliferative signaling mediated through the N-terminal cell surface. The antibody that inhibits α2M* binding to domain is unaffected,59 confirming that csGRP78 is a csGRP78 also inhibits binding and signaling of Cripto via 848 GONZALEZ-GRONOW ET AL. csGRP78.66 The sharing of similar transduction cascades upstream from GRP78 C-terminal region in cancer cells by Cripto and α2M* suggests that both of them bind to was tentatively identified as the functional binding site of the same domain on the N-terminal region of csGRP78. the O-linked carbohydrate recognizing antibody SAM- In addition, the autoantibodies to GRP78 produced in 6,34 which is internalized via endocytosis, inducing a cancer patients stimulate cell proliferation and protect deadly cascade, starting with cytochrome c release from cancer cells from apoptosis when they bind to csGRP78, mitochondria and caspase activation, thus leading to an possibly via the N-terminal region located between Leu98 uncontrolled lipid uptake and apoptosis in cancer cells.69 and Leu115, activating signaling pathways similar to those A specific antibody raised in sheep against the GRP78 35 2+ induced by α2M*. C-terminal regions induces a rise in cytosolic Ca , differ- As described above, the N-terminal region of csGRP78 ent to that observed with a GRP78 N-terminal region is also a receptor for the secreted form of Par-4 protein antibody in human prostate cancer cells.32 The same inducing cell apoptosis and plasminogen K5, a potent antibodies stain sections of human prostate intra- angiogenic inhibitor, that causes apoptosis of microvascular epithelial neoplasma, demonstrating the availability of endothelial cells.67 Although both these proteins induce both the csGRP78 N-terminal and C-terminal regions apoptosis, Par-4 elicits the extrinsic apoptotic pathway via expressed in this tissue.32 Furthermore, the signaling activation of caspase-3 and caspase-8,34 and K5 does it via pathways induced by activation of the human csGRP78 activation of caspase-7.35 The csGRP78 N-terminal region C-terminal region were validated using three different comprising amino acid residues 1–50 is also a receptor for goat antibodies specific against this region that induce the 12 amino acid angiogenic peptide ROY, which induces apoptosis in prostate and melanoma cancer cells.70 These in vitro angiogenesis under hypoxic conditions,33 and a antibodies induce up-regulation of p53, inhibition of NF- peptide derived from metalloproteinase 15 (ADAM15) that κB1 and NF-κB2 activation, suppression of Ras/MAPK, induces VEGF-independent angiogenesis on endothelial PI3K/Akt signaling, and down-regulation of NF-κB sig- cells under hypoxic conditions.33 Another angiogenic naling pathways.71 Furthermore, the up-regulation of p53 inhibitor, isthmin 1, also interacts with the N-terminal by these antibodies also promotes activation of caspase-3, region of csGRP78.68 Table 1 shows a summary of different caspase-7, and caspase-9,72 and down-regulation of IRE- agonists that bind to the N-terminal and C-terminal α, PERK, and ATF6α-dependent signaling.70 regions of csGRP78. A region in microplasminogen including amino acid residues Ser759–Phe778 is the Pg binding region to csGRP78 C-terminal region.9 This Pg segment is homologous to the 6 | MODULATION OF SIGNALING sequence Gly100–Phe119 of Dengue virus serotype 2 viral PATHWAYS TRANSDUCED BY THE protein coat,73 suggesting that csGRP78 C-terminal region cSGRP78 C-TERMINAL REGION is the receptor for microplasminogen and dengue virus COMPARTMENT serotype 2. The peptide CTVALPGGYVRVC, homologous to the Pg sequence Ser759–Phe778, is rapidly internalized The C-terminal region Gly639–Glu650, including a possibly and induces apoptosis in malignant cancer cells either O-glycosylated Thr643, localized only 12 amino acids alone or conjugated to taxol, demonstrating that csGRP78

TABLE 1 A summary of agonists that bind to the N-terminal or C-terminal regions of csGRP78

GRP78 N-terminal region (AA 1–300) GRP78 C-terminal region (AA 300–654)

Agonist Function References Agonist Function References

α 2M* Cell proliferation 8 MicroPg Fibrinolysis 9 Roy Angiogenesis 33 TF Anticoagulation 11 ADAM15 Angiogenesis 32 VDAC Pg binding partner 9 Pg K5 Antiangiogenic 9 Dengue virus Virus entry 39 T-cadherin Binding partner 36 Hepatitis virus Virus entry 41 Cripto Cell proliferation 13 α-GRP78 Abs Apoptotic 61 Par-4 Apoptotic 12 MHC-1 Binding partner 10 Borna virus Virus entry 40 MTJ-1 Binding partner 30 GRP78 Ab Anti-apoptotic 35 α-GRP78 mAb Apoptotic 58 GONZALEZ-GRONOW ET AL. 849 is a target for delivery of peptide-drug conjugates in can- 7 | SECRETED GRP78 cer.74 Similarly, the peptides WIFPWIQL and WDL AWMFRLPVG, containing GRP78 binding motifs,75 bind Pro-inflammatory cytokines induce ER stress in pancre- the csGRP78 C-terminal region in human prostate cancer atic beta cells inducing secretion and plasma membrane cells and are rapidly internalized.76 These peptides were translocation of GRP78. In stressed pancreatic beta cells, conjugated to the cell death-inducing antimicrobial peptide the secreted GRP78 acts as a proapoptotic ligand of

(KLAKLAK)2, and the resulting chimeric peptides can sup- csGRP78, promoting caspase mediated apoptosis that press tumor growth in xenograft and isogenic mouse triggers a self-destructive feedback loop in type 1 diabe- models of prostate and breast cancer.76 tes.45 Secreted GRP78 is also able to confer resistance to In summary, enough experimental evidence from our Bortezomib, a selective proteasome inhibitor, to several laboratory and others confirms that csGRP78 is a com- cell lines including endothelial, prostate cancer, murine partmentalized receptor with independent functions on melanoma, and myeloma cells,77 and stimulates differen- its N-terminal and C-terminal regions. As shown in tiation of bone marrow mesenchymal stem cells to Figure 2, the signaling pathways elicited by docking of cancer-associated fibroblasts.78 GRP78 is also secreted by agonists to the GRP78 N-terminus are mostly associated colon cancer cells through mechanisms involving with a positive effect on cell proliferation and survival, increased GRP78 acetylation that limits its sorting to dif- whereas docking of agonists to the GRP78 C-terminus ferent compartments, thereby facilitating its secretion via are associated with negative effects, leading to apoptosis exosomes in the tumor microenvironment,79 where the and cell death. acidic pH of the microenvironment accelerates the release of GRP78, promoting tumor progression in an autocrine/paracrine manner.79 This mechanism is con- trolled by the balance between the activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs), which affect the degree of acetylation of GRP78.80 GRP78 is acetylated at 11 lysine residues and control of its secretion is largely attributable to acetylation of K633, a residue whose deacetylation by HDAC6, a cytoplasmic class II HDAC, accounts for sorting of GRP78 into exosomes and its ultimate release in the tumor microenvironment of colon cancer cells.79 Secreted GRP78 utilizes csGRP78 as its receptor, and activates downstream PI3K/Akt and Wnt/β-catenin signaling to promote cancer cell proliferation.81 GRP78 secreted by tumor cells also acts as a chemokine to recruit macrophages to further promote cytoskeleton remodeling,82 and angiogenesis,77 which accelerate tumor growth. The ER stress-induced secretion of GRP78 has been suggested to play extracellular functions in the heart including augmentation of cardiac cell viability, immune- stimulatory cell signaling, and extracellular proteostasis.83 Furthermore, secreted GRP78 is also elevated in the aqueous humor of patients with diabetic macular edema, as a result from diabetic retinopathy under ER stress conditions,84 and its circulating level has been used as a prognostic marker directly correlated to immune cell death FIGURE 2 Signaling pathways induced by N-terminal or C- and ER stress during sepsis.85 terminal csGRP78 agonists. Agonists of the N-terminal region of csGRP78 (green arrow) activate several signaling pathways including PAK-2, PI3K, PBK/Akt, BcI-2, PDK1, and c-MYC positively affecting cell growth and proliferation. Conversely, 8 | THEROLEOFcsGRP78IN agonists of the C-terminal region of csGRP78 (red arrow) negatively NEUROLOGICAL DISORDERS affecting several signaling pathways including Ras/MAPK, PI3/Akt, Ire-α, PERK, ASTF6α, and NFκB1 that inhibit cell proliferation, A critical step observed in several immune-mediated leading to cell death and apoptosis. Details are described in the text neurological diseases, including multiple sclerosis, 850 GONZALEZ-GRONOW ET AL. neuromyelitis optica, neuropsychiatric systemic lupus receptor on the cell surface, where in association with erythematosus, and neuro-Behçet diseases, is the dis- tPA decreases ER stress and neuronal death during ruption of the BBB by circulating autoantibodies to cerebral ischemia independently of tPA thrombolytic GRP78, permitting the passage of pathogenic antibodies activity.87 Furthermore, csGRP78 is also a receptor for across the BBB to the brain.56 Such autoantibodies have neuroleukinthatexertsaxonalgrowthactivityinvitro also the potential to react with csGRP78 expressed at and in vivo, playing a key role in motor function recov- high levels in neurons and astrocytes of the temporal ery in spinal cord-injured mice.90 Astrocytes also cortex and hippocampus in the brain.7 In neurons, express csGRP78,91 that may be involved in the control csGRP78 is a receptor of alpha-synuclein (Syn), a small of the balance between plasmin formation and plasmin soluble protein expressed in the brain, that forms path- elimination in the brain parenchyma.92 Astrocytes are ological aggregates named Lewy bodies observed in sev- particularly interesting because they become reactive in eral neurodegenerative disorders such as Parkinson's several neurodegenerative disorders including disease, neurodegeneration with brain iron accumula- Parkinson's, Alzheimer's, Huntington's, and prion dis- tion, and Alzheimer's disease.86 In neurons, csGRP78 eases.90 In all these disorders, activation of the UPR has also functions as a receptor of tissue-type plasminogen been detected in brain tissue and specifically the PERK/ activator (tPA).87 The expression of tPA is regulated by eIF2α-P pathway regulated by GRP78,93 emphasizing GRP78,88 and the extent of the association between the importance of neuronal expression of csGRP78. tPA/GRP78 has been correlated with the secretion of Although csGRP78 has not been involved in tPA.89 Therefore, csGRP78 plays a dual role in mecha- amyotrophic lateral sclerosis (ALS), ER GRP78 over- nisms of neuroprotection by tPA, one by promoting its expression may play a neuroprotective role in this disease. expression and secretion, and the other by serving as its Many different mutations are associated with ALS, leading

FIGURE 3 Cross-talk of signaling pathways induced by activation of csGRP78. Binding of α2M* or autoantibodies to the csGRP78 N- terminal region compartment induces several signaling pathways including ERK 1/2, PI3-K, and Akt, leading to activation of NF-κB, which induces XIAP/Bcl-2 signaling, down-regulating antiapoptotic signaling, and promoting cell proliferation. Conversely, binding of antibodies to the csGRP78 C-terminal region up-regulate apoptotic signaling and antagonize α2M* pro-proliferative mechanisms, via suppression of Ras/MAPK and PI 3-kinase/AKT, promotion of caspase activation and activation of the p53 tumor suppressor protein which blocks NF-κB signaling pathways. At the center stage of these pathways, NF-κB functions as a molecular switch controlling what circuit will predominate, either that leading to cell proliferation and cell survival or that leading to apoptosis and cell death GONZALEZ-GRONOW ET AL. 851 to protein misfolding and aggregation. One of the proteins evaluated before any therapies are attempted, to avoid a suffering mutations is superoxide dismutase 1 (SOD1), negative reactivity with csGRP78, also expressed by astro- which forms aggregates in association with ER GRP78, as cytes and neurons in the brain. In summary, a rigorous observed in microsomes from spinal cords derived from multidisciplinary research effort focusing on agents mouse models of ALS.94 Furthermore, a GRP78 co- targeting csGRP78 will significantly contribute to the chaperone protein, the nucleotide exchange factor SIL development of more effective treatments not only for 1 (SIL 1), enhances ALS pathology when deficient, while cancer, but also for neurological disorders. its overexpression promotes significant neuroprotection and reduced SOD1 aggregation in motoneurons.95 These ACKNOWLEDGMENTS observations suggest that overexpression of GRP78 would We thank Mr. Steve Conlon, PhotoPath Medical Photog- mediate neuroprotection in ALS. rapher, Duke University Medical Center, who made the final computer illustrations shown in this manuscript. This work was supported by a grant from Fondo 9 | CONCLUDING REMARKS AND Nacional de Desarrollo Científico y Tecnologico de Chile, FUTURE PERSPECTIVES FONDECYT No 1130451.

While the role of csGRP78 in cancer is well established, CONFLICT OF INTEREST the regulation of its activity under different physiologic The authors declare no conflicts of interest. conditions and its many roles in disease still need to be clarified. Expanding the knowledge on the mechanisms by ORCID Richard C. Austin which csGRP78 acts will provide important information https://orcid.org/0000-0003-0127- not only on cancer but also in autoimmune diseases and 0865 Salvatore V. Pizzo https://orcid.org/0000-0001-6202- neurological disorders. In cancer, overexpression of csGRP78 makes it a potential target for tumor therapy.96,97 3118 Both GRP78 N-terminal and C-terminal regions function as a compartmentalized receptor with independent REFERENCES 1. Zhang LH, Zhang X. Roles of GRP78 in physiology and cancer. functions. Binding of α2M* or its autoantibodies to the J Cell Biochem. 2010;110:1299–1305. csGRP78 N-terminal region compartment induces NF-κB 2. Ma Y, Hendershot IM. The role of the unfolded protein signaling pathways activation, down-regulating apoptotic response in tumour development: Friend or foe? Nat Rev Can- 34 signaling, and promoting cell proliferation. Conversely, cer. 2004;4:966–977. ligation of csGRP78 with antibodies against its C- 3. Ibrahim IM, Abdelmalek DH, Elfiky AA. GRP78: A cell's terminal region up-regulate apoptotic signaling and response to stress. Life Sci. 2019;226:156–163. antagonize α2M* pro-proliferative mechanisms, via sup- 4. Tsai YL, Zhang Y, Tseng CC, Stanciauskas R, Pinaud F, pression of Ras/MAPK and PI 3-kinase/AKT, promotion Lee AS. Characterization and mechanism of stress-induced of caspase activation, and activation of the p53 tumor translocation of 78-kilodalton glucose-regulated protein – suppressor protein which blocks NF-κB signaling path- (GRP78) to the cell surface. J Biol Chem. 2015;290:8049 8064. 5. Lee AS, Bell J, Ting J. Biochemical characterization of the 94- ways.34 At the molecular level, a key regulator of the sig- and 78-kilodalton glucose-regulated proteins in hamster fibro- naling pathways of both csGRP78 compartments appears blasts. J Biol Chem. 1984;259:4616–4621. κ to be the NF- B signaling pathway, a mechanism known 6. Lee AS. GRP78 induction in cancer: Therapeutic and prognos- to control both cell proliferation and apoptosis in cancer tic implications. Cancer Res. 2007;67:3496–3499. cells.98 Figure 3 shows a summary of some steps that par- 7. Casas C. GRP78 at the Centre of the stage in cancer and ticipate in the regulation of the signaling pathways neuroprotection. Front Neurosci. 2017;11:177. involved after activation of both csGRP78 N- or C- 8. Misra UK, Gonzalez-Gronow M, Gawdi G, Hart JP, Johnson CE, Pizzo SV. The role of GRP78 in alpha terminal regions by specific agonists such as α2M* or a 2-macroglobulin-induced signal transduction. Evidence from csGRP78 antibody. RNA interference that the low density lipoprotein receptor- The use of csGRP78 as a functional target for thera- related protein is associated with, but not necessary for, peutic applications must consider that overexpression of GRP78-mediated signal transduction. J Biol Chem. 2002;277: GRP78 on tumor cells may cause enhanced secretion 42082–42087. of GRP78, which in addition to inducing the production 9. Gonzalez-Gronow M, Kaczowka SJ, Payne S, Wang F, of autoantibodies to GRP78, may also support tumor Gawdi G, Pizzo SV. Plasminogen structural domains exhibit growth.7 Because GRP78 autoantibodies disrupt the BBB different functions when associated with cell surface GRP78 or in autoimmune diseases and neurological disorders,56 the the voltage-dependent anion channel. J Biol Chem. 2007;282: possible use of exogenous GRP78 antibodies must be 32811–32820. 852 GONZALEZ-GRONOW ET AL.

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