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

2010; Elsevier BV; Volume: 285; Issue: 20 Linguagem: Inglês

10.1074/jbc.m109.087445

1083-351X

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

Signaling Pathways in Disease

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 Thr648 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. 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 Thr648 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. IntroductionEndoplasmic reticulum (ER) 2The abbreviations used are: ERendoplasmic reticulumaaamino acid(s)CNXcalnexinFACSfluorescence-activated cell sortingF-GRP78FLAG-GRP78TgthapsigarginPDIprotein-disulfide isomerasePBSphosphate-buffered salinerGRP78recombinant full-length GRP78. chaperones are essential for the normal function of the ER (1Ni M. Lee A.S. FEBS Lett. 2007; 581: 3641-3651Crossref PubMed Scopus (629) Google Scholar). One of the best characterized ER chaperones is the 78-kDa glucose-regulated protein (GRP78), which is also referred to as BiP or HSPA5. GRP78 is involved in many cellular processes, including translocating newly synthesized polypeptides across the ER membrane, facilitating the folding and assembly of proteins, targeting misfolded proteins for ER-associated protein degradation, regulating calcium homeostasis, and serving as an ER stress sensor (2Hendershot L.M. Mt. Sinai J. Med. 2004; 71: 289-297PubMed Google Scholar, 3Lee A.S. Methods. 2005; 35: 373-381Crossref PubMed Scopus (756) Google Scholar). GRP78 is a master regulator for ER stress due to its role as a major ER chaperone with antiapoptotic properties as well as its ability to control the activation of the unfolded protein response signaling. In the tumor microenvironment, tumor cells undergo ER stress due to hypoxia and nutrient deprivation. ER stress induction of GRP78 in cancer cells favors cell survival (4Fu Y. Lee A.S. Cancer Biol. Ther. 2006; 5: 741-744Crossref PubMed Scopus (215) Google Scholar, 5Lee A.S. Cancer Res. 2007; 67: 3496-3499Crossref PubMed Scopus (687) Google Scholar) and contributes significantly to tumor progression and drug resistance in both proliferating and dormant cancer cells, as well as tumor-associated endothelial cells (6Reddy R.K. Mao C. Baumeister P. Austin R.C. Kaufman R.J. Lee A.S. J. Biol. Chem. 2003; 278: 20915-20924Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar, 7Li J. Lee B. Lee A.S. J. Biol. Chem. 2006; 281: 7260-7270Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 8Ranganathan A.C. Zhang L. Adam A.P. Aguirre-Ghiso J.A. Cancer Res. 2006; 66: 1702-1711Crossref PubMed Scopus (254) Google Scholar, 9Pyrko P. Schönthal A.H. Hofman F.M. Chen T.C. Lee A.S. Cancer Res. 2007; 67: 9809-9816Crossref PubMed Scopus (346) Google Scholar, 10Virrey J.J. Dong D. Stiles C. Patterson J.B. Pen L. Ni M. Schönthal A.H. Chen T.C. Hofman F.M. Lee A.S. Mol. Cancer Res. 2008; 6: 1268-1275Crossref PubMed Scopus (130) Google Scholar, 11Baumeister P. Dong D. Fu Y. Lee A.S. Mol. Cancer Ther. 2009; 8: 1086-1094Crossref PubMed Scopus (78) Google Scholar).Traditionally, GRP78 is regarded as an ER lumen-localized chaperone protein due to the retrieval capacity through the KDEL retention motif present on its C terminus (12Munro S. Pelham H.R. Cell. 1986; 46: 291-300Abstract Full Text PDF PubMed Scopus (1041) Google Scholar). However, it has been reported that GRP78 can be detected in the nucleus and mitochondria (13Matsumoto A. Hanawalt P.C. Cancer Res. 2000; 60: 3921-3926PubMed Google Scholar, 14Sun F.C. Wei S. Li C.W. Chang Y.S. Chao C.C. Lai Y.K. Biochem. J. 2006; 396: 31-39Crossref PubMed Scopus (44) Google Scholar). Recently, an isoform of GRP78 generated by alternative splicing is localized to the cytosol (15Ni M. Zhou H. Wey S. Baumeister P. Lee A.S. PLoS ONE. 2009; 4: e6868Crossref PubMed Scopus (119) Google Scholar). Additionally, emerging evidence suggests that a subfraction of the GRP78 cellular pool can localize to the surface in specific cell types, in particular cancer cells (16Liu C. Bhattacharjee G. Boisvert W. Dilley R. Edgington T. Am. J. Pathol. 2003; 163: 1859-1871Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 17Arap M.A. Lahdenranta J. Mintz P.J. Hajitou A. Sarkis A.S. Arap W. Pasqualini R. Cancer Cell. 2004; 6: 275-284Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 18Liu Y. Steiniger S.C. Kim Y. Kaufmann G.F. Felding-Habermann B. Janda K.D. Mol. Pharmacol. 2007; 4: 435-447Crossref PubMed Scopus (98) Google Scholar, 19Gonzalez-Gronow M. Selim M.A. Papalas J. Pizzo S.V. Antioxid. Redox Signal. 2009; 11: 2299-2306Crossref PubMed Scopus (197) Google Scholar, 20Wang M. Wey S. Zhang Y. Ye R. Lee A.S. Antioxid. Redox Signal. 2009; 11: 2307-2316Crossref PubMed Scopus (377) Google Scholar). Global profiling of cell surface proteome of tumor cells revealed a relative abundance of heat shock chaperones and glucose-regulated proteins, including GRP78 (21Shin B.K. Wang H. Yim A.M. Le Naour F. Brichory F. Jang J.H. Zhao R. Puravs E. Tra J. Michael C.W. Misek D.E. Hanash S.M. J. Biol. Chem. 2003; 278: 7607-7616Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). The preferential expression of GRP78 on the surface of tumor cells, but not in normal organs, enables specific tumor targeting by circulating ligands as well as other cytotoxic agents for cancer therapy without harmful effect on normal tissues (17Arap M.A. Lahdenranta J. Mintz P.J. Hajitou A. Sarkis A.S. Arap W. Pasqualini R. Cancer Cell. 2004; 6: 275-284Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 18Liu Y. Steiniger S.C. Kim Y. Kaufmann G.F. Felding-Habermann B. Janda K.D. Mol. Pharmacol. 2007; 4: 435-447Crossref PubMed Scopus (98) Google Scholar, 22Kim Y. Lillo A.M. Steiniger S.C. Liu Y. Ballatore C. Anichini A. Mortarini R. Kaufmann G.F. Zhou B. Felding-Habermann B. Janda K.D. Biochemistry. 2006; 45: 9434-9444Crossref PubMed Scopus (145) Google Scholar). In another example, surface GRP78 mediates the antiangiogenic and proapoptotic activity of Kringle 5 through high affinity binding interaction of Kringle 5 with GRP78 exposed on the surface of stimulated endothelial cells and on hypoxic and cytotoxic stressed tumor cells (23Davidson D.J. Haskell C. Majest S. Kherzai A. Egan D.A. Walter K.A. Schneider A. Gubbins E.F. Solomon L. Chen Z. Lesniewski R. Henkin J. Cancer Res. 2005; 65: 4663-4672Crossref PubMed Scopus (205) Google Scholar, 24McFarland B.C. Stewart Jr., J. Hamza A. Nordal R. Davidson D.J. Henkin J. Gladson C.L. Cancer Res. 2009; 69: 5537-5545Crossref PubMed Scopus (45) Google Scholar).Although 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 (19Gonzalez-Gronow M. Selim M.A. Papalas J. Pizzo S.V. Antioxid. Redox Signal. 2009; 11: 2299-2306Crossref PubMed Scopus (197) Google Scholar, 20Wang M. Wey S. Zhang Y. Ye R. Lee A.S. Antioxid. Redox Signal. 2009; 11: 2307-2316Crossref PubMed Scopus (377) Google Scholar). It has been reported that GRP78 is an interactive partner of the low density lipoprotein receptor-related protein, and knockdown of GRP78 by small interfering RNA attenuates activated α2-macroglobulin-induced signal transduction, impacting survival and metastasis of prostate cancer cells (25Misra U.K. Gonzalez-Gronow M. Gawdi G. Hart J.P. Johnson C.E. Pizzo S.V. J. Biol. Chem. 2002; 277: 42082-42087Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 26Misra U.K. Gonzalez-Gronow M. Gawdi G. Wang F. Pizzo S.V. Cell. Signal. 2004; 16: 929-938Crossref PubMed Scopus (83) Google Scholar, 27Gonzalez-Gronow M. Cuchacovich M. Llanos C. Urzua C. Gawdi G. Pizzo S.V. Cancer Res. 2006; 66: 11424-11431Crossref PubMed Scopus (134) Google Scholar). Cripto, a multifunctional cell surface protein that is key to vertebrate embryogenesis and human tumor progression, was bound to cell surface GRP78, and blockade of this interaction prevented oncogenic Cripto signaling (28Shani G. Fischer W.H. Justice N.J. Kelber J.A. Vale W. Gray P.C. Mol. Cell. Biol. 2008; 28: 666-677Crossref PubMed Scopus (145) Google Scholar, 29Kelber J.A. Panopoulos A.D. Shani G. Booker E.C. Belmonte J.C. Vale W.W. Gray P.C. Oncogene. 2009; 28: 2324-2336Crossref PubMed Scopus (141) Google Scholar). Additionally, GRP78 associates with GPI-anchored T-cadherin on the surface of vascular endothelial cells, promoting their survival (30Philippova M. Ivanov D. Joshi M.B. Kyriakakis E. Rupp K. Afonyushkin T. Bochkov V. Erne P. Resink T.J. Mol. Cell. Biol. 2008; 28: 4004-4017Crossref PubMed Scopus (95) Google Scholar). GRP78 interacts with the major histocompatibility complex class I molecules and is implicated as a co-receptor for viral entry (31Triantafilou K. Fradelizi D. Wilson K. Triantafilou M. J. Virol. 2002; 76: 633-643Crossref PubMed Scopus (152) Google Scholar, 32Jindadamrongwech S. Thepparit C. Smith D.R. Arch. Virol. 2004; 149: 915-927Crossref PubMed Scopus (222) Google Scholar). Surface GRP78 is also required for the activation of an extrinsic apoptotic pathway mediated by extracellular Par-4 and TRAIL (33Burikhanov R. Zhao Y. Goswami A. Qiu S. Schwarze S.R. Rangnekar V.M. Cell. 2009; 138: 377-388Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar).Recently, a new 82-kDa tumor-specific variant of GRP78 containing an O-linked carbohydrate moiety specific to malignant cells was discovered (34Rauschert N. Brändlein S. Holzinger E. Hensel F. Müller-Hermelink H.K. Vollmers H.P. Lab. Investig. 2008; 88: 375-386Crossref PubMed Scopus (101) Google Scholar). This finding raises the possibility of targeting tumor cells via a specific cell surface variant of GRP78 (34Rauschert N. Brändlein S. Holzinger E. Hensel F. Müller-Hermelink H.K. Vollmers H.P. Lab. Investig. 2008; 88: 375-386Crossref PubMed Scopus (101) Google Scholar). Further, phage display-derived human monoclonal antibodies isolated by binding to the surface of live primary breast cancer cells recognize a modified form of surface GRP78 at the C terminus (35Jakobsen C.G. Rasmussen N. Laenkholm A.V. Ditzel H.J. Cancer Res. 2007; 67: 9507-9517Crossref PubMed Scopus (61) Google Scholar). Despite these advances, the extent and mechanism of relocalization of GRP78/BiP from the ER to the cell surface at the biochemical level is not well understood. Previously, through limited trypsin digestion and biochemical extractions of microsomes, we provided evidence that a subpopulation of GRP78 exists constitutively as a transmembrane protein, consistent with hydrophobicity predictions (6Reddy R.K. Mao C. Baumeister P. Austin R.C. Kaufman R.J. Lee A.S. J. Biol. Chem. 2003; 278: 20915-20924Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar). Because the ER membrane is a source for the plasma membrane, this could provide a plausible explanation for GRP78 cell surface localization. On the other hand, ER lumenal GRP78, by itself or through interacting with other proteins trafficking through the ER, could also cycle to the cell surface by escaping the ER retrieval mechanism. Here 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. The current studies provide quantitative analysis of GRP78 localization to the cell surface and reveal that this process involves multiple mechanisms. We demonstrate that ER stress can actively promote GRP78 surface expression, and overexpression of GRP78 can also lead to cell surface localization independent of ER stress. Further, based on analysis of domains of GRP78 that are exposed at the cell surface, we predict a topology for surface GRP78.DISCUSSIONIn view of the emerging importance of cell surface GRP78 in controlling cell signaling and viability, it is important to understand to what extent GRP78 is presented on the cell surface and the mechanism by which that is achieved. Studies on cell surface GRP78 expression performed in tissue culture show much variability, ranging from expression of GRP78 in various cancer cell lines to no expression in normal fibroblast cell lines (18Liu Y. Steiniger S.C. Kim Y. Kaufmann G.F. Felding-Habermann B. Janda K.D. Mol. Pharmacol. 2007; 4: 435-447Crossref PubMed Scopus (98) Google Scholar) to positive reactivity with GRP78 binding peptide motifs whether or not they are tumor cells (17Arap M.A. Lahdenranta J. Mintz P.J. Hajitou A. Sarkis A.S. Arap W. Pasqualini R. Cancer Cell. 2004; 6: 275-284Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). There are also conflicting reports of whether GRP78 is expressed on specific cell lines, such as the PC-3 prostate cancer cells (33Burikhanov R. Zhao Y. Goswami A. Qiu S. Schwarze S.R. Rangnekar V.M. Cell. 2009; 138: 377-388Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 39Misra U.K. Deedwania R. Pizzo S.V. J. Biol. Chem. 2006; 281: 13694-13707Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). The discrepancies are probably due to different techniques used to detect cell surface GRP78, which is present at very low amounts and in only a subpopulation of cells, and the divergent results could also represent intrinsic variabilities among cultured cell lines. Here we used cell surface biotinylation as the primary method for identification and isolation of cell surface GRP78, coupled with FACS analysis of living cells. Whereas previous studies primarily examined endogenous GRP78, using both polyclonal and monoclonal antibodies against GRP78, we examined in addition tagged versions of wild type and mutated GRP78, using epitope antibodies that recognize GRP78 with specificity and high affinity. Due to their high transfection efficiency, we have selected the 293T cells as the model system, with additional analysis performed in HeLa and MCF-7 cells for independent confirmation. Our studies reveal several novel findings toward understanding how GRP78, an ER lumen protein, is localized to the cell surface.We report here that in cells maintained under normal culture conditions, the amount of surface GRP78 is extremely low but nonetheless detectable with a high sensitivity assay. Substantially higher levels of GRP78 localize to the cell surface following ER stress. This is consistent with the notion that as the amount of intracellular GRP78 and other ER chaperones also bearing KDEL increases, the KDEL retrieval system may be overwhelmed such that a fraction of GRP78 escapes to the cell surface. We report here that the surface GRP78 does not simply increase in parallel with intracellular GRP78; rather, it is about 4-fold higher than the increase in intracellular GRP78, suggesting that ER stress may activate specific mechanisms for GRP78 surface localization and/or inactivate mechanisms for its ER retention.Nonetheless, ER stress is not obligatory for cell surface localization of GRP78. GRP78 surface localization is readily detected in non-stressed cells with ectopic expression of GRP78. Examination of unfolded protein response markers in the transfected cells confirmed that ER stress is not triggered. Thus, this offers an experimental system to study mechanisms for GRP78 cell localization without the complication of other factors influenced by ER stress. Here, using specific mutations of GRP78, we tested several hypotheses that could in principle account for cell surface localization of GRP78. First, we examined whether deletion of the C-terminal KDEL motif, which is known to cause secretion of GRP78 outside the cell, affects surface localization. Our results revealed that at low dosages, the fraction of surface to intracellular protein was higher for F-GRP78Δ compared with the full-length protein, suggesting that escape from the KDEL retrieval mechanism could enhance surface expression. However, why this trend was reversed remains to be determined. One speculation is that cell surface presentation of F-GRP78Δ is linked to secretion because at the lower dosages the fraction of secreted to intracellular F-GRP78Δ was also higher.The discovery of patient antisera specifically recognizing O-linked GRP78 on the surface of malignant cells (34Rauschert N. Brändlein S. Holzinger E. Hensel F. Müller-Hermelink H.K. Vollmers H.P. Lab. Investig. 2008; 88: 375-386Crossref PubMed Scopus (101) Google Scholar) and the discovery that C terminus modification of GRP78 accounts for cancer-specific antibody specificity (35Jakobsen C.G. Rasmussen N. Laenkholm A.V. Ditzel H.J. Cancer Res. 2007; 67: 9507-9517Crossref PubMed Scopus (61) Google Scholar) prompted us to test the hypothesis that the putative O-linked glycosylation site of GRP78 is within close proximity of the KDEL motif and that modification of this site could mask the KDEL motif and allow GRP78 to escape the KDEL retrieval system. Our results showed no significant change in the level or the electrophoretic mobility of surface GRP78 regardless of whether the putative glycosylation site was intact or mutated in three different cell types. It is possible that none of the cell lines that we tested possesses this O-linked glycosylation modification mechanism. It is noted that the O-linked glycosylated form of GRP78 represents a very minor fraction of total GRP78 (34Rauschert N. Brändlein S. Holzinger E. Hensel F. Müller-Hermelink H.K. Vollmers H.P. Lab. Investig. 2008; 88: 375-386Crossref PubMed Scopus (101) Google Scholar). Thus, if cell surface GRP78 also contains a very low amount of the glycosylated form, this could be a minor pathway below the detection sensibility of our assays.Previously, we reported the existence of a subpopulation of GRP78 that exists as an ER transmembrane protein with its N-terminal region exposed to the cytosol (6Reddy R.K. Mao C. Baumeister P. Austin R.C. Kaufman R.J. Lee A.S. J. Biol. Chem. 2003; 278: 20915-20924Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar). This was demonstrated by limited trypsin digestion of isolated microsomes, yielding a major resistant carboxyl band of about 35 kDa and a minor band of about 50 kDa. This was further confirmed with sodium carbonate extraction of the microsome membrane fractions, showing that GRP78 was present in both the membrane and the lumenal fractions (6Reddy R.K. Mao C. Baumeister P. Austin R.C. Kaufman R.J. Lee A.S. J. Biol. Chem. 2003; 278: 20915-20924Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar). Since ER membrane is a source of the plasma membrane, this form of GRP78 could be cycled to the cell surface. Consistent with this, we observed that calnexin, another ER transmembrane protein, is exposed to the cell surface, in agreement with an earlier report (37Okazaki Y. Ohno H. Takase K. Ochiai T. Saito T. J. Biol. Chem. 2000; 275: 35751-35758Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). In this model, the C-terminal region of GRP78 is expected to be exposed extracellularly. In support of this, studies using antibodies against the C terminus of GRP78 in FACS analysis detected surface GRP78 expression (18Liu Y. Steiniger S.C. Kim Y. Kaufmann G.F. Felding-Habermann B. Janda K.D. Mol. Pharmacol. 2007; 4: 435-447Crossref PubMed Scopus (98) Google Scholar, 33Burikhanov R. Zhao Y. Goswami A. Qiu S. Schwarze S.R. Rangnekar V.M. Cell. 2009; 138: 377-388Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Here we observed that the His epitope tagged at the C terminus of GRP78 is also exposed. However, evidence is accumulating that the N terminus of GRP78 is also exposed on the cell surface. Thus, using antibody that targets the N-terminal region of GRP78, it was demonstrated that the interaction between surface GRP78 and extracellular Par-4 and GPI-anchored Cripto and T-cadherin could be negatively affected (29Kelber J.A. Panopoulos A.D. Shani G. Booker E.C. Belmonte J.C. Vale W.W. Gray P.C. Oncogene. 2009; 28: 2324-2336Crossref PubMed Scopus (141) Google Scholar, 30Philippova M. Ivanov D. Joshi M.B. Kyriakakis E. Rupp K. Afonyushkin T. Bochkov V. Erne P. Resink T.J. Mol. Cell. Biol. 2008; 28: 4004-4017Crossref PubMed Scopus (95) Google Scholar, 33Burikhanov R. Zhao Y. Goswami A. Qiu S. Schwarze S.R. Rangnekar V.M. Cell. 2009; 138: 377-388Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Here using FACS analysis of ectopically expressed GRP78 bearing the FLAG epitope at the N terminus, we demonstrated directly that the N terminus of GRP78 is exposed. Furthermore, we discovered here that a middle domain of GRP78 is also exposed. We found no evidence that extracellular GRP78 binds stably to cell surface. Recent studies suggest that specific cell types may utilize different proteins for transporting GRP78 to the cell surface. For example, the ER transmembrane protein, MTJ-1, is implicated as the GRP78 carrier protein in macrophages (40Misra U.K. Gonzalez-Gronow M. Gawdi G. Pizzo S.V. J. Immunol. 2005; 174: 2092-2097Crossref PubMed Scopus (94) Google Scholar). Recently, the tumor suppressor Par-4 is reported to be required for GRP78 cell surface localization in PC-3 cells, although the molecular basis for translocation of Par-4 inside the ER remains to be determined (33Burikhanov R. Zhao Y. Goswami A. Qiu S. Schwarze S.R. Rangnekar V.M. Cell. 2009; 138: 377-388Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 41Lee A.S. Cancer Biol. Ther. 2009; 8: 2103-2105Crossref PubMed Scopus (20) Google Scholar). In conclusion, our studies address fundamental mechanisms for GRP78 cell surface localization and open up new areas of investigations for partner protein complexes for its surface localization because it is evident that surface GRP78 plays critical roles in cell signaling, proliferation, and survival and has great potential for therapeutic interventions. IntroductionEndoplasmic reticulum (ER) 2The abbreviations used are: ERendoplasmic reticulumaaamino acid(s)CNXcalnexinFACSfluorescence-activated cell sortingF-GRP78FLAG-GRP78TgthapsigarginPDIprotein-disulfide isomerasePBSphosphate-buffered salinerGRP78recombinant full-length GRP78. chaperones are essential for the normal function of the ER (1Ni M. Lee A.S. FEBS Lett. 2007; 581: 3641-3651Crossref PubMed Scopus (629) Google Scholar). One of the best characterized ER chaperones is the 78-kDa glucose-regulated protein (GRP78), which is also referred to as BiP or HSPA5. GRP78 is involved in many cellular processes, including translocating newly synthesized polypeptides across the ER membrane, facilitating the folding and assembly of proteins, targeting misfolded proteins for ER-associated protein degradation, regulating calcium homeostasis, and serving as an ER stress sensor (2Hendershot L.M. Mt. Sinai J. Med. 2004; 71: 289-297PubMed Google Scholar, 3Lee A.S. Methods. 2005; 35: 373-381Crossref PubMed Scopus (756) Google Scholar). GRP78 is a master regulator for ER stress due to its role as a major ER chaperone with antiapoptotic properties as well as its ability to control the activation of the unfolded protein response signaling. In the tumor microenvironment, tumor cells undergo ER stress due to hypoxia and nutrient deprivation. ER stress induction of GRP78 in cancer cells favors cell survival (4Fu Y. Lee A.S. Cancer Biol. Ther. 2006; 5: 741-744Crossref PubMed Scopus (215) Google Scholar, 5Lee A.S. Cancer Res. 2007; 67: 3496-3499Crossref PubMed Scopus (687) Google Scholar) and contributes significantly to tumor progression and drug resistance in both proliferating and dormant cancer cells, as well as tumor-associated endothelial cells (6Reddy R.K. Mao C. Baumeister P. Austin R.C. Kaufman R.J. Lee A.S. J. Biol. Chem. 2003; 278: 20915-20924Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar, 7Li J. Lee B. Lee A.S. J. Biol. Chem. 2006; 281: 7260-7270Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 8Ranganathan A.C. Zhang L. Adam A.P. Aguirre-Ghiso J.A. Cancer Res. 2006; 66: 1702-1711Crossref PubMed Scopus (254) Google Scholar, 9Pyrko P. Schönthal A.H. Hofman F.M. Chen T.C. Lee A.S. Cancer Res. 2007; 67: 9809-9816Crossref PubMed Scopus (346) Google Scholar, 10Virrey J.J. Dong D. Stiles C. Patterson J.B. Pen L. Ni M. Schönthal A.H. Chen T.C. Hofman F.M. Lee A.S. Mol. Cancer Res. 2008; 6: 1268-1275Crossref PubMed Scopus (130) Google Scholar, 11Baumeister P. Dong D. Fu Y. Lee A.S. Mol. Cancer Ther. 2009; 8: 1086-1094Crossref PubMed Scopus (78) Google Scholar).Traditionally, GRP78 is regarded as an ER lumen-localized chaperone protein due to the retrieval capacity through the KDEL retention motif present on its C terminus (12Munro S. Pelham H.R. Cell. 1986; 46: 291-300Abstract Full Text PDF PubMed Scopus (1041) Google Scholar). However, it has been reported that GRP78 can be detected in the nucleus and mitochondria (13Matsumoto A. Hanawalt P.C. Cancer Res. 2000; 60: 3921-3926PubMed Google Scholar, 14Sun F.C. Wei S. Li C.W. Chang Y.S. Chao C.C. Lai Y.K. Biochem. J. 2006; 396: 31-39Crossref PubMed Scopus (44) Google Scholar). Recently, an isoform of GRP78 generated by alternative splicing is localized to the cytosol (15Ni M. Zhou H. Wey S. Baumeister P. Lee A.S. PLoS ONE. 2009; 4: e6868Crossref PubMed Scopus (119) Google Scholar). Additionally, emerging evidence suggests that a subfraction of the GRP78 cellular pool can localize to the surface in specific cell types, in particular cancer cells (16Liu C. Bhattacharjee G. Boisvert W. Dilley R. Edgington T. Am. J. Pathol. 2003; 163: 1859-1871Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 17Arap M.A. Lahdenranta J. Mintz P.J. Hajitou A. Sarkis A.S. Arap W. Pasqualini R. Cancer Cell. 2004; 6: 275-284Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 18Liu Y. Steiniger S.C. Kim Y. Kaufmann G.F. Felding-Habermann B. Janda K.D. Mol. Pharmacol. 2007; 4: 435-447Crossref PubMed Scopus (98) Google Scholar, 19Gonzalez-Gronow M. Selim M.A. Papalas J. Pizzo S.V. Antioxid. Redox Signal. 2009; 11: 2299-2306Crossref PubMed Scopus (197) Google Scholar, 20Wang M. Wey S. Zhang Y. Ye R. Lee A.S. Antioxid. Redox Signal. 2009; 11: 2307-2316Crossref PubMed Scopus (377) Google Scholar). Global profiling of cell surface proteome of tumor cells revealed a relative abundance of heat shock chaperones and glucose-regulated proteins, including GRP78 (21Shin B.K. Wang H. Yim A.M. Le Naour F. Brichory F. Jang J.H. Zhao R. Puravs E. Tra J. Michael C.W. Misek D.E. Hanash S.M. J. Biol. Chem. 2003; 278: 7607-7616Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). The preferential expression of GRP78 on the surface of tumor cells, but not in normal organs, enables specific tumor targeting by circulating ligands as well as other cytotoxic agents for cancer therapy without harmful effect on normal tissues (17Arap M.A. Lahdenranta J. Mintz P.J. Hajitou A. Sarkis A.S. Arap W. Pasqualini R. Cancer Cell. 2004; 6: 275-284Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 18Liu Y. Steiniger S.C. Kim Y. Kaufmann G.F. Felding-Habermann B. Janda K.D. Mol. Pharmacol. 2007; 4: 435-447Crossref PubMed Scopus (98) Google Scholar, 22Kim Y. Lillo A.M. Steiniger S.C. Liu Y. Ballatore C. Anichini A. Mortarini R. Kaufmann G.F. Zhou B. Felding-Habermann B. Janda K.D. Biochemistry. 2006; 45: 9434-9444Crossref PubMed Scopus (145) Google Scholar). In another example, surface GRP78 mediates the antiangiogenic and proapoptotic activity of Kringle 5 through high affinity binding interaction of Kringle 5 with GRP78 exposed on the surface of stimulated endothelial cells and on hypoxic and cytotoxic stressed tumor cells (23Davidson D.J. Haskell C. Majest S. Kherzai A. Egan D.A. Walter K.A. Schneider A. Gubbins E.F. Solomon L. Chen Z. Lesniewski R. Henkin J. Cancer Res. 2005; 65: 4663-4672Crossref PubMed Scopus (205) Google Scholar, 24McFarland B.C. Stewart Jr., J. Hamza A. Nordal R. Davidson D.J. Henkin J. Gladson C.L. Cancer Res. 2009; 69: 5537-5545Crossref PubMed Scopus (45) Google Scholar).Although 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 (19Gonzalez-Gronow M. Selim M.A. Papalas J. Pizzo S.V. Antioxid. Redox Signal. 2009; 11: 2299-2306Crossref PubMed Scopus (197) Google Scholar, 20Wang M. Wey S. Zhang Y. Ye R. Lee A.S. Antioxid. Redox Signal. 2009; 11: 2307-2316Crossref PubMed Scopus (377) Google Scholar). It has been reported that GRP78 is an interactive partner of the low density lipoprotein receptor-related protein, and knockdown of GRP78 by small interfering RNA attenuates activated α2-macroglobulin-induced signal transduction, impacting survival and metastasis of prostate cancer cells (25Misra U.K. Gonzalez-Gronow M. Gawdi G. Hart J.P. Johnson C.E. Pizzo S.V. J. Biol. Chem. 2002; 277: 42082-42087Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 26Misra U.K. Gonzalez-Gronow M. Gawdi G. Wang F. Pizzo S.V. Cell. Signal. 2004; 16: 929-938Crossref PubMed Scopus (83) Google Scholar, 27Gonzalez-Gronow M. Cuchacovich M. Llanos C. Urzua C. Gawdi G. Pizzo S.V. Cancer Res. 2006; 66: 11424-11431Crossref PubMed Scopus (134) Google Scholar). Cripto, a multifunctional cell surface protein that is key to vertebrate embryogenesis and human tumor progression, was bound to cell surface GRP78, and blockade of this interaction prevented oncogenic Cripto signaling (28Shani G. Fischer W.H. Justice N.J. Kelber J.A. Vale W. Gray P.C. Mol. Cell. Biol. 2008; 28: 666-677Crossref PubMed Scopus (145) Google Scholar, 29Kelber J.A. Panopoulos A.D. Shani G. Booker E.C. Belmonte J.C. Vale W.W. Gray P.C. Oncogene. 2009; 28: 2324-2336Crossref PubMed Scopus (141) Google Scholar). Additionally, GRP78 associates with GPI-anchored T-cadherin on the surface of vascular endothelial cells, promoting their survival (30Philippova M. Ivanov D. Joshi M.B. Kyriakakis E. Rupp K. Afonyushkin T. Bochkov V. Erne P. Resink T.J. Mol. Cell. Biol. 2008; 28: 4004-4017Crossref PubMed Scopus (95) Google Scholar). GRP78 interacts with the major histocompatibility complex class I molecules and is implicated as a co-receptor for viral entry (31Triantafilou K. Fradelizi D. Wilson K. Triantafilou M. J. Virol. 2002; 76: 633-643Crossref PubMed Scopus (152) Google Scholar, 32Jindadamrongwech S. Thepparit C. Smith D.R. Arch. Virol. 2004; 149: 915-927Crossref PubMed Scopus (222) Google Scholar). Surface GRP78 is also required for the activation of an extrinsic apoptotic pathway mediated by extracellular Par-4 and TRAIL (33Burikhanov R. Zhao Y. Goswami A. Qiu S. Schwarze S.R. Rangnekar V.M. Cell. 2009; 138: 377-388Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar).Recently, a new 82-kDa tumor-specific variant of GRP78 containing an O-linked carbohydrate moiety specific to malignant cells was discovered (34Rauschert N. Brändlein S. Holzinger E. Hensel F. Müller-Hermelink H.K. Vollmers H.P. Lab. Investig. 2008; 88: 375-386Crossref PubMed Scopus (101) Google Scholar). This finding raises the possibility of targeting tumor cells via a specific cell surface variant of GRP78 (34Rauschert N. Brändlein S. Holzinger E. Hensel F. Müller-Hermelink H.K. Vollmers H.P. Lab. Investig. 2008; 88: 375-386Crossref PubMed Scopus (101) Google Scholar). Further, phage display-derived human monoclonal antibodies isolated by binding to the surface of live primary breast cancer cells recognize a modified form of surface GRP78 at the C terminus (35Jakobsen C.G. Rasmussen N. Laenkholm A.V. Ditzel H.J. Cancer Res. 2007; 67: 9507-9517Crossref PubMed Scopus (61) Google Scholar). Despite these advances, the extent and mechanism of relocalization of GRP78/BiP from the ER to the cell surface at the biochemical level is not well understood. Previously, through limited trypsin digestion and biochemical extractions of microsomes, we provided evidence that a subpopulation of GRP78 exists constitutively as a transmembrane protein, consistent with hydrophobicity predictions (6Reddy R.K. Mao C. Baumeister P. Austin R.C. Kaufman R.J. Lee A.S. J. Biol. Chem. 2003; 278: 20915-20924Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar). Because the ER membrane is a source for the plasma membrane, this could provide a plausible explanation for GRP78 cell surface localization. On the other hand, ER lumenal GRP78, by itself or through interacting with other proteins trafficking through the ER, could also cycle to the cell surface by escaping the ER retrieval mechanism. Here 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. The current studies provide quantitative analysis of GRP78 localization to the cell surface and reveal that this process involves multiple mechanisms. We demonstrate that ER stress can actively promote GRP78 surface expression, and overexpression of GRP78 can also lead to cell surface localization independent of ER stress. Further, based on analysis of domains of GRP78 that are exposed at the cell surface, we predict a topology for surface GRP78.