title: The Escherichia coli subtilase cytotoxin A subunit specifically cleaves cell-surface GRP78 protein and abolishes COOH-terminal-dependent signaling.
abstract: GRP78, a molecular chaperone with critical endoplasmic reticulum functions, is aberrantly expressed on the surface of cancer cells, including prostate and melanoma. Here it functions as a pro-proliferative and anti-apoptotic signaling receptor via NH(2)-terminal domain ligation. Auto-antibodies to this domain may appear in cancer patient serum where they are a poor prognostic indicator. Conversely, GRP78 COOH-terminal domain ligation is pro-apoptotic and anti-proliferative. There is no method to disrupt cell-surface GRP78 without compromising the total GRP78 pool, making it difficult to study cell-surface GRP78 function. We studied six cell lines representing three cancer types. One cell line per group expresses high levels of cell-surface GRP78, and the other expresses low levels (human hepatoma: Hep3B and HepG2; human prostate cancer: PC3 and 1-LN; murine melanoma: B16F0 and B16F1). We investigated the effect of Escherichia coli subtilase cytoxin catalytic subunit (SubA) on GRP78. We report that SubA specifically cleaves cell-surface GRP78 on HepG2, 1-LN, and B16F1 cells without affecting intracellular GRP78. B16F0 cells (GRP78(low)) have lower amounts of cleaved cell-surface GRP78. SubA has no effect on Hep3B and PC3 cells. The predicted 28-kDa GRP78 COOH-terminal fragment is released into the culture medium by SubA treatment, and COOH-terminal domain signal transduction is abrogated, whereas pro-proliferative signaling mediated through NH(2)-terminal domain ligation is unaffected. These experiments clarify cell-surface GRP78 topology and demonstrate that the COOH-terminal domain is necessary for pro-apoptotic signal transduction occurring upon COOH-terminal antibody ligation. SubA is a powerful tool to specifically probe the functions of cell-surface GRP78.
authors.affiliation: Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Rupa
authors.initials: R
authors.lastname: Ray
authors.forename: Gustaaf G
authors.initials: GG
authors.lastname: de Ridder
authors.forename: Jerry P
authors.initials: JP
authors.lastname: Eu
authors.forename: Adrienne W
authors.initials: AW
authors.lastname: Paton
authors.forename: James C
authors.initials: JC
authors.lastname: Paton
authors.forename: Salvatore V
authors.initials: SV
authors.lastname: Pizzo
mesh_terms: D000818:Animals; D000912:Antibodies, Neoplasm / Q000235:genetics / Q000378:metabolism; D001323:Autoantibodies / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D000091342:Endoplasmic Reticulum Chaperone BiP; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000494:pharmacology*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D008297:Male; D008545:Melanoma / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis / Q000187:drug effects*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D013381:Subtilisins / Q000494:pharmacology*
chemical_list: D000912:Antibodies, Neoplasm; D001323:Autoantibodies; D000091342:Endoplasmic Reticulum Chaperone BiP; D029968:Escherichia coli Proteins; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D043562:Receptors, G-Protein-Coupled; D013381:Subtilisins; C527234:subtilase cytotoxin, E coli