Isolation, Expression, and Characterization of Fully Functional Nontoxic BiP/GRP78 Mutants

Background: GRP78 is aberrantly expressed on the surface of many different tumor cells where it functions as a growth factor-like receptor. Results: SubA cleavage of cell-surface GRP78 abrogates signaling initiated by ligation of the GRP78 COOH-terminal. Conclusion: SubA specifically cleaves cell-surface GPR78. Significance: SubA is a powerful tool that can be used to specifically probe the functions of cell-surface GPR78.

Section: Methodsmentioning

confidence: 99%

The Escherichia coli Subtilase Cytotoxin A Subunit Specifically Cleaves Cell-surface GRP78 Protein and Abolishes COOH-terminal-dependent Signaling

Ray

Ridder

et al. 2012

“…Overproduction and Purification of Wild Type and G565R Mutant GRP78-18 ϫ 500-ml cultures of Escherichia coli strain BL21 DE3 harboring plasmid pHis-6BiP:G565R or pHis6-Bip (22)(23)(24) were grown in Luria broth supplemented with 100 g ml Ϫ1 ampicillin in 2-liter baffled glass Erhlenmeyer flasks at 37°C in an orbital incubator set at 160 rpm. At midlog growth phase, the cultures were made 0.2 mg ml Ϫ1 with isopropyl 1-thio-␤-D-galactopyranoside, and the incubation continued for a further 5 h when the cells were harvested by centrifugation at 10,000 ϫ g for 10 min.…”

Section: Methodsmentioning

confidence: 99%

“…Forms of GRP78-Overproduction of the wild type untagged GRP78 protein has previously been reported to be toxic to E. coli, whereas overproduction of a His 6 -tagged form is less toxic and overproduction of a His 6 -tagged G565R mutant form is nontoxic (22). The functional integrity of the G565R GRP78 was previously assessed by ATPase activity in the presence of the peptide LSVKFLT and the ability to interact with the J-domain of the DnaJ-like accessory protein MYJ1 and found to have near wild type values (22).…”

Section: Purification and Characterization Of The Wild Type And G565rmentioning

confidence: 99%

“…To do this, we chose to use a quantitative thermodynamic approach utilizing microcalorimetry. We report here for the first time a quantitative analysis of Ca 2ϩ , ADP, and ATP binding to wild type and a fully functional and widely used mutant (G565R) (22) of mouse GRP78 and show that Ca 2ϩ , ADP, and ATP bind cooperatively. Using differential scanning calorimetry (DSC), we show that in the absence of ADP or ATP GRP78 binds Ca 2ϩ weakly with a K D of ϳ0.7 mM.…”

mentioning

confidence: 90%

See 1 more Smart Citation

The Affinity of a Major Ca2+ Binding Site on GRP78 Is Differentially Enhanced by ADP and ATP

Lamb

Mee

et al. 2006

GRP78 is a major protein regulated by the mammalian endoplasmic reticulum stress response, and up-regulation has been shown to be important in protecting cells from challenge with cytotoxic agents. GRP78 has ATPase activity, acts as a chaperone, and interacts specifically with other proteins, such as caspases, as part of a mechanism regulating apoptosis. GRP78 is also reported to have a possible role as a Ca 2؉ storage protein. In order to understand the potential biological effects of Ca 2؉ and ATP/ADP binding on the biology of GRP78, we have determined its ligand binding properties. We show here for the first time that GRP78 can bind Ca 2؉ , ATP, and ADP, each with a 1:1 stoichiometry, and that the binding of cation and nucleotide is cooperative. These observations do not support the hypothesis that GRP78 is a dynamic Ca 2؉ storage protein. Furthermore, we demonstrate that whereas Mg 2؉ enhances GRP78 binding to ADP and ATP to the same extent, Ca 2؉ shows a differential enhancement. In the presence of Ca 2؉ , the K D for ATP is lowered ϳ11-fold, and the K D for ADP is lowered around 930-fold. The K D for Ca 2؉ is lowered ϳ40-fold in the presence of ATP and around 880-fold with ADP. These findings may explain the biological requirement for a nucleotide exchange factor to remove ADP from GRP78. Taken together, our data suggest that the Ca 2؉ -binding property of GRP78 may be part of a signal transduction pathway that modulates complex interactions between GRP78, ATP/ADP, secretory proteins, and caspases, and this ultimately has important consequences for cell viability.

“…Heat shock 70-kDa protein 5 (Hspa5), also known as binding immunoglobulin protein (Bip) or glucose-regulated protein 78 (Grp78), belongs to the heat shock protein 70 kDa family of molecular chaperones (24). It functions in endoplasmic reticulum (ER) homeostasis and is a key regulator of the unfolded protein stress response.…”

mentioning

confidence: 99%

Heat Shock 70-kDa Protein 5 (Hspa5) Is Essential for Pronephros Formation by Mediating Retinoic Acid Signaling

Shi

Wang

et al. 2015

Background:The function of Hspa5 in early embryonic development is not well understood. Results: Hspa5 is involved in mediating retinoic acid signaling and is required for pronephros. Conclusion: Hspa5 is essential for pronephros formation by mediating retinoic acid signaling. Significance: This is the first report on the cross-talk between physiological ER stress and transduction of retinoic acid signaling.