title: The affinity of a major Ca2+ binding site on GRP78 is differentially enhanced by ADP and ATP.
abstract: 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 Ca2+ storage protein. In order to understand the potential biological effects of Ca2+ 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 Ca2+, 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 Ca2+ storage protein. Furthermore, we demonstrate that whereas Mg2+ enhances GRP78 binding to ADP and ATP to the same extent, Ca2+ shows a differential enhancement. In the presence of Ca2+, the KD for ATP is lowered approximately 11-fold, and the KD for ADP is lowered around 930-fold. The KD for Ca2+ is lowered approximately 40-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 Ca2+-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.
authors.affiliation: Institute of Cell and Molecular Biosciences, Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom.
authors.forename: Heather K
authors.initials: HK
authors.lastname: Lamb
authors.forename: Christopher
authors.initials: C
authors.lastname: Mee
authors.forename: Weiming
authors.initials: W
authors.lastname: Xu
authors.forename: Lizhi
authors.initials: L
authors.lastname: Liu
authors.forename: Sylvie
authors.initials: S
authors.lastname: Blond
authors.forename: Alan
authors.initials: A
authors.lastname: Cooper
authors.forename: Ian G
authors.initials: IG
authors.lastname: Charles
authors.forename: Alastair R
authors.initials: AR
authors.lastname: Hawkins
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002151:Calorimetry; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016018:Least-Squares Analysis; D008274:Magnesium / Q000378:metabolism; D051379:Mice; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012044:Regression Analysis; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D008274:Magnesium; D002118:Calcium