The affinity of a major Ca2+ binding site on GRP78 is differentially enhanced by ADP and ATP

被引:48
作者
Lamb, HK
Mee, C
Xu, WM
Liu, LZ
Blond, S
Cooper, A
Charles, IG
Hawkins, AR
机构
[1] Newcastle Univ, Sch Med, Inst Cell & Mol Biosci, Newcastle Upon Tyne NE2 4HH, Tyne & Wear, England
[2] UCL, Wolfson Inst Biomed Res, London WC1E 6AU, England
[3] Univ Illinois, Ctr Pharmaceut Biotechnol, Dept Med Chem & Pharmacognosy, Chicago, IL USA
[4] Univ Glasgow, Dept Chem, Glasgow G12 8QQ, Lanark, Scotland
基金
英国生物技术与生命科学研究理事会; 英国医学研究理事会;
关键词
D O I
10.1074/jbc.M503964200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 K-D for ATP is lowered similar to 11-fold, and the K-D for ADP is lowered around 930-fold. The K-D for Ca2+ is lowered similar to 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.
引用
收藏
页码:8796 / 8805
页数:10
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