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Title: Mapping of the ATP-binding sites on inositol 1,4,5-trisphosphate receptor type 1 and type 3 homotetramers by controlled proteolysis and photoaffinity labeling
Authors: Maes, K
Missiaen, Ludwig
Parys, Jan
De Smet, Patrick
Sienaert, Ilse
Waelkens, Etienne
Callewaert, Geert
De Smedt, Humbert #
Issue Date: May-2001
Series Title: Journal of Biological Chemistry vol:276 issue:5 pages:3492-7
Abstract: Submillimolar ATP concentrations strongly enhance the inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release, by binding specifically to ATP-binding sites on the IP(3) receptor (IP(3)R). To locate those ATP-binding sites on IP(3)R1 and IP(3)R3, both proteins were expressed in Sf9 insect cells and covalently labeled with 8-azido-[alpha-(32)P]ATP. IP(3)R1 and IP(3)R3 were then purified and subjected to a controlled proteolysis, and the labeled proteolytic fragments were identified by site-specific antibodies. Two fragments of IP(3)R1 were labeled, each containing one of the previously proposed ATP-binding sites with amino acid sequence GXGXXG (amino acids 1773-1780 and 2016-2021, respectively). In IP(3)R3, only one fragment was labeled. This fragment contained the GXGXXG sequence (amino acids 1920-1925), which is conserved in the three IP(3)R isoforms. The presence of multiple interaction sites for ATP was also evident from the IP(3)-induced Ca(2+) release in permeabilized A7r5 cells, which depended on ATP over a very broad concentration range from micromolar to millimolar.
ISSN: 0021-9258
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Physiology Section (-)
Laboratory of Molecular and Cellular Signaling
Laboratory of Protein Phosphorylation and Proteomics
Laboratory of Phosphoproteomics (-)
# (joint) last author

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