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.
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.
Maes
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Laboratorium voor Fysiologie and the Laboratorium voor Biochemie, K.U. Leuven Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. Karlien.Maes@med.kuleuven.ac.be
Missiaen
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Parys
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De Smet
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Sienaert
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Waelkens
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Callewaert
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De Smedt
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D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D002478:Cells, Cultured; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007313:Insecta; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases; D002118:Calcium