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 (IP3)-induced Ca2+ release, by binding specifically to ATP-binding sites on the IP3 receptor (IP3R). To locate those ATP-binding sites on IP3R1 and IP3R3, both proteins were expressed in Sf9 insect cells and covalently labeled with 8-azido-[α-32P]ATP. IP3R1 and IP3R3 were then purified and subjected to a controlled proteolysis, and the labeled proteolytic fragments were identified by site-specific antibodies. Two fragments of IP3R1 were labeled, each containing one of the previously proposed ATP-binding sites with amino acid sequence GX GXX G (amino acids 1773–1780 and 2016–2021, respectively). In IP3R3, only one fragment was labeled. This fragment contained the GX GXX G sequence (amino acids 1920–1925), which is conserved in the three IP3R isoforms. The presence of multiple interaction sites for ATP was also evident from the IP3-induced Ca2+ release in permeabilized A7r5 cells, which depended on ATP over a very broad concentration range from micromolar to millimolar.