Mapping of the ATP-binding sites on inositol 1,4,5-trisphosphate receptor type 1 and type 3 homotetramers by controlled proteolysis and photoaffinitylabeling

Submillimolar ATP concentrations strongly enhance the inositol 1,4,5-trisph osphate (IP3)-induced Ca2+ release, by binding specifically to ATP-binding sites on the IP3, receptor (IP3R). To locate those ATP-binding sites on IP( 3)R1 and IP(3)R3, both proteins were expressed in Sf9 insect cells and cova lently labeled with 8-azido-[alpha -32P]ATP. IP(3)R1 and IP(3)R3 were then purified and subjected to a controlled proteolysis, and the labeled proteol ytic fragments were identified by site-specific antibodies. Two fragments o f 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 20 16-2021, respectively). In IP(3)R3, only one fragment was labeled. This fra gment contained the GXGXXG sequence (amino acids 1920-1925), which is conse rved in the three IP3R isoforms, The presence of multiple interaction sites for ATP was also evident from the IP3-induced Ca2+ release in permeabilize d A7r5 cells, which depended on ATP over a very broad concentration range f rom micromolar to millimolar.