Inositol 1,4,5-Trisphosphate Receptors: Labeling the Inositol 1,4,5-Trisphosphate Binding Site with Photoaffinity Ligands

We have photolabeled the inositol 1,4,5-trisphosphate (IP₃) receptor and probed the IP₃ ligand binding site using two novel photoaffinity ligands, [¹²⁵I] (azidosalicyl)aminopropyl-IP₃ ([¹²⁵I] ASA-IP₃) and [³H] (benzoyldihydrocinnamyl)aminopropyl-IP₃ ([³H]BZDC-IP₃). Both ligands have high affinity for the IP₃ receptor and, when photoactivated, label the IP₃ receptor protein with appropriate inositol phosphate selectivity. The high specific activity of [¹²⁵I] ASA-IP₃ allowed identification of a single photolabeling site within the IP₃R by two-dimensional peptide analysis. Substantially higher levels of incorporation into the receptor are achieved with [³H]BZDC-IP₃ (50–60% efficiency) than with [¹²⁵I] ASA-IP₃ (3%), facilitating the use of [³H]BZDC-IP₃ as a better ligand for the high-efficiency labeling and purification of IP₃R-labeled peptides. Peptides were generated from photolabeled IP₃ receptor by trypsin digestion and purified by high-pressure liquid chromatography (HPLC). A single purified [³H]BZDC-IP₃-labeled peptide, corresponding to IP₃R amino acids 476–501, was sequenced and shown to match specific sequences in the N-terminal 20% of the IP₃ receptor, an area suggested on the basis of mutagenesis studies to contain the IP₃ recognition site.