Inositol 1,4,5-trisphosphate receptors: Labeling the inositol 1,4,5-trisphosphate binding site with photoaffinity ligands

• Published in: Biochemistry (Easton) Vol. 32; no. 7; pp. 1719 - 1726
• Main Authors: Mourey, Robert J, Estevez, Virginia A, Marecek, James F, Barrow, Roxanne K, Prestwich, Glenn D, Snyder, Solomon H
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 23.02.1993
• Subjects: Affinity Labels - metabolism; Amino Acid Sequence; Animals; Binding Sites; Biological and medical sciences; Calcium Channels; Cell receptors; Cell structures and functions; Chromatography, High Pressure Liquid; Fundamental and applied biological sciences. Psychology; inositol 1,4,5-trisphosphate; Inositol 1,4,5-Trisphosphate - analogs & derivatives; Inositol 1,4,5-Trisphosphate - metabolism; Inositol 1,4,5-Trisphosphate Receptors; Iodine Radioisotopes; Kinetics; labelling; ligands; Miscellaneous; Molecular and cellular biology; Molecular Sequence Data; Peptide Fragments - metabolism; photoaffinity; Photochemistry; Rats; receptors; Receptors, Cell Surface - metabolism; Receptors, Cytoplasmic and Nuclear; recognition; sites; Tritium; Trypsin - metabolism; Ligand binding; Vertebrata; Membrane receptor; Mammalia; Rat; Rodentia; Biomembrane; Binding site; Inositol phosphate; Photoaffinity labelling; Brain (vertebrata)
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00058a004

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.