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

Robert J. Mourey, Virginia A. Estevez, James F. Marecek, Roxanne K. Barrow, Glenn D. Prestwich, Solomon H. Snyder

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1719-1726
Number of pages8
JournalBiochemistry
Volume32
Issue number7
DOIs
StatePublished - Jan 1 1993

ASJC Scopus subject areas

  • Biochemistry

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