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.
Department of Neuroscience, Johns Hopkins Medical Institutions, Baltimore, Maryland 21205.
R J
RJ
Mourey
V A
VA
Estevez
J F
JF
Marecek
R K
RK
Barrow
G D
GD
Prestwich
S H
SH
Snyder
D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015220:Calcium Channels; D002851:Chromatography, High Pressure Liquid; D015544:Inositol 1,4,5-Trisphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007457:Iodine Radioisotopes; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010777:Photochemistry; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D014316:Tritium; D014357:Trypsin / Q000378:metabolism*
D000345:Affinity Labels; D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D014316:Tritium; D015544:Inositol 1,4,5-Trisphosphate; D014357:Trypsin