Nicotinic Acid Adenine Dinucleotide 2'-Phosphate (NAADP) Binding Proteins in T-Lymphocytes.
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Although several channels, including two-pore channels (TPC), ryanodine receptor (RYR) and mucolipin (TRP-ML1) have been implicated in NAADP regulation of calcium signaling, the NAADP receptor has not been identified. In this study, the photoaffinity probe, [32P]-5-azido-NAADP ([32P]-5-N3-NAADP), was used to study NAADP binding proteins in extracts from NAADP responsive Jurkat T-lymphocytes. [32P]-5-N3-NAADP photolabeling of Jurkat S100 cytosolic fractions resulted in the labeling of at least ten distinct proteins. Several of these S100 proteins, including a doublet at 22/23 kDa and small protein at 15 kDa displayed selectivity for NAADP as the labeling was protected by inclusion of unlabeled NAADP, whereas the structurally similar NADP required much higher concentrations for protection. Interestingly, the labeling of several S100 proteins (60, 45, 33 and 28 kDa) was stimulated by low concentrations of unlabeled NAADP, but not by NADP. The effect of NAADP on the labeling of the 60 kDa protein was biphasic, peaking at 100 nM with a five-fold increase and displaying no change at 1 µM NAADP. Several proteins were also photolabeled when the P100 membrane fraction from Jurkat cells was examined. Similar to the results with S100, a 22/23 kDa doublet and a 15 kDa protein appeared to be selectively labeled. NAADP did not increase the labeling of any P100 proteins as it did in the S100 fraction. The photolabeled S100 and P100 proteins were successfully resolved by two-dimensional gel electrophoresis. [32P]-5-N3-NAADP photolabeling and two-dimensional electrophoresis should represent a suitable strategy in which to identify and characterize NAADP binding proteins.
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN 55455, USA.
Timothy F
TF
Walseth
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN 55455, USA.
Yaping
Y
Lin-Moshier
The Calcium Signalling Group, Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
Karin
K
Weber
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN 55455, USA.
Jonathan S
JS
Marchant
Department of Medicinal and Biological Chemistry, University of Toledo, Toledo, OH 43614, USA.
James T
JT
Slama
The Calcium Signalling Group, Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany ; Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
Andreas H
AH
Guse
Jurkat T Cells; NAADP; Photoaffinity Labeling; [32P]5-N3-NAADP