Nicotinic Acid Adenine Dinucleotide 2 -Phosphate (NAADP) Binding Proteins in T-Lymphocytes

Abstract

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.

TPC1 Knockout Knocks Out TPC1

“…1C and D). This result under- scores prior conclusions (11)(12)(13). Repetition of our published photolabeling experiments with Tpcn1 T159 samples characterized by Ruas and colleagues (15) is expected to yield a similar result.…”

“…Photolabeling studies using a radioactive NAADP-derived probe (5N 3 -[ 32 P]NAADP) have suggested that NAADP may not directly bind to TPCs but may stimulate channel activity via small NAADP-binding proteins (ϳ23 kDa) associated with the TPC complex (11)(12)(13)(14). One piece of evidence supporting this idea is the preservation of photolabeling in TPC knockout mice (11).…”

Activity of nicotinic acid substituted nicotinic acid adenine dinucleotide phosphate (NAADP) analogs in a human cell line: Difference in specificity between human and sea urchin NAADP receptors

“…However, it is important to note that the Ca 2+ release studies are done in cellular homogenates of sea urchin eggs and with intact cells for the mammalian receptor. Recently, photoaffinity labeling of NAADP targets in homogenates of sea urchin eggs and of the mammalian cell lines SKBR3 and Jurkat showed different molecular masses of the photolabeled proteins [32, 46, 47]. This suggests that differences are inherent in the receptors themselves.…”

Function and dysfunction of two-pore channels

“…Finally, insensitivity of TPCs to NAADP might reflect loss of crucial accessory factors. In this context, photo-affinity labeling studies suggest that NAADP does not bind directly to TPCs but rather to small molecular weight binding proteins that associate with TPCs (65)(66)(67).…”