Signal Transduction
Photoaffinity Labeling of High Affinity Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-Binding Proteins in Sea Urchin Egg*

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Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [32P-5-azido]nicotinic acid adenine dinucleotide phosphate ([32P-5N3]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [32P-5N3]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N3-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [32P-5N3]NAADP binding was saturable and displayed high affinity (Kd ∼10 nm) in both binding and photolabeling experiments. [32P-5N3]NAADP photolabeling was irreversible in a high K+ buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [32P-5N3]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs.

Calcium Intracellular Release
Calcium Signaling
NAADP
Nucleoside Nucleotide Analogs
Photoaffinity Labeling
Two-pore Channels

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*

This work was supported, in whole or in part, by National Institutes of Health Grant GM088790 (to J. S. M.). This work was also supported by University of Minnesota Academic Health Center Seed Grant 2101.95 (to T. F. W.) and a Wellcome Trust Programme Grant (to A. G. and J. P.).

This article contains supplemental Figs. 1S and 2S.

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Supported by a doctoral dissertation fellowship from the University of Minnesota Graduate School.