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

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...

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Bibliographic Details
Published in:The Journal of biological chemistry 2012-01, Vol.287 (4), p.2308-2315
Main Authors: Walseth, Timothy F., Lin-Moshier, Yaping, Jain, Pooja, Ruas, Margarida, Parrington, John, Galione, Antony, Marchant, Jonathan S., Slama, James T.
Format: Article
Language:English
Subjects:
Animals
Binding Sites
Calcium Channels - metabolism
Calcium Intracellular Release
Calcium Signaling
NAADP
NADP - analogs & derivatives
NADP - metabolism
Nucleoside Nucleotide Analogs
Ovum - metabolism
Photoaffinity Labeling
Photoaffinity Labels - chemistry
Protein Binding
Signal Transduction
Strongylocentrotus purpuratus - metabolism
Two-pore Channels
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Summary: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. Background: Nicotinic acid adenine dinucleotide phosphate (NAADP) regulates calcium release from internal acidic stores via two-pore channels (TPCs). Results: A novel photosensitive probe (5-azido-NAADP) identified high affinity NAADP binding sites that interact with, but are distinct from, TPCs. Conclusion: High affinity NAADP-binding proteins complex with TPCs. Significance: This work provides new mechanistic insights into how NAADP regulates calcium release via TPCs.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M111.306563