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J Biol Chem
2012 Jan 20;2874:2308-15. doi: 10.1074/jbc.M111.306563.
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Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg.
Walseth TF
,
Lin-Moshier Y
,
Jain P
,
Ruas M
,
Parrington J
,
Galione A
,
Marchant JS
,
Slama JT
.
???displayArticle.abstract??? 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 [(32)P-5-azido]nicotinic acid adenine dinucleotide phosphate ([(32)P-5N(3)]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [(32)P-5N(3)]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N(3)-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [(32)P-5N(3)]NAADP binding was saturable and displayed high affinity (K(d) ∼10 nM) in both binding and photolabeling experiments. [(32)P-5N(3)]NAADP photolabeling was irreversible in a high K(+) buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [(32)P-5N(3)]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.
FIGURE 1. Structure of 5-azido-NAADP. The * denotes the phosphorus labeled with 32P.
FIGURE 2. [32P-5N3]NAADP photoaffinity labeling is UV-dependent and blocked by unlabeled NAADP. S. purpuratus egg homogenates were incubated with [32P-5N3]NAADP for 90 min in the absence (lanes 1 and 2) or presence (lane 3) of 1 μm NAADP. Lane 1 represents a control that was not subjected to UV light. Lanes 2 and 3 were subjected to UV light. Image is a phosphor image of the resulting gel (see “Experimental Procedures” for standard conditions).
FIGURE 3. Specificity of [32P-5N3]NAADP photolabeling in sea urchin egg homogenates. Photolabeling was performed in the absence or presence of the indicated concentrations of various competitors. A, phosphor image of a gel examining the effect of NAADP, NAD, NAAD, nicotinamide mononucleotide (NMN), nicotinic acid (NA), and nicotinamide (nico). The concentrations of NAADP range from 0.1 to 10,000 nm in 10-fold increments. B, phosphor image of a gel examining the effect of 5N3-NAADP and NADP. The concentration of unlabeled 5N3-NAADP ranges from 1 to 10,000 nm in 10-fold increments. C, competition of [32P-5N3]NAADP photolabeling (45-, 40-, and 30-kDa bands combined) by unlabeled NAADP and 5N3-NAADP. Data represent the mean ± S.E. (n = 4) of densitometric analyses of experiments similar to those shown in panels A and B. D, the effect of various nucleotides on [32P-5N3]NAADP photolabeling (45-, 40-, and 30-kDa bands). Data represent the mean ± S.E. (n = 4) of densitometric analyses of experiments similar to those shown in panels A and B. cADPR, cyclic ADP-ribose.
FIGURE 4. [32P-5N3]NAADP photolabeling and binding display saturable kinetics. A, photolabeling of sea urchin egg homogenate performed at increasing concentrations of [32P-5N3]NAADP in the absence (−) or presence of (+) 1 μm NAADP. A phosphor image of acrylamide gel is shown. B, saturation kinetics of [32P-5N3]NAADP photolabeling. Total labeling (closed circles), nonspecific labeling (in the presence of 1 μm NAADP, closed squares), and specific labeling (open circles) were determined from densitometric analyses of phosphor images of experiments similar to the one shown in panel A (n = 3). DLU, digital light units. C, saturation kinetics of [32P-5N3]NAADP binding. The conditions for binding were the same as used for photoaffinity labeling. Total binding (closed circles), nonspecific binding (in the presence of 1 μm NAADP, closed squares), and specific binding (open circles) were determined by a conventional filtration binding assay.
FIGURE 5. [32P-5N3]NAADP photolabeling displays apparent irreversibility in high K+. A and B, S. purpuratus egg homogenates were incubated with 1 nm [32P-5N3]NAADP for 90 min in either high K+ (250 mm, A) or low K+ (25 mm, B). Unlabeled 500 nm NAADP was added to the incubation at 0, 2, 5, 15, and 30 min. All samples were photolabeled after 90 min in the presence of [32P-5N3]NAADP. Control samples were incubated with [32P-5N3]NAADP only. Phosphor images of the acrylamide gels are shown. C, densitometric analyses of experiments similar to those shown in panels A and B (n = 4).
FIGURE 6. TPC immunoblot analyses of egg homogenates. S. purpuratus egg homogenates photolabeled in the presence of 1 μm unlabeled 5N3-NAADP were subjected to immunoblotting as described under “Experimental Procedures” using affinity-purified antibodies against TPC1 (lane 2), TPC2 (lane 3), and TPC3 (lane 4). Lane 1 is a phosphor image of the same egg homogenate photolabeled with [32P-5N3]NAADP under standard conditions.
FIGURE 7. Antibodies to sea urchin TPC1and TPC3 specifically immunoprecipitate proteins photoaffinity-labeled with [32P-5N3]NAADP. The first four lanes (left to right) are immunoprecipitated fractions from experiments using control antibody-, anti-TPC1-, anti-TPC2-, and anti-TPC3-coupled agarose beads, respectively. The far right lane (Input lane) is the input extract used for the immunoprecipitation experiments and represents CHAPS solubilized egg extract photolabeled in the absence 5 μm NAADP. The lane labeled Input + 1μm NAADP represents CHAPS-solubilized egg extract photolabeled in the absence of 5 μm NAADP. A phosphor image of the gel is shown.
