TY - JOUR
T1 - Analogues of the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist Ned-19 indicate two binding sites on the NAADP receptor
AU - Rosen, Daniel
AU - Lewis, Alexander M.
AU - Mizote, Akiko
AU - Thomas, Justyn M.
AU - Aley, Parvinder K.
AU - Vasudevan, Sridhar R.
AU - Parkesh, Raman
AU - Galione, Antony
AU - Izumi, Minoru
AU - Ganesan, A.
AU - Churchill, Grant C.
PY - 2009/12/11
Y1 - 2009/12/11
N2 - Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca2+-releasing messenger. Biological data suggest that its receptor has two binding sites: one high-affinity locking site and one low-affinity opening site. To directly address the presence and function of these putative binding sites, we synthesized and tested analogues of the NAADP antagonist Ned-19. Ned-19 itself inhibits both NAADP-mediated Ca2a release and NAADP binding. A fluorometry bioassay was used to assess NAADP-mediated Ca2+ release, whereas a radioreceptor assay was used to assess binding to theNAADPreceptor (only at the high-affinity site). In Ned-20, the fluorine is para rather than ortho as in Ned-19. Ned-20 does not inhibit NAADP-mediated Ca2+ release but inhibits NAADP binding. Conversely, Ned-19.4 (a methyl ester of Ned-19) inhibits NAADP-mediated Ca2+ release but cannot inhibit NAADP binding. Furthermore, Ned-20 prevents the self-desensitization response characteristic ofNAADP in sea urchin eggs, confirming that this response is mediated by a high-affinity allosteric site to which NAADP binds in the radioreceptor assay. Collectively, these data provide the first direct evidence for two binding sites (one high- and one lowaffinity) on the NAADP receptor.
AB - Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca2+-releasing messenger. Biological data suggest that its receptor has two binding sites: one high-affinity locking site and one low-affinity opening site. To directly address the presence and function of these putative binding sites, we synthesized and tested analogues of the NAADP antagonist Ned-19. Ned-19 itself inhibits both NAADP-mediated Ca2a release and NAADP binding. A fluorometry bioassay was used to assess NAADP-mediated Ca2+ release, whereas a radioreceptor assay was used to assess binding to theNAADPreceptor (only at the high-affinity site). In Ned-20, the fluorine is para rather than ortho as in Ned-19. Ned-20 does not inhibit NAADP-mediated Ca2+ release but inhibits NAADP binding. Conversely, Ned-19.4 (a methyl ester of Ned-19) inhibits NAADP-mediated Ca2+ release but cannot inhibit NAADP binding. Furthermore, Ned-20 prevents the self-desensitization response characteristic ofNAADP in sea urchin eggs, confirming that this response is mediated by a high-affinity allosteric site to which NAADP binds in the radioreceptor assay. Collectively, these data provide the first direct evidence for two binding sites (one high- and one lowaffinity) on the NAADP receptor.
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U2 - 10.1074/jbc.M109.016519
DO - 10.1074/jbc.M109.016519
M3 - Article
C2 - 19826006
AN - SCOPUS:71749118608
VL - 284
SP - 34930
EP - 34934
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 50
ER -