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Analogues of the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist ned-19 indicate two binding sites on the NAADP receptor

Analogues of the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist ned-19 indicate two binding sites on the NAADP receptor
Analogues of the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist ned-19 indicate two binding sites on the NAADP receptor
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca(2+)-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 Ca(2+) release and NAADP binding.

A fluorometry bioassay was used to assess NAADP-mediated Ca(2+) release, whereas a radioreceptor assay was used to assess binding to the NAADP receptor (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 Ca(2+) release but inhibits NAADP binding. Conversely, Ned-19.4 (a methyl ester of Ned-19) inhibits NAADP-mediated Ca(2+) release but cannot inhibit NAADP binding. Furthermore, Ned-20 prevents the self-desensitization response characteristic of NAADP 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 low-affinity) on the NAADP receptor.
0021-9258
34930-34934
Rosen, Daniel
c71e8db1-029c-403c-969d-2866f43e073e
Lewis, Alexander M.
653ad7ca-984c-4c2a-9e1b-0452f69c96f2
Mizote, Akiko
d858d2e2-e648-48f0-a2f8-e8a9c203b7c9
Thomas, Justyn M.
f48ee69b-b969-4a4d-ac7f-e22ff729fcd7
Aley, Parvinder K.
47682c89-e7f3-481a-9176-23bb4053ba36
Vasudevan, Sridhar R.
bff5882f-aff0-42b9-adbe-a05512003ee2
Parkesh, Raman
53bf51c3-a36f-49db-b073-5be15e526a8a
Galione, Anthony
8b37d2a7-fd5a-4666-ac6c-276a9a9ad328
Izumi, Minoru
09f507ed-6c5c-42ba-b127-13532414c78f
Ganesan, A.
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Churchill, Grant C.
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Rosen, Daniel
c71e8db1-029c-403c-969d-2866f43e073e
Lewis, Alexander M.
653ad7ca-984c-4c2a-9e1b-0452f69c96f2
Mizote, Akiko
d858d2e2-e648-48f0-a2f8-e8a9c203b7c9
Thomas, Justyn M.
f48ee69b-b969-4a4d-ac7f-e22ff729fcd7
Aley, Parvinder K.
47682c89-e7f3-481a-9176-23bb4053ba36
Vasudevan, Sridhar R.
bff5882f-aff0-42b9-adbe-a05512003ee2
Parkesh, Raman
53bf51c3-a36f-49db-b073-5be15e526a8a
Galione, Anthony
8b37d2a7-fd5a-4666-ac6c-276a9a9ad328
Izumi, Minoru
09f507ed-6c5c-42ba-b127-13532414c78f
Ganesan, A.
62aa5a87-9308-4383-8686-99726b6bcfb9
Churchill, Grant C.
1bfd3b90-5986-4019-978a-c3ee7720bb52

Rosen, Daniel, Lewis, Alexander M., Mizote, Akiko, Thomas, Justyn M., Aley, Parvinder K., Vasudevan, Sridhar R., Parkesh, Raman, Galione, Anthony, Izumi, Minoru, Ganesan, A. and Churchill, Grant C. (2009) Analogues of the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist ned-19 indicate two binding sites on the NAADP receptor. The Journal of Biological Chemistry, 284 (50), 34930-34934. (doi:10.1074/jbc.M109.016519).

Record type: Article

Abstract

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca(2+)-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 Ca(2+) release and NAADP binding.

A fluorometry bioassay was used to assess NAADP-mediated Ca(2+) release, whereas a radioreceptor assay was used to assess binding to the NAADP receptor (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 Ca(2+) release but inhibits NAADP binding. Conversely, Ned-19.4 (a methyl ester of Ned-19) inhibits NAADP-mediated Ca(2+) release but cannot inhibit NAADP binding. Furthermore, Ned-20 prevents the self-desensitization response characteristic of NAADP 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 low-affinity) on the NAADP receptor.

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Published date: 11 December 2009

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Local EPrints ID: 160137
URI: http://eprints.soton.ac.uk/id/eprint/160137
ISSN: 0021-9258
PURE UUID: b84be917-652a-4c3c-b62d-b5ea7413c499

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Date deposited: 12 Jul 2010 09:02
Last modified: 16 Jul 2019 23:54

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Contributors

Author: Daniel Rosen
Author: Alexander M. Lewis
Author: Akiko Mizote
Author: Justyn M. Thomas
Author: Parvinder K. Aley
Author: Sridhar R. Vasudevan
Author: Raman Parkesh
Author: Anthony Galione
Author: Minoru Izumi
Author: A. Ganesan
Author: Grant C. Churchill

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