Nicotinic acid adenine dinucleotide phosphate is an evolutionarily conserved second messenger, which mobilizes Ca 2+ from acidic stores. The molecular identity of the NAADP receptor has yet to be defined. In pursuit of isolating and identifying NAADP-binding proteins, we synthesized and characterized a bifunctional probe that incorporates both a photoactivatable crosslinking azido moiety at the 5-position of the nicotinic ring and a ‘clickable’ ethynyl moiety to the 8-adenosyl position in NAADP. Microinjection of this 5N 3 -8-ethynyl-NAADP into cultured U2OS cells induced robust Ca 2+ responses. Higher concentrations of 5N 3 -8-ethynyl were required to elicit Ca 2+ release or displace 32 P-NAADP in radioligand binding experiments in sea urchin egg homogenates. In human cell extracts, incubation of 32 P-5N 3 -8-ethynyl-NAADP followed by UV irradiation resulted in selective labeling of 23 kDa and 35 kDa proteins and photolabeling of these proteins was prevented when incubated in the presence of unlabeled NAADP. Compared to the monofunctional 32 P-5N 3 -NAADP, the clickable 32 P-5N 3 -8-ethynyl-NAADP demonstrated less labeling of the 23 kDa and 35 kDa proteins (~3-fold) but provided an opportunity for further enrichment through the ‘clickable’ ethynyl moiety. No proteins were specifically labeled by 32 P-5N 3 -8-ethynyl-NAADP in sea urchin egg homogenate. These experiments demonstrate that 5N 3 -8-ethynyl-NAADP is biologically active and selectively labels putative NAADP-binding proteins in mammalian systems, evidencing a ‘bifunctional’ probe with utility for isolating NAADP-binding proteins.
|Original language||English (US)|
|Number of pages||9|
|Journal||Biochimica et Biophysica Acta - Molecular Cell Research|
|State||Published - Jul 2019|
Bibliographical noteFunding Information:
J.T.S. and T.F.W. acknowledge financial support from NIH Institute of General Medical Sciences Grant Number GM100444 . J.S.M and G.S.G. received support from NIH GM088790 . Work at the University of Toledo was partially supported by The University of Toledo Foundation Cancer Research Support Account.
- Clickable NAADP analog
- Intracellular calcium release
- Nicotinic acid adenine dinucleotide phosphate (NAADP)
- Nucleotide analogs
- Photoaffinity labeling