Abstract
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.