5-Azido-8-ethynyl-NAADP: A bifunctional, clickable photoaffinity probe for the identification of NAADP receptors

Publication date: Available online 4 December 2018Source: Biochimica et Biophysica Acta (BBA) - Molecular Cell ResearchAuthor(s): Gihan S. Gunaratne, Peiling Su, Jonathan S. Marchant, James T. Slama, Timothy F. WalsethAbstractNicotinic acid adenine dinucleotide phosphate is an evolutionarily conserved second messenger, which mobilizes Ca2+ 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 5N3-8-ethynyl-NAADP into cultured U2OS cells induced robust Ca2+ responses. Higher concentrations of 5N3-8-ethynyl were required to elicit Ca2+ release or displace 32P-NAADP in radioligand binding experiments in sea urchin egg homogenates. In human cell extracts, incubation of 32P-5N3-8-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 32P-5N3-NAADP, the clickable 32P-5N3-8-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...
Source: Biochimica et Biophysica Acta (BBA) Molecular Cell Research - Category: Molecular Biology Source Type: research