Quantitative Proteomic Analysis of the Putative NAADP-TPC Signaling Complex

Abstract

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent known Ca2+-mobilizing second messenger which uniquely mobilizes Ca2+ from acidic endolysosomal organelles. Despite the importance of NAADP-evoked Ca2+ signaling, the molecular identities of the NAADP receptors and Ca2+-release channels involved in this process have yet to be unequivocally defined. Accumulated evidence indicates that the poorly understood two-pore channels (TPCs) participate in NAADP-evoked Ca2+ release. However, how TPCs are involved in NAADP-signaling remains largely unknown or debatable. A more unified hypothesis is that TPCs are not directly involved in NAADP binding but are part of the NAADP receptor/Ca2+-release channel complex, in which the molecular identities of other key proteins remain unknown. To dissect the molecular components of the putative NAADP-TPC signaling complex, we performed affinity purification using both TPCs and NAADP as baits to purify the signaling complex in HEK-293 and SKBR3 cells. We employed stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomic approach to specifically identify the TPC and NAADP interacting proteins in these cells. Upon systematic investigation, we have identified large and comprehensive datasets of TPC-interactome and NAADP-interactome.