Project Summary: Intracellular Ca2+ signaling via changes in cytosolic Ca2+ concentration controls a wide range of cellular and physiologic processes. Ca2+ mobilization from intracellular stores mediated by second messengers plays a critical role in regulation of cytosolic Ca2+ levels. Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+-mobilizing second messenger identified to date; it uniquely mobilizes Ca2+ from acidic endolysosomal organelles. NAADP has been shown to be effective in evoking Ca2+ release in a multitude of different mammalian cells and defects in NAADP signaling are now being implicated in many diseases. Despite the importance of NAADP-evoked Ca2+ signaling, the molecular basis of NAADP-evoked Ca2+ release remains largely unclear. With immobilized NAADP–based affinity purification and quantitative proteomic analyses of NAADP and TPC interacting proteins, we identified Lsm12 to be a shared interacting partner of NAADP, TPC1, and TPC2. Lsm12 directly binds to NAADP via its Lsm domain, colocalizes with TPC2, and mediates the apparent association of NAADP to isolated TPC2 or TPC2-containing membranes. Lsm12 is essential and immediately participates in NAADP-evoked TPC activation and Ca2+ mobilization. Our findings thus reveal a putative RNA-binding protein functioning as an NAADP receptor and a TPC regulatory protein and provide a new molecular basis for understanding the mechanisms of NAADP signaling. Our further studies showed that Lsm12 has multifaceted function by affecting TPC channel gating properties and functioning in non-TPC dependent NAADP signaling. We hypothesize that: 1) Lsm12 as an NAADP receptor achieves its high selectivity and affinity to NAADP than NADP via its Lsm domain; 2) Lsm12 mediates TPC channel activation by NAADP via protein-protein interactions and/or dephosphorylation; and 3) Lsm12 can regulate multiple ion channels and mediate NAADP-evoked intracellular Ca2+ elevation via shared mechanisms. To test our hypotheses, we will pursue the following 3 specific aims. Aim 1. Determine the molecular mechanism of the Lsm domain in NAADP binding. Aim 2. Determine the molecular mechanisms of Lsm12-mediated TPC activation by NAADP. Aim 3. Determine the multifaceted function of Lsm12 in NAADP-evoked Ca2+ signaling. Findings from the proposed research will elucidate the molecular mechanisms and function of NAADP/Lsm12- mediated Ca2+ signaling and facilitate the development of new drugs for this important Ca2+ signaling process.