PROJECT SUMMARY Increases in cellular Ca2+ resulting from activation of intracellular Ca2+ channels regulate many physiological events. Dysfunction of intracellular Ca2+ signaling pathways is involved in many disease states. To appreciate how cell functions are controlled by Ca2+ signals, and how pathological cues subvert their function, we must understand how the `functional architecture' of intracellular ion channels controls Ca2+ signaling dynamics. In this proposal, we investigate the mechanistic basis of Ca2+ release mediated by the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP). NAADP potently releases Ca2+ from endosomes and lysosomes by activating a family of cation channels called two-pore channels (TPCs). These signals locally regulate trafficking of endolysosomal cargoes and can be amplified into whole cell Ca2+ signals by Ca2+- induced Ca2+ release through neighboring Ca2+ channels on the endoplasmic reticulum (ER). However, the molecular basis of NAADP-evoked Ca2+ release is unclear as no NAADP binding site has been identified on the TPC protein. Rather NAADP acts via an unidentified NAADP binding protein (NAADP-BP) associated with the TPC complex to mediate TPC activation. Here, a multi-PI team (Marchant, Patel, Slama): (i) designed a novel bifunctional probe to enable a photolabeling strategy that has identified the elusive NAADP-BP as Jupiter Microtubule Associated Homolog 2 (JPT-2), and (ii) optimized novel TPC activatory ligands that mimic NAADP action. These tools provide opportunity to finally decipher the molecular basis of NAADP action. This proposal coalesces medicinal chemistry, molecular analyses, and high-resolution imaging expertise to resolve: (1) How NAADP binds to JPT2, and how JPT2 associates with TPCs. We will define the nucleotide binding characteristics of JPT2, and the role of JPT2 in conferring NAADP sensitivity to TPCs (2) The functional impact of JPT2 on NAADP-evoked Ca2+ signals. We will probe the essentiality and cell biology of JPT2, resolving its role in endogenous NAADP-evoked Ca2+ signals and targeting of TPCs to specialized microdomains (membrane contact sites) that couple to endoplasmic reticulum Ca2+ channels. (3) How NAADP sensitivity is regulated physiologically. Identification of JPT2 provides a molecular handle to investigate how cellular NAADP sensitivity is physiologically controlled by regulation of JPT2 stability. We show JPT2 and TPCs interact with tankyrase, a poly-ADP-ribosyltransferase. We hypothesize that JPT2 is a tankyrase substrate, with cellular JPT2 levels controlled by ADP ribosylation.