Abstract
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing second messenger which uniquely mobilizes Ca2+ from acidic endolysosomal organelles. However, the molecular identity of the NAADP receptor remains unknown. Given the necessity of the endolysosomal two-pore channel (TPC1 or TPC2) in NAADP signaling, we performed affinity purification and quantitative proteomic analysis of the interacting proteins of NAADP and TPCs. We identified a Sm-like protein Lsm12 complexed with 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 from acidic stores. These findings reveal a putative RNA-binding protein to function as an NAADP receptor and a TPC regulatory protein and provides a molecular basis for understanding the mechanisms of NAADP signaling.
Highlights
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing second messenger which uniquely mobilizes Ca2+ from acidic endolysosomal organelles
Given the essential role of two-pore channels (TPCs) in NAADP signaling, we hypothesize that the NAADP receptor is part of a TPC-containing NAADP signaling multiprotein complex (Supplementary Fig. 1) in which the receptor interacts with the channels either directly as an accessory protein as previously proposed[23,24] or indirectly via a different mechanism
Upon transient expression of human TPC1 (TPC1-eGFP-FLAG) and TPC2 (TPC2-eGFPFLAG) in HEK293 cells, we used immobilized NAADP to pull down the NAADP-interacting proteins and an anti-FLAG antibody to pull down the TPC1 and TPC2 and their interacting proteins (Fig. 1a)
Summary
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing second messenger which uniquely mobilizes Ca2+ from acidic endolysosomal organelles. Lsm[12] is essential and immediately participates in NAADP-evoked TPC activation and Ca2+ mobilization from acidic stores. These findings reveal a putative RNA-binding protein to function as an NAADP receptor and a TPC regulatory protein and provides a molecular basis for understanding the mechanisms of NAADP signaling. Other researchers reported that TPCs were fully insensitive to NAADP and Na +-selective channels with very limited Ca2+ permeability in conventional patch-clamp recordings of exogenous mammalian TPCs expressed on whole enlarged endolysosomes[17], plasma membranes[17,18], or plant vacuoles[19,20]. The molecular identity of the NAADP receptor remains elusive and the molecular basis of NAADP-evoked Ca2+ release remains controversial and poorly understood
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