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Purified TPC isoforms form NAADP receptors with distinct roles for Ca(2+) signaling and endolysosomal trafficking.
Ruas M
,
Rietdorf K
,
Arredouani A
,
Davis LC
,
Lloyd-Evans E
,
Koegel H
,
Funnell TM
,
Morgan AJ
,
Ward JA
,
Watanabe K
,
Cheng X
,
Churchill GC
,
Zhu MX
,
Platt FM
,
Wessel GM
,
Parrington J
,
Galione A
.
Abstract
Intracellular Ca(2+) signals constitute key elements in signal transduction. Of the three major Ca(2+) mobilizing messengers described, the most potent, nicotinic acid adenine dinucleotide phosphate (NAADP) is the least well understood in terms of its molecular targets [1]. Recently, we showed that heterologous expression of two-pore channel (TPC) proteins enhances NAADP-induced Ca(2+) release, whereas the NAADP response was abolished in pancreatic beta cells from Tpcn2 gene knockout mice [2]. However, whether TPCs constitute native NAADP receptors is unclear. Here we show that immunopurified endogenous TPC complexes possess the hallmark properties ascribed to NAADP receptors, including nanomolar ligand affinity [3-5]. Our study also reveals important functional differences between the three TPC isoforms. Thus, TPC1 and TPC2 both mediate NAADP-induced Ca(2+) release, but the subsequent amplification of this trigger Ca(2+) by IP(3)Rs is more tightly coupled for TPC2. In contrast, TPC3 expression suppressed NAADP-induced Ca(2+) release. Finally, increased TPC expression has dramatic and contrasting effects on endolysosomal structures and dynamics, implicating a role for NAADP in the regulation of vesicular trafficking. We propose that NAADP regulates endolysosomal Ca(2+) storage and release via TPCs and coordinates endoplasmic reticulum Ca(2+) release in a role that impacts on Ca(2+) signaling in health and disease [6].
Figure 1. Endogenous SpTPC Protein Complexes Bind to [32P]NAADP with Hallmark Properties of the NAADP Receptor(A) Schematic representation of protocol used for membrane preparations from Strongylocentrotus purpuratus egg homogenates via differential centrifugation. The following abbreviations are used: H, homogenate; S, supernatant; P, pellet.(B) Levels of specific [32P]NAADP binding in membrane preparations (n = 3). Panels below graph correspond to immunoblots probed with affinity-purified SpTPC isoform-specific antibodies showing high molecular weight forms of SpTPCs.(C) Specific [32P]NAADP binding for SpTPC1 and SpTPC3 immunoprecipitates (IPs) from S10P100 fractions with anti-SpTPC1 and anti-SpTPC3 sera. Values (n = 6) determined by Cerenkov counting were normalized against control IP with nonimmune serum.(D) Specific [32P]NAADP binding for SpTPC2 IP from P100 fractions with anti-SpTPC2 serum. Values (n = 11) obtained by phosphorimaging were normalized against control IP with preimmune serum.(E) Inhibition of [32P]NAADP binding to S10P100 membranes or SpTPC1 or SpTPC3 IPs by increasing concentrations of NAADP (n = 3). Values were normalized to amount of bound [32P]NAADP in absence of competitor.(F) Same as in (E), but with NADP as competitor (n = 3).(G) Bound [32P]NAADP to S10P100 membranes or SpTPC1 or SpTPC3 IPs after washes with buffer with K+ (GluIM) or without K+ (HEPES), in the absence or presence of 10 μM NAADP. Values (n = 3) of remaining radioactivity were normalized to values obtained for GluIM washes in the absence of NAADP.Data are represented as mean ± standard error of the mean (SEM); p > 0.05 (not significant, NS), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S1.
Figure 3. Intracellular Localization of SpTPCs(A) Colocalization of mCherry.SpTPC with acidic organelles (LysoTracker Green) in live HEK293 cells expressing mCherry.SpTPCs. Scale bar corresponds to 10 μm.(B) Anti-SpTPC3 immunofluorescence of S. purpuratus eggs with or without peptide block for assessment of immunostaining specificity. Scale bar corresponds to 10 μm, or 2 μm for higher magnification panel.(C) Localization of SpTPCs.mCherry expressed in oocytes of starfish Asterina miniata and detected by mCherry fluorescence or by immunofluorescence with corresponding anti-SpTPC antibodies. Scale bar corresponds to 20 μm. Noninjected controls were not labeled with anti-SpTPC antibodies (data not shown). In oocytes expressing SpTPC3.mCherry, the germinal vesicle is out of the plane of focus. mCherry.SpTPCs showed similar cortical localization (data not shown). Oocytes expressing mCherry alone showed a signal across the entire cell (data not shown). Of note is the presence of an SpTPC1 signal in the nucleus. Indeed, SpTPC1 has a putative nuclear localization signal (amino acids 327–334). Its physiological significance may represent an important avenue for further analysis of TPC function, especially in light of reports describing NAADP signaling in nucleus [33, 34]. See also Figure S3.
Figure 4. Two-Pore Channel Overexpression Causes Changes in Endolysosomal Trafficking and Morphology(A) Lipid endocytosis and recycling in HA.SpTPC-overexpressing HEK293 cells assessed by Alexa Fluor 568-cholera toxin B subunit (CtxB; green). Nuclei are labeled with Hoechst 33342 (Hoechst, blue). Scale bar corresponds to 5 μm. Endolysosomal localization was confirmed by colocalization with endocytosed high molecular weight rhodamine dextran (data not shown); Golgi localization by a single perinuclear distribution is not overlapping with the dextran.(B) Summary of results from experiments in (A) corresponding to three separate experiments with a minimum of 50 cells analyzed per experiment.(C) Pattern of lysosomal staining by LysoTracker Green (LysoTracker, green) in HA.SpTPC-overexpressing HEK293 cells, in the absence or presence of NAADP receptor antagonist Ned-19 (10 μM, 12 hr). Nuclei were labeled with Hoechst 33342 (Hoechst, blue). Scale bars correspond to 5 μm.(D) Summary of results from experiments in (C) corresponding to three separate experiments, with a minimum of 50 cells analyzed per experiment. To allow meaningful comparison between images, we standardized LysoTracker loading conditions and imaging protocols for all cell types. pH differences between cell types were negligible, as assessed by fluorescein-dextran fluorescence (data not shown).(E) Lysosomal storage disease phenotype in HA.HsTPC2-overexpressing HEK293 cells visualized by electron microscopy. Inset is a magnification of one region of a HA.HsTPC2-overexpressing cell. Scale bars correspond to 200 nm. The following abbreviations are used: Nuc, nucleus; MLB, multiple lamellar inclusion body; L, lysosome.Data are represented as mean ± SEM. See also Figure S4.
Aarhus,
Activation and inactivation of Ca2+ release by NAADP+.
1996, Pubmed,
Echinobase
Aarhus,
Activation and inactivation of Ca2+ release by NAADP+.
1996,
Pubmed
,
Echinobase
Bezin,
Regulation of nuclear Ca2+ signaling by translocation of the Ca2+ messenger synthesizing enzyme ADP-ribosyl cyclase during neuronal depolarization.
2008,
Pubmed
Billington,
Characterization of NAADP(+) binding in sea urchin eggs.
2000,
Pubmed
,
Echinobase
Brailoiu,
Essential requirement for two-pore channel 1 in NAADP-mediated calcium signaling.
2009,
Pubmed
Brailoiu,
An ancestral deuterostome family of two-pore channels mediates nicotinic acid adenine dinucleotide phosphate-dependent calcium release from acidic organelles.
2010,
Pubmed
,
Echinobase
Britten,
The single-copy DNA sequence polymorphism of the sea urchin Strongylocentrotus purpuratus.
1978,
Pubmed
,
Echinobase
Calcraft,
NAADP mobilizes calcium from acidic organelles through two-pore channels.
2009,
Pubmed
Cancela,
Coordination of agonist-induced Ca2+-signalling patterns by NAADP in pancreatic acinar cells.
1999,
Pubmed
,
Echinobase
Churchill,
NAADP mobilizes Ca(2+) from reserve granules, lysosome-related organelles, in sea urchin eggs.
2002,
Pubmed
,
Echinobase
Churchill,
Prolonged inactivation of nicotinic acid adenine dinucleotide phosphate-induced Ca2+ release mediates a spatiotemporal Ca2+ memory.
2001,
Pubmed
,
Echinobase
Churchill,
Spatial control of Ca2+ signaling by nicotinic acid adenine dinucleotide phosphate diffusion and gradients.
2000,
Pubmed
,
Echinobase
Davis,
Ca(2+) signaling occurs via second messenger release from intraorganelle synthesis sites.
2008,
Pubmed
,
Echinobase
Dickinson,
Modulation of NAADP (nicotinic acid-adenine dinucleotide phosphate) receptors by K+ ions: evidence for multiple NAADP receptor conformations.
2003,
Pubmed
,
Echinobase
Galione,
NAADP, a new intracellular messenger that mobilizes Ca2+ from acidic stores.
2006,
Pubmed
,
Echinobase
Galione,
The NAADP receptor: new receptors or new regulation?
2005,
Pubmed
,
Echinobase
Genazzani,
Nicotinic acid-adenine dinucleotide phosphate mobilizes Ca2+ from a thapsigargin-insensitive pool.
1996,
Pubmed
,
Echinobase
Genazzani,
Unique inactivation properties of NAADP-sensitive Ca2+ release.
1996,
Pubmed
,
Echinobase
Gerasimenko,
NAADP mobilizes Ca2+ from a thapsigargin-sensitive store in the nuclear envelope by activating ryanodine receptors.
2003,
Pubmed
Kuronita,
A role for the lysosomal membrane protein LGP85 in the biogenesis and maintenance of endosomal and lysosomal morphology.
2002,
Pubmed
Lee,
A derivative of NADP mobilizes calcium stores insensitive to inositol trisphosphate and cyclic ADP-ribose.
1995,
Pubmed
,
Echinobase
Lee,
Functional visualization of the separate but interacting calcium stores sensitive to NAADP and cyclic ADP-ribose.
2000,
Pubmed
,
Echinobase
Lim,
NAADP+ initiates the Ca2+ response during fertilization of starfish oocytes.
2001,
Pubmed
,
Echinobase
Lloyd-Evans,
Niemann-Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium.
2008,
Pubmed
Luzio,
The role of calcium and other ions in sorting and delivery in the late endocytic pathway.
2007,
Pubmed
Menteyne,
Generation of specific Ca(2+) signals from Ca(2+) stores and endocytosis by differential coupling to messengers.
2006,
Pubmed
Morgan,
Investigating cADPR and NAADP in intact and broken cell preparations.
2008,
Pubmed
,
Echinobase
Morgan,
Fertilization and nicotinic acid adenine dinucleotide phosphate induce pH changes in acidic Ca(2+) stores in sea urchin eggs.
2007,
Pubmed
,
Echinobase
Naylor,
Identification of a chemical probe for NAADP by virtual screening.
2009,
Pubmed
,
Echinobase
Parkesh,
Cell-permeant NAADP: a novel chemical tool enabling the study of Ca2+ signalling in intact cells.
2008,
Pubmed
,
Echinobase
Piper,
CUPpling calcium to lysosomal biogenesis.
2004,
Pubmed
Shilling,
Activation by serotonin of starfish eggs expressing the rat serotonin 1c receptor.
1990,
Pubmed
,
Echinobase
Sillence,
Storage diseases: new insights into sphingolipid functions.
2003,
Pubmed
Sodergren,
The genome of the sea urchin Strongylocentrotus purpuratus.
2006,
Pubmed
,
Echinobase
Zong,
The two-pore channel TPCN2 mediates NAADP-dependent Ca(2+)-release from lysosomal stores.
2009,
Pubmed