HN1L/JPT2: A signaling protein that connects NAADP generation to Ca2+ microdomain formation.

NAADP-evoked Ca2+ release through type 1 ryanodine receptors (RYR1) is a major mechanism underlying the earliest signals in T cell activation, which are the formation of Ca2+ microdomains. In our characterization of the molecular machinery underlying NAADP action, we identified an NAADP-binding protein, called hematological and neurological expressed 1-like protein (HN1L) [also known as Jupiter microtubule-associated homolog 2 (JPT2)]. Gene deletion of Hn1l/Jpt2 in human Jurkat and primary rat T cells resulted in decreased numbers of initial Ca2+ microdomains and delayed the onset and decreased the amplitude of global Ca2+ signaling. Photoaffinity labeling demonstrated direct binding of NAADP to recombinant HN1L/JPT2. T cell receptor/CD3-dependent coprecipitation of HN1L/JPT2 with RYRs and colocalization of these proteins suggest that HN1L/JPT2 connects NAADP formation with the activation of RYR channels within the first seconds of T cell activation. Thus, HN1L/JPT2 enables NAADP to activate Ca2+ release from the endoplasmic reticulum through RYR.

NAD binding by human CD38 analyzed by Trp189 fluorescence.

The NAD-glycohydrolase/ADP-ribosyl cyclase CD38 catalyzes the metabolism of nicotinamide adenine dinucleotide (NAD) to the Ca2+ mobilizing second messengers ADP-ribose (ADPR), 2'-deoxy-ADPR, and cyclic ADP-ribose (cADPR). In the present study, we investigated binding and metabolism of NAD by a soluble fragment of human CD38, sCD38, and its catalytically inactive mutant by monitoring changes in endogenous tryptophan (Trp) fluorescence. Addition of NAD resulted in a concentration-dependent decrease in sCD38 fluorescence that is mainly caused by the Trp residue W189. Amplitude of the fluorescence decrease was fitted as one-site binding curve revealing a dissociation constant for NAD of 29 µM. A comparable dissociation constant was found with the catalytically inactive sCD38 mutant (KD 37 µM NAD) indicating that binding of NAD is not significantly affected by the mutation. The NAD-induced decrease in Trp fluorescence completely recovered in case of sCD38. Kinetics of recovery was slowed down with decreasing temperature and sCD38 concentration and increasing NAD concentration demonstrating that recovery in fluorescence is proportional to the enzymatic activity of sCD38. Accordingly, recovery in fluorescence was not observed with the catalytically inactive mutant. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

2'-Deoxyadenosine 5'-diphosphoribose is an endogenous TRPM2 superagonist.

Transient receptor potential melastatin 2 (TRPM2) is a ligand-gated Ca2+-permeable nonselective cation channel. Whereas physiological stimuli, such as chemotactic agents, evoke controlled Ca2+ signals via TRPM2, pathophysiological stimuli such as reactive oxygen species and genotoxic stress result in prolonged TRPM2-mediated Ca2+ entry and, consequently, apoptosis. To date, adenosine 5'-diphosphoribose (ADPR) has been assumed to be the main agonist for TRPM2. Here we show that 2'-deoxy-ADPR was a significantly better TRPM2 agonist, inducing 10.4-fold higher whole-cell currents at saturation. Mechanistically, this increased activity was caused by a decreased rate of inactivation and higher average open probability. Using high-performance liquid chromatography (HPLC) and mass spectrometry, we detected endogenous 2'-deoxy-ADPR in Jurkat T lymphocytes. Consistently, cytosolic nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2) and nicotinamide adenine dinucleotide (NAD)-glycohydrolase CD38 sequentially catalyzed the synthesis of 2'-deoxy-ADPR from nicotinamide mononucleotide (NMN) and 2'-deoxy-ATP in vitro. Thus, 2'-deoxy-ADPR is an endogenous TRPM2 superagonist that may act as a cell signaling molecule.

Dynamics of G protein effector interactions and their impact on timing and sensitivity of G protein-mediated signal transduction.

G protein coupled receptors regulate numerous cellular functions primarily via coupling to heterotrimeric G proteins and subsequent regulation of effector proteins such as ion channels, enzymes or GTP exchange factors for small G proteins. The dynamics of interactions between G protein subunits and effectors have been difficult to study particularly in a cellular context. The introduction of Förster resonance energy transfer methods into the field of GPCR research led to interesting insights into the temporal patterns of interactions between G protein subunits and their effectors. In this review we specifically focus on the interaction of Gαi subunits with adenylyl cyclases and of Gαq subunits with p63RhoGEF or G protein coupled receptor kinases type 2. Comparing the dynamics of these interactions revealed remarkable differences between different G protein effectors regarding the ability to be modulated by members of the regulator of G protein signalling protein family as well as the sensitivity towards receptor activation.

Influence of gαq on the dynamics of m3-acetylcholine receptor-g-protein-coupled receptor kinase 2 interaction.

G-protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase with an important function in the desensitization of G-protein-coupled receptors. Based on its ability to bind G-protein ßγ subunits as well as activated Gαq subunits, it can be considered as an effector for G-proteins. The recruitment of GRK2 to activated receptors is well known to be mediated by Gßγ together with negatively charged membrane phospholipids. In the current study, we address the role of Gαq on the interaction of GRK2 with activated Gq-protein-coupled receptors. Therefore, we established new Förster resonance energy transfer (FRET)-based assays to study the interaction of GRK2 with the M3-acetylcholine (M3-ACh) receptor as well as Gq-protein subunits with high spatiotemporal resolution in single living human embryonic kidney 293T cells. M3-ACh receptor stimulation with 10 µM acetylcholine resulted in distinct changes in FRET, which reflects interaction of the respective proteins. GRK2 mutants with reduced binding affinity toward Gαq [GRK2(D110A)] and Gßγ [GRK2(R587Q)] were used to determine the specific role of Gq-protein-binding by GRK2. Comparison of absolute FRET amplitudes demonstrated that Gαq enhances the extent and stability of the GRK2-M3-ACh receptor interaction, and that not only Gßγ but also Gαq can target GRK2 to the membrane. This reveals an important role of Gαq in efficient recruitment of GRK2 to M3-ACh receptors. Furthermore, interactions between Gαq and GRK2 were associated with a prolongation of the interaction between GRK2 and the M3-ACh receptor and enhanced arrestin recruitment by these receptors, indicating that Gαq influences signaling and desensitization.