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.

Transient receptor potential melastatin 2 (TRPM2) is a ligand-gated Ca-permeable nonselective cation channel. Whereas physiological stimuli, such as chemotactic agents, evoke controlled Ca signals via TRPM2, pathophysiological stimuli such as reactive oxygen species and genotoxic stress result in prolonged TRPM2-mediated Ca 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.

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 bg subunits as well as activated Ga q 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 Gbg together with negatively charged membrane phospholipids. In the current study, we address the role of Ga q on the interaction of GRK2 with activated G q -protein-coupled receptors. Therefore, we established new Förster resonance energy transfer (FRET)-based assays to study the interaction of GRK2 with the M 3 -acetylcholine (M 3 -ACh) receptor as well as G q -protein subunits with high spatiotemporal resolution in single living human embryonic kidney 293T cells. M 3 -ACh receptor stimulation with 10 mM acetylcholine resulted in distinct changes in FRET, which reflects interaction of the respective proteins. GRK2 mutants with reduced binding affinity toward Ga q [GRK2(D110A)] and Gbg [GRK2(R587Q)] were used to determine the specific role of G q -protein-binding by GRK2. Comparison of absolute FRET amplitudes demonstrated that Ga q enhances the extent and stability of the GRK2-M 3 -ACh receptor interaction, and that not only Gbg but also Ga q can target GRK2 to the membrane. This reveals an important role of Ga q in efficient recruitment of GRK2 to M 3 -ACh receptors. Furthermore, interactions between Ga q and GRK2 were associated with a prolongation of the interaction between GRK2 and the M 3 -ACh receptor and enhanced arrestin recruitment by these receptors, indicating that Ga q influences signaling and desensitization.