Andreas H. Guse

Regulation of calcium signalling in T lymphocytes by the second messenger cyclic ADP-ribose

Cyclic ADP-ribose (cADPR) is a natural compound that mobilizes calcium ions in several eukaryotic cells. Although it can lead to the release of calcium ions in T lymphocytes, it has not been firmly established as a second messenger in these cells. Here, using high-performance liquid chromatography analysis, we show that stimulation of the T-cell receptor/CD3 (TCR/CD3) complex results in activation of a soluble ADP-ribosyl cyclase and a sustained increase in intracellular levels of cADPR. There is a causal relation between increased cADPR concentrations, sustained calcium signalling and activation of T cells, as shown by inhibition of TCR/CD3-stimulated calcium signalling, cell proliferation and expression of the early- and late-activation markers CD25 and HLA-DR by using cADPR antagonists. The molecular target for cADPR, the type-3 ryanodine receptor/calcium channel, is expressed in T cells. Increased cADPR significantly and specifically stimulates the apparent association of [3H]ryanodine with the type-3 ryanodine receptor, indicating a direct modulatory effect of cADPR on channel opening. Thus we show the presence, causal relation and biological significance of the major constituents of the cADPR/calcium-signalling pathway in human T cells.

PrPc capping in T cells promotes its association with the lipid raft proteins reggie-1 and reggie-2 and leads to signal transduction

The cellular prion protein (PrPc) resides in lipid rafts, yet the type of raft and the physiological function of PrPc are unclear. We show here that cross‐linking of PrPc with specific antibodies leads to 1) PrPc capping in Jurkat and human peripheral blood T cells; 2) to cocapping with the intracellular lipid raft proteins reggie‐1 and reggie‐2; 3) to signal transduction as seen by MAP kinase phosphorylation and an elevation of the intracellular Ca2+ concentration; 4) to the recruitment of Thy‐1, TCR/CD3, fyn, lck and LAT into the cap along with local tyrosine phosphorylation and F‐actin polymerization, and later, internalization of PrPc together with the reggies into limp‐2 positive lysosomes. Thus, PrPc association with reggie rafts triggers distinct transmembrane signal transduction events in T cells that promote the focal concentration of PrPc itself by guiding activated PrPc into preformed reggie caps and then to the recruitment of important interacting signaling molecules.

NAADP: A Universal Ca2+ Trigger

NAADP elicits an initial release of calcium, which is subsequently amplified through the action of other calcium messengers. Cells possess multiple calcium ion (Ca2+) stores and multiple messenger molecules to mobilize them. These include d-myo-inositol 1,4,5-trisphosphate (IP3), cyclic adenosine diphosphoribose (cADPR), and the most recently identified Ca2+-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), which acts on a wide spectrum of cells, from plant cells to mammalian cells. Accumulating evidence indicates that NAADP targets both acidic (lysosome-like) Ca2+ stores and endoplasmic reticular stores. Recent studies in invertebrate and mammalian cells suggest that NAADP provides an initiating Ca2+ signal, which is amplified by cADPR- or IP3-dependent mechanisms (or both) through Ca2+-induced Ca2+ release. Diverse stimuli activate a rapid rise of endogenous NAADP concentration, resulting in severalfold increases of NAADP over basal values within seconds. The enzyme CD38 can catalyze both the synthesis and hydrolysis of NAADP, making it ideal for effecting the rapid metabolism of NAADP. The crystal structure of CD38 and the structures of its various substrate complexes have now been determined, clarifying the mechanism of its multifunctional catalysis. We anticipate that these advances will lead to the unmasking of all the key components of the Ca2+ signaling pathway mediated by NAADP. Intracellular signal transduction is fundamental to the ability of multicellular organisms to convert incoming signals into meaningful cellular responses. Calcium ions (Ca2+) released from intracellular stores transduce numerous physiological functions, from fertilization (at the beginning of life) to muscle contraction, induction of cell differentiation and proliferation, and finally apoptotic cell death. The free cytosolic Ca2+ concentration is much lower than that in the extracellular space or in intracellular stores, such as the endoplasmic reticulum (ER). Moreover, the free cytosolic Ca2+ concentration is tightly controlled through the activity of ion carrier proteins and adenosine 5′-triphosphate (ATP)–driven Ca2+ pumps, and through the open probability of Ca2+ channels. The latter is modulated by various mechanisms, including regulation by small signaling molecules generated in response to incoming extracellular signals, which are called second messengers. NAADP is the most recently identified and also the most potent messenger molecule known to mobilize Ca2+ stores in cells. Here, we summarize recent advances concerning NAADP, including the Ca2+ stores it targets, the receptor(s) with which it interacts, its Ca2+-signaling functions, and its metabolism, thereby unraveling a universal cellular signaling pathway.

Second messenger function and the structure–activity relationship of cyclic adenosine diphosphoribose (cADPR)

Cyclic ADP‐ribose (cADPR) is a Ca2+ mobilizing second messenger found in various cell types, tissues and organisms. Receptor‐mediated formation of cADPR may proceed via transmembrane shuttling of the substrate NAD and involvement of the ectoenzyme CD38, or via so far unidentified ADP‐ribosyl cyclases located within the cytosol or in internal membranes. cADPR activates intracellular Ca2+ release via type 2 and 3 ryanodine receptors. The exact molecular mechanism, however, remains to be elucidated. Possibilities are the direct binding of cADPR to the ryanodine receptor or binding via a separate cADPR binding protein. In addition to Ca2+ release, cADPR also evokes Ca2+ entry. The underlying mechanism(s) may comprise activation of capacitative Ca2+ entry and/or activation of the cation channel TRPM2 in conjunction with adenosine diphosphoribose. The development of novel cADPR analogues revealed new insights into the structure–activity relationship. Substitution of either the northern ribose or both the northern and southern ribose resulted in much simpler molecules, which still retained significant biological activity.

Activation of T Cell Calcium Influx by the Second Messenger ADP-ribose

Stimulation of Jurkat T cells by high concentrations of concanavalin A (ConA) induced an elevation of the endogenous adenosine diphosphoribose (ADPR) concentration and an inward current significantly different from the Ca2+ release-activated Ca2+ current (ICRAC). Electrophysiological characterization and activation of a similar current by infusion of ADPR indicated that the ConA-induced current is carried by TRPM2. Expression of TRPM2 in the plasma membrane of Jurkat T cells was demonstrated by reverse transcription-PCR, Western blot, and immunofluorescence. Inhibition of ADPR formation reduced ConA-mediated, but not store-operated, Ca2+ entry and prevented ConA-induced cell death of Jurkat cells. Moreover, gene silencing of TRPM2 abolished the ADPR- and ConA-mediated inward current. Thus, ADPR is a novel second messenger significantly involved in ConA-mediated cell death in T cells.

Functional Ryanodine Receptor Expression Is Required for NAADP-mediated Local Ca2+ Signaling in T-lymphocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing nucleotide involved in T cell Ca2+ signaling (Berg, I., Potter, B. V. L., Mayr, G. W., and Guse, A. H. (2000) J. Cell Biol. 150, 581–588). The objective of this study was to analyze whether the first subcellular Ca2+ signals obtained upon NAADP stimulation of T-lymphocytes depend on the functional expression of ryanodine receptors. Using combined microinjection and high resolution confocal calcium imaging, we demonstrate here that subcellular Ca2+ signals, characterized by amplitudes between ∼30 and 100 nm and diameters of ∼0.5 μm, preceded global Ca2+ signals. Co-injection of the ryanodine receptor antagonists ruthenium red and ryanodine together with NAADP abolished the effects of NAADP, whereas the d-myo-inositol 1,4,5-trisphosphate antagonist heparin and the Ca2+ entry blocker SKF&96365 were without effect. This pharmacological approach was confirmed by a molecular knock-down approach. Jurkat T cell clones with largely reduced expression of ryanodine receptors did not respond to microinjections of NAADP. Taken together, our data suggest that the Ca2+ release channel sensitive to NAADP in T-lymphocytes is the ryanodine receptor.

Cyclic ADP-ribose: A Novel Ca2+-Mobilising Second Messenger

Cyclic ADP-ribose (cADPR) was discovered as a potent Ca2+-mobilising natural compound in sea urchin eggs. Recently, cADPR was reported to stimulate Ca2+ signalling in several higher eukaryotic cell systems (e.g., smooth and cardiac muscle cells, neuronal cells, adrenal chromaffin cells, macrophages, pancreatic acinar cells and T-lymphocytes). The following aspects of the role of cADPR as a Ca2+-mobilising second messenger are reviewed: coupling of metabolism of cADPR to stimulation of receptors in the plasma membrane, properties and pharmacology of Ca2+ release by cADPR and the involvement of cADPR in Ca2+ entry.

ADP-ribosylation at R125 gates the P2X7 ion channel by presenting a covalent ligand to its nucleotide binding site

ADP‐ribosylation is a post‐translational modification regulating protein function in which amino acid‐specific ADP‐ribosyltransferases (ARTs) transfer ADP‐ribose from NAD onto specific target proteins. Attachment of the bulky ADP‐ribose usually inactivates the target by sterically blocking its interaction with other proteins. P2X7, an ATP‐gated ion channel with important roles in inflammation and cell death, in contrast, is activated by ADP‐ribosylation. Here, we report the structural basis for this gating and present the first molecular model for the activation of a target protein by ADP‐ribosylation. We demonstrate that the ecto‐enzyme ART2.2 ADP‐ribosylates P2X7 at arginine 125 in a prominent, cysteine‐rich region at the interface of 2 receptor subunits. ADP‐ribose shares an adenine‐ribonculeotide moiety with ATP. Our results indicate that ADP‐ribosylation of R125 positions this common chemical framework to fit into the nucleotide‐binding site of P2X7 and thereby gates the channel.—Adriouch, S., Bannas, P., Schwarz, N., Fliegert, R., Guse, A. H., Seman, M., Haag, F., Koch‐Nolte, F. ADP‐ribosylation at R125 gates the P2X7 ion channel by presenting a covalent ligand to its nucleotide binding site. FASEB J. 22, 861–869 (2008)

Chemotaxis of Mouse Bone Marrow Neutrophils and Dendritic Cells Is Controlled by ADP-Ribose, the Major Product Generated by the CD38 Enzyme Reaction

The ectoenzyme CD38 catalyzes the production of cyclic ADP-ribose (cADPR) and ADP-ribose (ADPR) from its substrate, NAD+. Both products of the CD38 enzyme reaction play important roles in signal transduction, as cADPR regulates calcium release from intracellular stores and ADPR controls cation entry through the plasma membrane channel TRPM2. We previously demonstrated that CD38 and the cADPR generated by CD38 regulate calcium signaling in leukocytes stimulated with some, but not all, chemokines and controls leukocyte migration to inflammatory sites. However, it is not known whether the other CD38 product, ADPR, also regulates leukocyte trafficking In this study we characterize 8-bromo (8Br)-ADPR, a novel compound that specifically inhibits ADPR-activated cation influx without affecting other key calcium release and entry pathways. Using 8Br-ADPR, we demonstrate that ADPR controls calcium influx and chemotaxis in mouse neutrophils and dendritic cells activated through chemokine receptors that rely on CD38 and cADPR for activity, including mouse FPR1, CXCR4, and CCR7. Furthermore, we show that the calcium and chemotactic responses of leukocytes are not dependent on poly-ADP-ribose polymerase 1 (PARP-1), another potential source of ADPR in some leukocytes. Finally, we demonstrate that NAD+ analogues specifically block calcium influx and migration of chemokine-stimulated neutrophils without affecting PARP-1-dependent calcium responses. Collectively, these data identify ADPR as a new and important second messenger of mouse neutrophil and dendritic cell migration, suggest that CD38, rather than PARP-1, may be an important source of ADPR in these cells, and indicate that inhibitors of ADPR-gated calcium entry, such as 8Br-ADPR, have the potential to be used as anti-inflammatory agents.

Ca2+ Entry Induced by Cyclic ADP-ribose in Intact T-Lymphocytes

Cyclic ADP-ribose (cADPr) is a potent Ca2+-mobilizing natural compound (Lee, H. C., Walseth, T. F., Bratt, G. T., Hayes, R. N., and Clapper, D. L. (1989) J. Biol. Chem. 264, 1608-1615) which has been shown to release Ca2+ from an intracellular store of permeabilized T-lymphocytes (Guse, A. H., Silva, C. P., Emmrich, F., Ashamu, G., Potter, B. V. L., and Mayr, G. W. (1995) J. Immunol. 155, 3353-3359). Microinjection of cADPr into intact single T lymphocytes dose dependently induced repetitive but irregular Ca2+ spikes which were almost completely dependent on the presence of extracellular Ca2+. The Ca2+ spikes induced by cADPr could be blocked either by co-injection of cADPr with the specific antagonist 8-NH2-cADPr, by omission of Ca2+ from the medium, or by superfusion of the cells with Zn2+ or SK-F 96365. Ratiometric digital Ca2+ imaging revealed that single Ca2+ spikes were initiated at several sites (“hot spots”) close to the plasma membrane. These hot spots then rapidly formed a circular zone of high Ca2+ concentration below the plasma membrane which subsequently propagated like a closing optical diaphragm into the center of the cell. Taken together these data indicate a role for cADPr in Ca2+ entry in T-lymphocytes.