MINIREVIEW Second messenger function and the structure–activity relationship of cyclic adenosine diphosphoribose (cADPR) Andreas H. Guse University Medical Center Hamburg-Eppendorf, Center of Experimental Medicine, Institute of Biochemistry and Molecular Biology I, Cellular Signal Transduction, Hamburg, Germany The cyclic ADP-ribose ⁄ Ca 2+ signalling pathway Cyclic ADP-ribose (cADPR) was discovered in 1987 as aCa 2+ mobilizing metabolite of the well-known co- enzyme b-nicotinamide adenine dinucleotide (NAD) by Lee and coworkers [1]. The cyclic structure of cADPR was initially predicted to originate from an N-glycosyl linkage between the anomeric carbon of the ribose, which in the precursor NAD is linked to nicotinamide, and the amino ⁄ imino group at C6 of the adenine moiety [2]. Spectroscopic data [3] and finally a crystal structure revealed cyclization between the anomeric C1 of this ribose moiety (commonly termed ‘northern ribose’ while the ribose linked to N9 of adenine is called the ‘southern’ ribose; Fig. 1) and the N1 of the adenine ring [4]. Besides d-myo-inositol 1,4,5-trisphosphate (InsP 3 ) and nicotinic acid adenine dinucleotide phosphate (NAADP; reviewed in [4a]), cADPR is one of the prin- cipal Ca 2+ -releasing second messengers involved in cel- lular Ca 2+ homeostasis. Changes in the cellular Ca 2+ homeostasis are among the fundamental signalling pro- cesses in multicellular organisms. Such changes occur in response to extracellular signals, e.g. hormones, mediators, cell–cell contacts or physical stimuli, and represent one of the most important, powerful and ver- satile intracellular signal transducers. Changes in the Correspondence A. H. Guse, University Medical Center Hamburg-Eppendorf, Center of Experimental Medicine, Institute of Biochemistry and Molecular Biology I: Cellular Signal Transduction, Martinistr. 52, 20246 Hamburg, Germany Fax: +49 40 42803 9880 Tel: +49 40 42803 2828 E-mail: guse@uke.uni-hamburg.de (Received 10 March 2005, accepted 05 July 2005) doi:10.1111/j.1742-4658.2005.04863.x Cyclic ADP-ribose (cADPR) is a Ca 2+ 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 Ca 2+ release via type 2 and 3 ryanodine receptors. The exact molecular mechanism, however, remains to be elucida- ted. Possibilities are the direct binding of cADPR to the ryanodine receptor or binding via a separate cADPR binding protein. In addition to Ca 2+ release, cADPR also evokes Ca 2+ entry. The underlying mechanism(s) may comprise activation of capacitative Ca 2+ 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. Abbreviations ADPRC, ADP-ribosyl cyclase; 8-Br-N1-cIDPR, 8-bromo-cyclic inosine diphosphoribose; cADPcR, cyclic ADP carbocyclic ribose; cADPR, cyclic adenosine diphosphoribose; cADPR-BP, cADPR binding protein; cArisDPR, cyclic aristeromycin diphosphoribose; N1-cIDPR, N1-coupled cyclic inosine diphosphoribose; cIDP-DE, N1-[(phosphoryl-O-ethoxy)-methyl]-N9-[(phosphoryl-O-ethoxy)-methyl]-hypoxanthine-cyclic pyro- phosphate; cIDPRE, N1-ethoxymethyl-cIDPR; CRAC, Ca 2+ release activated Ca 2+ channel; FKBP, FK506 binding protein; InsP 3 , D-myo-inositol 1,4,5-trisphosphate; NAADP, nicotinic acid adenine dinucleotide phosphate; RyR, ryanodine receptor; TRP, transient receptor potential. 4590 FEBS Journal 272 (2005) 4590–4597 ª 2005 FEBS cellular Ca 2+ homeostasis finally result in meaningful physiological response of the cell. Thus, intracellular Ca 2+ signalling is one of the most important transduc- tion systems to integrate physiological responses of multicellular organisms. Because the free cytosolic and nuclear Ca 2+ concen- tration ([Ca 2+ ] i ) is kept fairly low (approximately 50–100 nm) by ATP-driven Ca 2+ pumps located in both the plasma membrane and intracellular mem- branes (reviewed in [5]), rapid increases of [Ca 2+ ] i can be achieved by increasing the open probability of Ca 2+ channels, either localized in the membranes of intracellular Ca 2+ stores or in the plasma membrane. Such Ca 2+ entry channels in the plasma membrane and Ca 2+ release channels in intracellular membranes have been reviewed in the past [6–11]. Review articles dealing with the cADPR ⁄ Ca 2+ signalling system, the topic of this article, have also been published in the last two years [12–16]. Thus, I will not repeat in detail the topics presented in those reviews, but I will briefly describe the hallmarks of the cADPR ⁄ Ca 2+ signalling system. Subsequently I will spend more time in discuss- ing recent findings related to the biological activity of cADPR analogues and some clues regarding the struc- ture–activity relationship of cADPR. The cADPR ⁄ Ca 2+ signalling system is active in diverse cellular systems, including smooth, skeletal and cardiac muscle, neuronal and neuronal-related cells, hemopoietic cells, acinar cells, and oocytes (for a more complete list see [15]). Because the cADPR ⁄ Ca 2+ sig- nalling system was also observed in protozoa and plant cells, it appears to be a phylogenetically old and con- served system. As for the InsP 3 ⁄ Ca 2+ signalling sys- tem, in several cell types extracellular stimuli activate cADPR-forming enzymes called ADP-ribosyl cyclases Fig. 2. Receptor-mediated formation, metabolism and sites of action of cADPR. Dotted lines indicate minor pathway or relations not gener- ally accepted or proven. Cx43, connexin 43; CRAC, Ca 2+ release-activated Ca 2+ channel; cADPRH, cADPR-hydrolase. Fig. 1. Structure of cADPR. A. H. Guse Second messenger function of cADPR FEBS Journal 272 (2005) 4590–4597 ª 2005 FEBS 4591 (ADPRC) and thereby induce the formation of cADPR (Fig. 2). G-protein coupling and Tyr phos- phorylation have been implicated in ADPRC activa- tion [17,18]. The enzymes responsible for the synthesis of cADPR are still a matter of debate. An ADPRC that acts mainly as a cyclizing enzyme has been purified and cloned more than 10 years ago from the ovotestis of Aplysia californica [19,20]. Mammalian homologues of this enzyme are the membrane proteins CD38 and CD157 (reviewed in [21]). After their discovery it was surprising to note that their catalytic sites are located outside of the cell (or in intracellular vesicles), but obviously not in direct contact to the substrate NAD and the intracellular Ca 2+ release channel sensitive to cADPR, the ryanodine receptor (RyR). This situation has been described as the ‘topological paradox’ of the cADPR ⁄ Ca 2+ signalling system [22]. De Flora and coworkers have worked out a potential solution for this problem. They found that NAD can leave the cell via connexin 43 hemichannels (Fig. 2; [23]). Outside the cell (or inside CD38 containing vesicles) NAD is then converted, at least in part, to cADPR. Evidence was presented that both CD38 and nucleoside trans- porters act as cADPR-transporting proteins (Fig. 2; [24]). This system in principle represents a solution for the topological paradox. However, connexin 43 hemi- channels appear to be open for NAD export only at [Ca 2+ ] i  100 nm, indicating that this system is un- likely to operate when [Ca 2+ ] i is elevated above nor- mal basal levels [25]. On the other hand, ectoenzymes producing cADPR (such as CD38 and CD157) and transport systems for cADPR in the plasma membrane open the possibility that cADPR acts as a paracrine signalling molecule (reviewed in [14]). Indeed, recently a potentially very important example for such a para- crine intercellular signalling molecule was described. CD157-(BST-1)-positive bone marrow stromal cells via production of extracellular cADPR induced the expan- sion of human hemopoietic progenitor cells [26]. A crucial point in this intercellular signalling pathway appears to be the expression of concentrative nucleo- side transporters in the hemopoietic progenitors since this allows uptake of a sufficient amount of cADPR into the target cells [26]. In addition to the relatively complicated system for cADPR synthesis described above, several reports sug- gest expression of either cytosolic or membrane-bound enzymes not related to CD38 or CD157 [18,27–31]. None of these enzymes have been identified on the molecular level so far; however, some (or all) of them might be located at cellular sites more suitable for rapid formation of intracellular cADPR. Ca 2+ release by cADPR via ryanodine receptors Whatever these enzymes turn out to be, receptor-medi- ated formation of cADPR obviously takes place in many cell types and cADPR acts on the type 2 and ⁄ or type 3 RyR. This interaction was initially demonstra- ted by the sensitivity of cADPR-mediated Ca 2+ release to pharmacological inhibitors of RyR, such as ruthen- ium red or inhibitory concentrations of ryanodine [32] and has since been confirmed in many cell systems. Moreover, molecular knock-down of type 3 RyR in T-lymphocytes resulted in a significant reduction of cADPR-induced Ca 2+ release, also suggesting such an interaction [33]. However, the exact molecular mechanisms under- lying this interaction are poorly studied. In the first study to identify a cADPR receptor, [ 32 P]8-N 3 -cADPR was used to covalently label putative cADPR binding proteins (cADPR-BP) in sea urchin eggs [34]. As pro- teins of 100 and 140 kDa were labelled, it was conclu- ded that either proteolytic fragments of RyR were labelled or that a distinct cADPR-BP mediated the effects at the RyR (Fig. 2). A putative direct binding site of cADPR at the RyR has not been described so far. In contrast, in a limited number of cell systems FK506 binding protein 12.6 (FKBP 12.6, calstabin2) was found to bind cADPR and to mediate responsive- ness of RyR towards cADPR [35,36]. The data sup- port a model in which binding of FKBP 12.6 to RyR decreases its open probability, whereas binding of cADPR or FK506 to FKBP 12.6 weakens the inter- action between FKBP 12.6 and RyR, thereby resulting in an increased open probability of RyR. Other studies have shown that in specific cell sys- tems additional proteins must be present, e.g. that cal- modulin effectively decreases the EC 50 for cADPR in sea urchin egg homogenates [37] or that Tyr phos- phorylation of the RyR enhances its responsiveness to cADPR [38]. Ca 2+ entry by cADPR In addition to Ca 2+ release via RyR, cADPR has been demonstrated to activate Ca 2+ entry [18,39,40]. Ini- tially, it was shown that microinjection of cADPR into Jurkat T-cells induced long-lasting trains of Ca 2+ spikes that were blocked by addition of Zn 2+ or SKF96365 [39]. Preincubation with the specific cADPR antagonist 7-deaza-8-Br-cADPR abolished long-lasting Ca 2+ signalling evoked by T-cell receptor ⁄ CD3 ligation [18]. Evidence for cADPR involvement in calcium entry was also obtained in neutrophils [40]. The chemotatic Second messenger function of cADPR A. H. Guse 4592 FEBS Journal 272 (2005) 4590–4597 ª 2005 FEBS peptide fMLP induced biphasic calcium signalling – calcium release followed by calcium entry – in neu- trophils from wild type mice. The calcium entry phase was blocked by 8-Br-cADPR, a cADPR antagonist. Furthermore, fMLP did not elicit the calcium entry response in neutrophils from Cd38 – ⁄ – mice. The Cd38 – ⁄ – neutrophils lack the ability to produce cADPR [40]. These data suggest that cADPR, in addition to Ca 2+ release, also promotes Ca 2+ entry. What are the underlying mechanisms? A mechan- ism generally assumed to play a role in nonexcitable cells is the capacitative Ca 2+ entry mechanism [reviewed in 7,41,42]. Ca 2+ currents with very low amplitude activated by store-depletion have been detected in several nonexcitable cells types [43,44]. Evidence for activation of store-operated Ca 2+ entry secondary to cADPR-mediated Ca 2+ release (Fig. 2) was obtained in RyR knock-down Jurkat T-cells in which the long-lasting phase of Ca 2+ signalling was partially reduced in amplitude [33]. Moreover, appli- cation of cADPR into InsP 3 receptor-deficient DT40 cells evoked CRAC-like plasma membrane currents [45]. Extracellular addition of the novel membrane- permeant cADPR agonists N1-ethoxymethyl-cIDPR (cIDPRE) and N1-[(phosphoryl-O-ethoxy)-methyl]- N9-[(phosphoryl-O-ethoxy)-methyl]-hypoxanthine-cyclic pyrophosphate (cIDP-DE) to intact T-cells employing aCa 2+ -free ⁄ Ca 2+ -reintroduction protocol also sug- gests capacitative Ca 2+ entry secondary to Ca 2+ release evoked by cADPR [46,47]. In recent years, the plasma membrane ion channel transient receptor potential – melastatin-like (TRPM2) has gained atten- tion because it is activated by adenosine diphospho- ribose (ADPR), which is synthesized from NAD by CD38-type ADPRC and which is also a breakdown product of cADPR (Fig. 2). TRPM2 is a Ca 2+ - and Na + -permeable cation channel that is mainly expressed in the brain and in cells of the immune sys- tem [48–50]. The nudix box in the cytosolic C-ter- minal region of TRPM2, a conserved motif of enzymes with nucleotide pyrophosphatase activity, appears to bind ADPR and regulate TRPM2 [48,49,51]. Very recently, it was shown that cADPR can also activate TRPM2 [52]. Activation of TRPM2 by cADPR alone resulted in very small currents and was observed only at very high cADPR concentra- tions (EC 50 ¼ 700 lm; [52]); such concentrations likely are not present in cells, as determination of cADPR usually resulted in low micromolar concen- trations (e.g. [18]). Most interestingly, a likely physio- logical concentration of 10 lm cADPR shifted the EC 50 for ADPR from 12 lm to 90 nm [52]. Thus, cADPR appears to be a potent coregulator for Ca 2+ (and Na + ) entry via TRPM2. The situation, however, appears to be more complex because physiological concentrations of AMP inhibit the effect of ADPR on TRPM2 channel gating. The individual contribu- tion of each of these nucleotides to the regulation of Ca 2+ entry under physiological conditions, e.g. with- out washout of endogenous intracellular compounds, will require further investigation in the future. Structure–activity relationship of cADPR In-depth reviews covering the chemistry and biological activity of many cADPR analogues have been pub- lished [16,53–55] and the reader interested in more complete coverage of the subject may refer to these review articles. However, I will focus on an interesting series of agonistic cADPR analogues recently devel- oped. When analysing the Ca 2+ -mobilizing properties of derivatives modified in the northern ribose of cADPR in permeabilized T-cells, it was observed that replacement of the hydroxyl group at C2¢¢ [for clarity atoms of the ‘northern ribose’ will be marked as dou- ble prime (¢¢) while atoms in the southern ribose will be marked as single prime (¢)] by an amino group was almost without effect on the EC 50 of Ca 2+ release (Fig. 3; [56]). This indicates that at this side of the molecule either the polar interactions with its interact- ing protein were fully replaced by the amino group or that no or only minor ligand protein interactions took place. Astonishingly, another modification of the nor- thern ribose, cyclic ADP carbocyclic ribose (cADPcR; Fig. 3), showed weaker Ca 2+ release activity indicating that the oxygen atom of the northern ribose is indeed important for Ca 2+ release [56]. This situation is unique for the northern ribose since replacement of the oxygen by a carbocyclic bridge in the southern ribose in the molecule termed cyclic aristeromycin diphos- phoribose (cArisDPR, Fig. 3; [57]) did not significantly alter its Ca 2+ releasing potential in permeabilized T-cells [56]. These data indicate that both ribose moie- ties might be suitable targets for additional and more radical modifications. Besides modifications in the ribose moieties, novel analogues modified in the nucleobase show that the base hypoxanthine can replace adenine without loss of biological activity [58]. This is true for N1-cID- PR, a cyclic molecule in which the cyclic bond is made between the anomeric C1 of the northern ribose and N1 of inosine (Fig. 4), while N7-cIDPR showed no Ca 2+ release activity in sea urchin egg homogenates [59]. Indeed, Ca 2+ release activity of inosine derivatives was first described for a series of A. H. Guse Second messenger function of cADPR FEBS Journal 272 (2005) 4590–4597 ª 2005 FEBS 4593 cIDPR analogues that were cyclized between N1 of inosine and C2 of the northern ribose [60]. In addi- tion, 8-Br-N1-cIDPR induced Ca 2+ signalling in intact T-cells [61,62]. This finding was surprising since 8-Br-N1-cADPR is a well-known antagonist of cADPR [63]. Fig. 3. Ca 2+ -releasing activity of some southern and northern ribose modified cADPR analogues. Fig. 4. Ca 2+ -releasing activity of some cIDPR analogues. Second messenger function of cADPR A. H. Guse 4594 FEBS Journal 272 (2005) 4590–4597 ª 2005 FEBS A combination of nucleobase and ribose modifica- tions led to the development of an N1-ethoxymethyl- cIDPR (cIDPRE) in which the northern ribose was replaced by an ether strand mimicking the C1-O- C4 ⁄ C5 part of the original ribose (Fig. 4; [46]). Despite this enormous modification the compound was a par- tial agonist in permeabilized T-cells and induced both local and global Ca 2+ signalling in intact T-cells [46]. 8-Azido- and 8-NH 2 -cIDPRE performed similarly (Fig. 4) whereas the halogenated compounds 8-Br- and 8-Cl-cIDPRE were almost without effect (Fig. 4; [46]). An even stronger modification of the original molecule cADPR was achieved by substitution of both the nor- thern and southern ribose by ether strands, resulting in N1-[(phosphoryl-O-ethoxy)-methyl]-N9-[(phosphoryl- O-ethoxy)-methyl]-hypoxanthine-cyclic pyrophosphate (cIDP-DE; Fig. 4). This compound was a partial agon- ist in permeabilized cells and, when applied extracellu- larly to intact T-cells, induced biphasic Ca 2+ signalling comparable to T-cell receptor⁄ CD3 stimulation [47]. The biological activity of cIDP-DE is not restricted to T-lymphocytes; extracellular addition to intact mouse cardiac myocytes revealed activation of subcellular Ca 2+ signalling and the induction of global Ca 2+ waves, which occurred in an oscillatory manner [47]. In terms of structure–activity relationship these data indicate that the northern and southern riboses are pri- marily necessary as linkers between the base adenine (or hypoxanthine) and the diphospho-bridge, as they can be replaced by much simpler ether strands. These ether strands mimic the distance between the nucleo- base and the diphospho-bridge, but on the other hand likely are involved in polar interactions with the cADPR receptor protein. Certainly, the natural lin- kers, the northern and southern ribose moieties, do a better job, as can be seen from the quantitative com- parison with cADPR [47], probably by allowing more interactions, but the new analogues open the possibil- ity for the development of further, perhaps even more simple compounds with biological activity. Such com- pounds might be more suitable for pharmaceutical applications as compared to the cADPR analogues available so far. Conclusion Although the molecular mechanism of receptor-medi- ated formation of cADPR is still mysterious in many aspects, significant advancements were achieved by demonstrating that the topological paradox of extracel- lular ⁄ intravesicular CD38 can be circumvented by spe- cific transport processes of the substrate NAD and the second messenger cADPR. In addition, the description of novel, non-CD38-like ADPRC may be a good start- ing point for their identification in the near future. The use of novel inosine-based cyclic nucleotides signifi- cantly added to our understanding of the structure– activity relationship of cADPR. Finally, a potential new mechanism underlying Ca 2+ entry mediated by cADPR may, in addition to capacitative Ca 2+ entry, involve gating of TRPM2 in conjunction with ADPR. Acknowledgements I am grateful to my coworkers and collaboration part- ners for their continuous support. Thanks are also expressed to Tim Walseth (Minneapolis, USA) for crit- ically reading the manuscript. Research in my lab is supported by grants from the Deutsche Forschungs- gemeinschaft (no. GU 360 ⁄ 7-3 ⁄ 9-1 ⁄ 9-2/10-1), the Hertie-Foundation (no. 1.01.1 ⁄ 04 ⁄ 010, jointly with Alexander Flu ¨ gel, Martinsried, Germany), the Well- come Trust (research collaboration grant no. 068065 jointly with Barry Potter, Bath, UK) and the Deutsche Akademische Austauschdienst (no. 423 ⁄ vrc-PPP-sr, jointly with Li-he Zhang, Beijing, China). 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