ReviewCalcium signaling

NAADP: A Universal Ca2+ Trigger

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Science Signaling  04 Nov 2008:
Vol. 1, Issue 44, pp. re10
DOI: 10.1126/scisignal.144re10


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 Ca2+ Signaling

The exchange of information between cells is fundamental to the function of multicellular organisms. Because many extracellular signaling molecules are not membrane permeant, intracellular signal transduction is required. Ca2+ signaling involves the translation of a signal input into changes in the free cytosolic and nucleoplasmic Ca2+ concentration ([Ca2+]i). Changes in [Ca2+]i can occur locally, globally, or both. Both local and global Ca2+ signals are characterized by complex spatiotemporal patterns, such as oscillations or waves. Ca2+ release from intracellular stores and Ca2+ inflow from the extracellular space both increase [Ca2+]i, whereas decreases in [Ca2+]i are achieved by Ca2+ pumps in both intracellular and plasma membranes and by binding of Ca2+ ions to Ca2+-binding proteins. Multiple Ca2+ channels in plasma membrane and intracellular membranes, multiple Ca2+ pumps, and multiple Ca2+-binding proteins have evolved to mediate these processes. In addition, three second messengers that elicit Ca2+ release, termed nicotinic acid adenine dinucleotide phosphate (NAADP), d-myo-inositol 1,4,5-trisphosphate (IP3), and cyclic adenosine diphosphoribose (cADPR), have been identified to date. All three second messengers mobilize Ca2+ from endogenous Ca2+ stores. In addition, depletion of intracellular Ca2+ stores stimulates Ca2+ entry through Ca2+ channels in what is known as the capacitative mechanism, which involves the CRACM1 (also known as Orai1)–STIM1 complex. Here, we concentrate on one of the three Ca2+-mobilizing second messengers, NAADP. Intriguingly, recent data suggest that NAADP may serve as a universal Ca2+ trigger of cells by evoking an initial release of Ca2+, which is then amplified by Ca2+-induced Ca2+ release (CICR).

NAADP: The Most Powerful Ca2+-Mobilizing Second Messenger Known

NAADP was discovered in 1987 as an impurity in commercial preparations of nicotinamide adenine dinucleotide phosphate (NADP) that could release Ca2+ sequestered in sea urchin egg homogenates independently of IP3 or cADPR (1). The structure of NAADP was determined in 1995 (2) and shown to be essentially identical to NADP, except that the -NH2 group of the nicotinamide ring of NADP is substituted by an -OH group (Fig. 1). This structural alteration converts NADP from an inactive nucleotide to the most potent Ca2+-mobilizing second messenger identified to date, being effective in the low nanomolar range (1).

Fig. 1

Structures of NAADP and NADP. The red circles highlight the only structural difference between them.

The Organelle and Receptor Sensitive to NAADP

The initial work on identifying the NAADP target organelle and receptor was conducted in sea urchin eggs. In this cell system, the search for proof of functional independence of the Ca2+-release systems activated by NAADP, cADPR, and IP3 culminated in experiments involving the separation and functional visualization of an endomembrane system specifically sensitive to NAADP (3). This involved stratifying the organelles of live sea urchin eggs into defined layers by gentle centrifugation and then using Ca2+ imaging to visualize Ca2+ release. This technique enabled the visualization of NAADP-mediated Ca2+ release emanating from an organellar layer distinct from that responsive to IP3 and cADPR (3). Biochemical and pharmacological characterization of this endomembrane system identified it as an acidic, lysosome-related organelle (4). Accordingly, in sea urchin eggs, either elimination of acidic stores by osmotic swelling or blocking acidic store refilling by inhibiting the vacuolar H+–adenosine triphosphatase (ATPase) with bafilomycin A abrogated NAADP-induced Ca2+ signaling (4). Moreover, cell fractionation by density gradient centrifugation showed that the acidic Ca2+ stores sensitive to NAADP are separable from the microsomes.

These results are consistent with cross-desensitization experiments done in intact eggs and homogenates with all three Ca2+-mobilizing molecules: IP3, cADPR, and NAADP (1, 5). Each of these molecules desensitizes Ca2+ release to itself but not to the other two messengers, indicating that the NAADP receptor is distinct from the receptors for IP3 and cADPR (1, 5). Another unique property of the sea urchin NAADP receptor in egg is self-inactivation by subthreshold concentrations of NAADP (6, 7). This property is not observed in other Ca2+-release mechanisms, such as those mediated by cADPR and IP3. Receptor-binding studies indicate that binding sites for NAADP in the egg membrane have high affinity, with a dissociation constant (Kd) of about 200 pM, and high specificity with respect to structurally related dinucleotides, such as NADP and NADPH (8). NAADP binding is not affected by lumenal or cytosolic Ca2+ concentration (9, 10) and shows little dependence on pH (10). Displacement of labeled NAADP by unlabeled NAADP is not possible if the labeled NAADP is added first, indicating an irreversible mode of binding to the receptor (10) reminiscent of the self-inactivation seen with NAADP-dependent Ca2+ release. This irreversible mode of binding depends on K+ and is not observed when K+ is replaced by Na+ (11). Solubilization of the egg NAADP receptor irreversibly labeled with [32P]NAADP provides a rough estimate of its molecular mass of ~408 to 471 kD (12) and reveals a critical dependence of NAADP binding on phospholipids (13). Taken together, these data indicate the presence of a previously unidentified receptor, localized to acidic organelles related to lysosomes, that is responsible for mediating the Ca2+ signaling function of NAADP in sea urchin eggs (Fig. 2).

Fig. 2

Schematic presentation of the NAADP–Ca2+ signaling pathway. Upon cell stimulation by an extracellular ligand, NAADP is formed and acts either on TRP-ML1 [inset taken from Kiselyov et al. (74) and modified] localized to acidic stores, or on type 1 RyR [inset taken from Zissimopoulos and Lai (75) and modified] localized to the ER. Transmembrane assignment of type 1 RyR is based on the 10 transmembrane model (76). Action on type 1 or 2 RyR may also proceed through an additional NAADP-binding protein. Initial trigger Ca2+ released through TRP-ML1 may be amplified through Ca2+-induced Ca2+ release through RyR (or IP3 receptors localized to ER; not shown). Ca2+ entry through the plasma membrane induced by NAADP may involve TRPM2 activation by adenosine diphosphoribose (TRPM2 also results in Na+ entry) or may occur secondary to store depletion and activation of CRACM1 (Orai1) by the capacitative mechanism.

Although the results regarding the NAADP receptor and target organelles in sea urchin eggs are clear, the situation appears to be more complex in mammalian cells. Evidence consistent with that from sea urchin eggs has been obtained (1418), culminating in the proposed identification of the mammalian ion channel activated by NAADP as the transient receptor potential–mucolipin 1 channel (TRP-ML1) (19) present in lysosomes (Fig. 2). Consistent with this interpretation, purified liver and smooth muscle lysosomes reconstituted into lipid planar bilayers showed channel activities responsive to NAADP in a concentration-dependent manner (19, 20) that were specifically diminished in open probability by antibody directed against TRP-ML1, indicating that current activation by NAADP indeed proceeds by way of TRP-ML1 (19, 20). NAADP action on these channels is biphasic, so that the open probability decreases with very high concentrations of NAADP, consistent with the self-desensitization seen in eggs. Furthermore, gene silencing of TRP-ML1 diminished both endothelin-1–induced Ca2+signaling in intact arterial smooth muscle cells and NAADP activation of TRP-ML1 reconstituted in lipid planar bilayers (20). However, the identification of TRP-ML1 as the channel activated by NAADP is tentative and based on a commercial antibody directed against TRP-ML1 and gene silencing by siRNA. More characterizations of the antibody and additional experiments are needed before this conclusion can be regarded as definitive.

Several studies have described an alternative scenario favoring the endoplasmic reticulum (ER) as the target organelle and a ryanodine receptor (RyR) as the NAADP receptor (Fig. 2). The first report demonstrating NAADP-mediated Ca2+ release from the ER used plant microsomes of nonvacuolar origin from red beet and cauliflower (21). The first indication that NAADP may be targeting ER-type Ca2+ stores in mammalian cells came from studies on nuclei isolated from pancreatic acinar cells, in which NAADP released Ca2+ from the nuclear envelope, a Ca2+ pool that forms a continuum with the ER and is sensitive to thapsigargin, a specific inhibitor of the Ca2+-ATPase (pump) in the ER (22, 23). NAADP-dependent Ca2+ release in this system is sensitive to RyR inhibitors, such as ryanodine and ruthenium red, suggesting involvement of RyRs in the response (23). In astrocytes, depletion of Ca2+ stores in the ER diminishes NAADP-mediated Ca2+ signaling, whereas permeabilization of lysosomes or inhibition of vacuolar H+-ATPase does not (24). Further evidence for the ER being at least one of the Ca2+ stores targeted by NAADP comes from experiments with permeabilized pancreatic acinar cells, in which both ER and acidic stores can be mobilized by all three Ca2+ messengers: NAADP, cADPR, and IP3. The responses to NAADP are blocked by RyR inhibitors (25). Moreover, in these cells, the acidic stores do not appear to possess Golgi or lysosomal characteristics (25). Likewise, in human Jurkat T cells, NAADP releases Ca2+ from a thapsigargin-sensitive, ER-type store rather than from a lysosomal Ca2+ store (26). Furthermore, in T cells, NAADP-mediated local and global Ca2+ signaling is sensitive to both RyR gene silencing through RNA interference or its pharmacological inhibition, suggesting a major role for RyRs in the NAADP signaling pathway (27, 28). Even small, local Ca2+ signals observed in the first few hundred milliseconds after microinjection of NAADP did not occur if RyR expression was suppressed or if RyR function was blocked pharmacologically (28), indicating that RyRs are directly targeted by NAADP (Fig. 2). However, NAADP-evoked Ca2+-release events may be too small or too fast (or both) to be detected directly by currently available methods of confocal Ca2+ imaging. Perhaps, NAADP may trigger Ca2+-induced Ca2+ release through RyRs secondary to the primary Ca2+-release events. Thus, it is difficult to distinguish between direct versus indirect effects of NAADP on RyRs at present.

The first evidence for a direct effect of NAADP on the RyR was obtained with heart sarcoplasmic reticulum membranes incorporated into lipid planar bilayers. Under these conditions, low micromolar concentrations of NAADP activate single channel currents with characteristics of type 2 RyRs (29). When highly purified type 1 RyRs are incorporated into lipid planar bilayers, NAADP can activate them even at nanomolar concentrations (30). However, in this system neither the biphasic response to NAADP nor self-inactivation by subthreshold concentrations of NAADP is apparent, indicating a mode of activation different from that seen in the sea urchin egg and several mammalian cell systems, including the reconstituted lysosomal channel sensitive to NAADP (19).

In conclusion, different proteins, including type 1 and type 2 RyR localized to ER (or sarcoplasmic reticulum) and TRP-ML1 localized to lysosomes, have been proposed as NAADP targets (Fig. 2). Either evolution has developed an NAADP-binding domain present in both these channels, or another unidentified protein mediates binding of NAADP to both channels. Future mapping of the binding site(s) will unambiguously reveal the true nature of the direct NAADP target.

Some reports also describe evidence for NAADP-mediated Ca2+ entry across the plasma membrane (27, 31, 32). In primary neutrophils, micromolar concentrations of NAADP coactivates the TRP melastatin-like type 2 channel (TRPM2) in the presence of subthreshold concentrations of ADP-ribose (31). However, it remains to be demonstrated that Ca2+ entry observed in response to NAADP is direct and not secondary to store-depletion–activated Ca2+ entry.

NAADP Concentrations in Intact Cells

NAADP acts at low nanomolar concentrations in cells, which initially made it difficult to measure endogenous NAADP concentrations. Such measurements are now possible with the development of highly selective and sensitive assays. Basal NAADP concentrations have been determined in many cell types, including human erythrocytes, rat hepatocytes, and Escherichia coli, and the values obtained range from about 2 to 16 nM (33). The insulin-secreting β-cell line MIN6 was the first cell type in which a rise in NAADP was observed in response to an extracellular stimulus (34). In these cells, stimulation with a high glucose concentration elicits about a twofold increase in NAADP concentration (34). In pancreatic acinar cells, cholecystokinin (CCK) induces an increase in NAADP concentration within 5 to 10 s, followed by a decline in NAADP concentration within 60 s of stimulation (35). A similarly rapid time course is observed after histamine stimulation of human myometrial cells (36). In Jurkat cells, stimulation of the T cell receptor–CD3 complex results in NAADP production within 10 to 20 s and a decrease within the first minute, which is followed by a second phase of increased NAADP concentration that is sustained for 5 to 20 min (37). Taken together, these data demonstrate the fundamental characteristic of a second messenger: rapid formation in response to a stimulus.

NAADP as a Ca2+ Trigger

It is clear from the above that cells possess multiple Ca2+ stores and multiple messenger molecules for their mobilization. A fundamental question concerns how the Ca2+ signals elicited by these different messengers interact and integrate with each other. A convincing view emerging from recent studies is that NAADP functions as a trigger to elicit an initial Ca2+ signal that is then amplified by cADPR- or IP3-dependent responses (or both) through CICR. This view was first proposed on the basis of results from pancreatic acinar cells, in which spatially and temporally complex Ca2+ changes are induced by various agonists such as CCK and acetylcholine (ACh). It is generally believed that the Ca2+ response to ACh is mediated by the IP3 pathway (38). The Ca2+ response to CCK, by contrast, is mediated by NAADP. Thus, prior self-desensitization of the NAADP receptors by high concentrations of NAADP selectively eliminates Ca2+ signals induced by CCK but not those induced by ACh, indicating that the former is mediated by NAADP (39, 40). Nevertheless, blockade of either cADPR or IP3 receptors with specific antagonists inhibits CCK-induced Ca2+ signals, suggesting that CCK-mediated signals are triggered by NAADP but require either cADPR- or IP3-mediated Ca2+ release or both for amplification (40). This notion was confirmed by measurements of endogenous NAADP concentrations, which show that CCK, but not ACh, induces a rapid increase in NAADP concentration that precedes an increase in cADPR concentration (35).

This triggering function of NAADP has also been found in pancreatic islet cells. Glucagon-like peptide-1 (GLP-1) elicits an increase in intracellular Ca2+ and insulin secretion in islet cells, a process blocked by inhibitors of the NAADP pathway (41). Similar to the situation in acinar cells, NAADP concentrations in the islets increase rapidly after treatment with GLP-1, and this change precedes temporally an increase in cADPR concentration. Indeed, in this case, NAADP directly increases cADPR concentration, demonstrating its causal and triggering role in mediating GLP-1 signaling (41). The results are in general agreement with those described previously in the islet cells in response to stimulation by glucose and CCK summarized above (14, 34). There is, however, one notable difference between the Ca2+ response in islet and acinar cells. In islets, Ca2+ changes induced by GLP-1 are not affected by inhibitors of cADPR and IP3 signaling, indicating that Ca2+ released from NAADP-sensitive stores in the islets is substantial enough for detection even without further amplification. This dichotomic dependence of NAADP-induced Ca2+ signals on subsequent amplification may account for the variable sensitivity of the NAADP response to inhibitors of Ca2+ release from the ER stores observed in different cells. In general, the results from the pancreatic cells largely support the role of acidic stores as being the organelles targeted by NAADP.

Pancreatic cells are not unique in their response to NAADP; similar responses are seen in Jurkat T lymphocytes. Activation of the T cell receptor–CD3 complex by the OKT3 antibody elicits a rapid increase in NAADP concentration that occurs in seconds and coincides with the initial phase, the so-called pacemaker phase, of T cell Ca2+ signaling (37). An increase in the cADPR concentration, by contrast, is much delayed and requires hundreds of seconds to reach its peak (42). Like the response to CCK in pancreatic acinar cells, the OKT3-induced Ca2+ changes in Jurkat cells are amplified and are thus sensitive to inhibitors of cADPR- and IP3-dependent signaling (42). The OKT3-induced NAADP increase is sensitive to genistein, a general inhibitor of tyrosine kinases. Inhibiting the increase in NAADP concentration by pretreatment with genistein depresses changes in Ca2+ concentration as well, consistent with NAADP serving a causal and initiating role in T cell Ca2+ signaling (37). One common feature of the agonist-induced increase in NAADP concentration in these three cell systems is that it is rapid and transient, suggesting that activation of NAADP synthesis and its removal are coordinated and may be directly coupled.

Direct visualization of the Ca2+-triggering function of NAADP has been achieved by Ca2+ imaging experiments in both arterial smooth muscle cells and T cells. Infusion or microinjection of NAADP into these cells induces an increase in Ca2+ concentration, which starts in spatially restricted trigger zones near the periphery of the cells and in the case of smooth muscle cells then propagates, after a delay of tens of seconds, as a global Ca2+ wave across the entire cell that initiates contraction (43). The wave, but not the initial Ca2+ signal, depends on Ca2+ amplification through release from the sarcoplasmic reticulum. Organelle staining indicates that the initial trigger zones of smooth muscle cells coincide with the distribution patterns of lysosomes, consistent with lysosomal Ca2+ stores being targets of NAADP. This NAADP signaling pathway in smooth muscle is important in regulating the vasoconstricting effect of endothelin-1. Thus, inhibitors of NAADP signaling selectively block the contraction induced by this potent vasoconstrictor, but not that activated by prostaglandin-F2a (15). Likewise, endothelin-1, and not prostaglandin-F2a, stimulates an increase in NAADP concentration in these cells. In T cells, the first local Ca2+ signals upon microinjection of NAADP were detected after a few hundred milliseconds; also, in contrast to the scenario in smooth muscle cells, the initial spatially restricted Ca2+ signaling events were fully dependent on RyR expression (28).

NAADP also serves as a Ca2+ trigger in invertebrate cells. During fertilization of sea urchin eggs, the cells in which the Ca2+ signaling action of NAADP was first described (2), a global Ca2+ wave starts at the sperm-egg fusion site and propagates across the egg. Preceding this fertilization wave, there is a cortical “Ca2+ flash” mediated by NAADP, which occurs immediately after cell fusion (44). The NAADP concentration in the egg increases after fertilization; a part of this increase is thought to be delivered by the fertilizing sperm, because increases in NAADP concentration occur in sperm after contact with the jelly components surrounding the egg (44). An increase in cADPR concentration coincides temporally with the global Ca2+ wave (45, 46), whereas an increase in IP3 concentration lags behind the wave by tens of seconds (47). A similar pattern is seen in the starfish oocyte, another invertebrate cell. Microinjection of NAADP into the starfish oocyte induces membrane depolarization and a cortical Ca2+ flash, similar to those observed immediately after sperm-oocyte fusion. Desensitization of the NAADP mechanism before fertilization blocks both processes (48). The generality of NAADP functioning as a Ca2+ trigger thus spans across the phylogenic spectrum, from invertebrate to mammalian cells.

NAADP Metabolism

As described above, agonist-induced increases in cellular NAADP concentration in many cell systems occurs rapidly and transiently, indicating that both NAADP synthesis and its degradation are tightly regulated and coordinated. To date, the only enzymes known to catalyze the synthesis of NAADP are CD38 and its Aplysia homolog, the ADP-ribosyl cyclase (49). Both enzymes produce NAADP by catalyzing the exchange of the base, nicotinamide, of NADP with nicotinic acid. This reaction is highly efficient and preferentially occurs at acidic pH (49). It has been noted previously that the acidic dependence of NAADP synthesis and the acidic Ca2+ stores it targets may not be simply a coincidence and may suggest possible colocalization of the synthesizing enzyme with the stores (50, 51). Other possible reactions leading to NAADP synthesis have long been suggested, such as phosphorylation of NAAD or deamidation of NADP (2), but, to date, no enzymatic reaction other than the base-exchange reaction and no enzymes other than CD38 and the Aplysia cyclase have ever been shown to produce NAADP (52). In sea urchin sperm, an NAADP-synthesizing enzyme activity has recently been described (53), which involves the base-exchange reaction and could be mediated by an enzyme homologous to CD38.

CD38 is a multifunctional protein, initially identified as a lymphocyte antigen by monoclonal antibody typing (54, 55), which has since been found in virtually all tissues and cells examined. As a protein that interacts with CD4, CD38 has been implicated in exerting a protective effect on lymphocytes against HIV infection by competing against the CD4 target protein, gp120, of HIV (56). As an enzyme, CD38 catalyzes not only the synthesis of NAADP through the base-exchange reaction but also the cyclization of NAD to produce cADPR (57). Thus, CD38 produces two Ca2+ messengers that target different intracellular Ca2+ stores [reviewed in (58, 59)]. In some cells, particularly lymphocytes, CD38 is expressed on the surface with the catalytic domain exposed to the outside. Because the substrate, NAD, and its signaling products, cADPR and NAADP, are present and functioning inside the cell, this creates a topological paradox. Much has been done toward resolving the paradox, and readers are referred to a recent review on the subject (60).

CD38 also catalyzes the hydrolysis of NAADP to ADP-ribose phosphate, as well as that of cADPR to ADP-ribose (57, 61). NAADP can be hydrolyzed by nucleotide pyrophosphatase and alkaline phosphatase (50), as well as by CD38. The action of CD38 is, however, highly specific for NAADP, whereas phosphatases act on all nucleotides. An NAADP dephosphorylating activity apparently specific for NAADP has also been described (62).

Substantial progress has been achieved in understanding how CD38 can catalyze the synthesis and hydrolysis of two structurally and functionally distinct Ca2+ messengers. CD38 has now been crystallized and its structure solved to 1.5 Å resolution (63, 64). A single active pocket near the central cleft of the molecule has been shown by both site-directed mutagenesis (65) and crystal substrate complexes (64, 66, 67) to catalyze all the reactions of CD38. This site can accommodate structurally similar substrates such as NAD and nicotinamide guanosine dinucleotide (NGD), as well as distinct ones, such as cADPR, nicotinamide, and ADP-ribose.

NAD enters the active-site pocket with the nicotinamide end first and interacts with the site through both hydrophobic stacking with Trp189 and hydrogen bonding with Glu146 and Asp155 (Fig. 3) (66). The catalytic residue, Glu226, and other critical residues responsible for complexing with various substrates have been identified.

Fig. 3

NAD bound at the active site of CD38. The crystal structure of the extramembranous domain of human CD38 complexed with NAD was obtained by soaking NAD with crystals of an inactive mutant of CD38, E226Q 61.

Intriguingly, the high-resolution crystal structure of the complex of CD38 and NAD shows that the Glu226 is 3.2 Å from the anomeric C-1′ carbon of the nicotinamide ribose, too far for direct nucleophilic attack. Instead, the catalytic residue forms two hydrogen bonds with the hydroxyl groups of the nicotinamide ribose (66). This interaction appears to strain the ribose ring. Likewise, strong interactions between the nicotinamide and the active site lead to a rotation of the plane of the carboxamide group by 22° from the plane of the pyridine ring, straining the nicotinamide moiety as well. It is proposed that these distortions are the main driving force for the cleavage of the glycosidic bond between the nicotinamide and the ribose, rather than a direct attack on the anomeric C-1′ of the nicotinamide ribose (66).

After release of the nicotinamide group, an intermediate is formed, which is stabilized by hydrogen bonding between Glu226 and the ribosyl OH groups. This intermediate is likely to be linked to the enzyme noncovalently (66, 68). The adenine ring of NAD can fold back, and the nucleophilic attack of the intermediate by the N-1 nitrogen of the ring will lead to cyclization and the formation of cADPR. Alternatively, if the intermediate is attacked by either water or nicotinic acid, ADP-ribose or NAADP will be formed, respectively. This scheme can thus account for how a single active site of CD38 can catalyze three different reactions: cyclization, hydrolysis, or base exchange (68, 69).

The multifunctionality of CD38 makes it an ideal candidate for regulating the endogenous concentration of NAADP. Indeed, it is the only known enzyme that can synthesize as well as hydrolyze NAADP (49, 61), ensuring that the degradation pathway is at the precise location of NAADP synthesis and thereby facilitating its efficient removal. This is consistent with the observation that agonist-induced changes in NAADP concentration usually occur in the form of a rapid spike. However, it remains to be determined whether CD38 is actually responsible for the endogenous metabolism of NAADP.

In CD38 knockout mice, NAADP concentration in tissue appears unchanged from that in wild-type mice (41, 36). In contrast, cADPR concentration is greatly reduced in the knockout mice (70). In the pancreatic islets of the knockout mice, GLP-1 can still induce increases in NAADP (as described above), although the extent of the increase appears depressed and the pattern of subsequent Ca2+ changes is different (41).

It is possible that ADP-ribosyl cyclases other than CD38 can catalyze the base-exchange reaction and synthesize NAADP. CD157 is similar in amino acid sequence to CD38 and also possesses cyclase activity (71). However, the enzymatic activity of CD157 is hundreds of times less than that of CD38 and CD157 is also a glycosylphosphatidylinositol-linked extracellular antigen, with characteristics not conducive for functioning as an intracellular signaling enzyme that produces second messengers inside the cells. Whether CD157 can take an alternate form in the CD38 knockout mice, because of compensative pressure, remains to be determined. In this context, it would be of interest to investigate mice that lack both CD38 and CD157.

It is also possible that some unidentified enzyme can catalyze the base-exchange reaction and synthesize NAADP. This has previously been proposed (41, 72) and, in sea urchin eggs, three different isoforms of intracellular ADP-ribosyl cyclase have been described (73). This unidentified enzyme, if it exists, must have a sequence quite different from those of CD38 and CD157, as repeated genome-wide searches have yielded no other homolog.

In the past decade, much progress has been made in elucidating the Ca2+ signaling function of NAADP. Discovered as a contaminant in batches of NADP that could mobilize intracellular Ca2+ stores in sea urchin eggs (1, 2), its second-messenger function has now been documented in numerous cells, from plant cells to mammalian cells. Recent advances should soon bring about the molecular identification of many of the key components of this intriguing Ca2+ signaling pathway.


Research on NAADP in the authors’ labs has been supported by the Deutsche Forschungsgemeinschaft (grant GU 360/7-1 to 7-5 to A.H.G.), Werner-Otto-Foundation (to A.H.G.), Gemeinnützige Hertie-Stiftung (jointly to A.H.G. and A. Flügel, Munich, Germany), and General Research Fund of Hong Kong (grants HKU769107M and HKU768408M to H.C.L.).

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