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-

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

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

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

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

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+

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

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

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

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-

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