Tài liệu Báo cáo khoa học: Second messenger function and the structure–activity relationship of cyclic adenosine diphosphoribose (cADPR) doc

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:
(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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