The orphan receptor GPR17 identified as a new dual uracil nucleotides/cysteinyl-leukotrienes receptor

Abstract

Nucleotides and cysteinyl-leukotrienes (CysLTs) are unrelated signaling molecules inducing multiple effects through separate G-protein-coupled receptors: the P2Y and the CysLT receptors. Here we show that GPR17, a Gi-coupled orphan receptor at intermediate phylogenetic position between P2Y and CysLT receptors, is specifically activated by both families of endogenous ligands, leading to both adenylyl cyclase inhibition and intracellular calcium increases. Agonist-response profile, as determined by [(35)S]GTPgammaS binding, was different from that of already known CysLT and P2Y receptors, with EC(50) values in the nanomolar and micromolar range, for CysLTs and uracil nucleotides, respectively. Both rat and human receptors are highly expressed in the organs typically undergoing ischemic damage, that is, brain, heart and kidney. In vivo inhibition of GPR17 by either CysLT/P2Y receptor antagonists or antisense technology dramatically reduced ischemic damage in a rat focal ischemia model, suggesting GPR17 as the common molecular target mediating brain damage by nucleotides and CysLTs. In conclusion, the deorphanization of GPR17 revealed a dualistic receptor for two endogenous unrelated ligand families. These findings may lead to dualistic drugs of previously unexplored therapeutic potential.

Full text

The orphan receptor GPR17 identified as a new
dual uracil nucleotides/cysteinyl-leukotrienes
receptor
Paolo Ciana
1,6
, Marta Fumagalli
1,6
, Maria
Letizia Trincavelli
2,6
, Claudia Verderio
3
,
Patrizia Rosa
3
, Davide Lecca
1
, Silvia
Ferrario
1
, Chiara Parravicini
1
, Vale
´
rie
Capra
1
, Paolo Gelosa
1
, Uliano Guerrini
1
,
Silvia Belcredito
1
, Mauro Cimino
4
, Luigi
Sironi
1
, Elena Tremoli
1,5
, G Enrico Rovati
1
,
Claudia Martini
2
and Maria P Abbracchio
1,
*
1
Department of Pharmacological Sciences, University of Milan, Milan,
Italy,
2
Department of Psychiatry, Neurobiology, Pharmacology and
Biotechnology, University of Pisa, Pisa, Italy,
3
Department of Medical
Pharmacology, CNR Institute of Neuroscience, Cellular and Molecular
Pharmacology, University of Milan, Milan, Italy,
4
Institute of
Pharmacology and Pharmacognosy, University of Urbino, Urbino, Italy,
5
Monzino Cardiologic Center IRCCS, Milan, Italy
Nucleotides and cysteinyl-leukotrienes (CysLTs) are unre-
lated signaling molecules inducing multiple effects through
separate G-protein-coupled receptors: the P2Y and the CysLT
receptors. Here we show that GPR17, a Gi-coupled orphan
receptor at intermediate phylogenetic position between
P2Y and CysLT receptors, is specifically activated by both
families of endogenous ligands, leading to both adenylyl
cyclase inhibition and intracellular calcium increases.
Agonist-response profile, as determined by [
35
S]GTPcSbind-
ing, was different from that of already known CysLT
and P2Y receptors, with EC
50
values in the nanomolar
and micromolar range, for CysLTs and uracil nucleotides,
respectively. Both rat and human receptors are highly
expressed in the organs typically undergoing ischemic
damage, that is, brain, heart and kidney. In vivo inhibition
of GPR17 by either CysLT/P2Y receptor antagonists or
antisense technology dramatically reduced ischemic
damage in a rat focal ischemia model, suggesting GPR17
as the common molecular target mediating brain damage by
nucleotides and CysLTs. In conclusion, the deorphanization
of GPR17 revealed a dualistic receptor for two endogenous
unrelated ligand families. These findings may lead to dua-
listic drugs of previously unexplored therapeutic potential.
The EMBO Journal (2006) 25, 4615–4627. doi:10.1038/
sj.emboj.7601341; Published online 21 September 2006
Subject Categories: signal transduction; neuroscience
Keywords: cysteinyl-leukotrienes; extracellular nucleotides;
G-protein-coupled receptors; GPR17; neuroinflammation
Introduction
The seven transmembrane domain G-protein-coupled recep-
tors (GPCRs) constitute the largest membrane receptor
family, and, owing to their crucial role in cell-to-cell
communication and involvement in a variety of physiological
phenomena, represent the most common target of pharma-
ceutical drugs (Howard et al, 2001). The progress of human
genome sequencing has revealed the existence of several
hundred orphan GPCRs (Fredriksson et al, 2003), that is,
molecularly identified receptors that still lack a defined
physiologically relevant ligand. The ‘deorphanization’ of
these receptors and the identification of their roles is expected
to clarify novel regulatory mechanisms of physiological phe-
nomena and to unveil novel drug targets (Howard et al,2001;
Fredriksson et al, 2003; Mori et al, 2005).
In addition to established metabolic roles, adenine (ATP,
ADP), uracil (UTP, UDP) and sugar nucleotides (e.g., UDP-
glucose and UDP-galactose) are universal and phylogeneti-
cally ancient signaling molecules involved in a multitude of
biological processes, from embryogenesis to adult homeo-
stasis (Burnstock and Knight, 2004). Actions of extracellular
nucleotides on target cells are mediated by specific mem-
brane receptors: the seven ligand-gated purinergic P2X
channels and the eight G-protein-coupled P2Y receptors
(the P2Y
1,2,4,6,11,12,13,14
receptors) (Abbracchio et al, 2003;
Burnstock and Knight, 2004). Owing to their involvement in
the regulation of many physiological phenomena, dysfunc-
tions of nucleotides and their receptors have been associated
to various human diseases, including immune and ischemic/
inflammatory conditions (Abbracchio et al, 2003; Burnstock
and Knight, 2004). Conversely, cysteinyl-leukotrienes
(CysLTs, such as LTC
4
and LTD
4
) are inflammatory lipid
mediators generated by 5-lipoxygenase metabolism of arachi-
donic acid (Samuelsson, 2000) acting through G-protein-
coupled CysLT
1
and CysLT
2
receptors (Brink et al, 2003)
and implicated in bronchial asthma (Drazen, 2003), stroke
(Ciceri et al, 2001) and cardiovascular diseases (Brink et al,
2003).
Recent data highlight the existence of a functional cross-
talk between the nucleotide and the CysLT systems in orches-
trating inflammatory responses (Capra et al, 2005). Both
types of mediators accumulate at the sites of inflammation,
and inflammatory cells often co-express both P2Y and CysLT
receptors. In rat microglia, the brain immune cells involved in
the response to cerebral hypoxia and trauma, the activation
of P2Y
1
and CysLT receptors mediate co-release of nucleotides
and CysLTs (Ballerini et al, 2005), which might, in turn,
contribute to neuroinflammation and neurodegeneration.
In human monocyte/macrophage-like cells, CysLT
1
receptor
function is regulated by extracellular nucleotides via hetero-
logous desensitization (Capra et al, 2005), and, in the same
cells, montelukast and pranlukast, two selective CysLT
1
receptor antagonists (Brink et al, 2003), functionally interact
Received: 3 April 2006; accepted: 22 August 2006; published online:
21 September 2006
*Corresponding author. Department of Pharmacological Sciences,
University of Milan, via Balzaretti 9, Milan 20133, Italy.
Tel.: þ 390 250 318 310; Fax: þ 390 250 318 284;
E-mail: mariapia.abbracchio@unimi.it
6
These authors contributed equally to this work
The EMBO Journal (2006) 25, 4615–4627
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2006 European Molecular Biology Organization
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with P2Y receptor signaling pathways (Mamedova et al,
2005). Moreover, there are close structural and phylogenetic
relationships between P2Yand CysLT receptors, which cluster
together into the ‘purine receptor cluster’ of the rhodopsin
family of GPCRs, which also includes a large number of
orphan GPCRs (Fredriksson et al, 2003). Mellor et al (2001)
proposed that, in human mast cells, both the CysLT
1
receptor
and a yet unidentified elusive receptor upregulated by treat-
ment with the pro-inflammatory cytokine interleukin-4 (IL-4)
were responsive to both CysLTs and UDP. Here, we chal-
lenged the hypothesis that GPR17, an orphan receptor at an
intermediate phylogenetic position between P2Y and CysLT
receptor families, may represent the yet unidentified elusive
receptor responding to both nucleotides and CysLTs. We
show that the heterologous expression of GPR17 in a variety
of different cell lines results in the appearance of highly
specific and concentration-dependent responses to both
families of signaling molecules. We also show that in vivo
knockdown of GPR17 by either CysLT/P2Y receptor antago-
nists or by antisense technology markedly prevents evolution
of ischemic brain damage in a rat focal ischemia model,
suggesting GPR17 as a common molecular target mediating
the inflammatory effects induced in vivo by nucleotides and
CysLTs. We thus propose that novel pharmacological agents
targeting both signaling components of dualistic receptors
may prove very effective in halting or preventing human
diseases, such as inflammation (Mellor et al, 2001) and
ischemia (the present study).
Results
GPR17, a close relative of both P2Y and CysLT receptors,
is highly expressed in organs undergoing ischemic
damage
The relationships among P2Y, CysLT receptors and GPR17 are
shown in Figure 1A. Phylogenetically, CysLT
1
and CysLT
2
receptors cluster together, whereas P2Y receptors cluster in
two phylogenetically distinct subgroups, one encompassing
P2Y
1,2,4,6,11
and the other encompassing P2Y
12,13,14
(Abbracchio et al, 2003). GPR17 is equally distant from the
P2Y
12,13,14
subgroup and the CysLT
1
and CysLT
2
group, and
thus, its ligand specificity cannot be predicted simply based
on its phylogenetic position. As a first step to the ‘deorpha-
nization’ of GPR17, we cloned and analyzed the coding
sequences from the human and rat receptors. The previously
unidentified rat ortholog (GenBank accession no. DQ777767)
displayed an 89% amino-acid identity with hGPR17
(Figure 1B). The hydropathic profile of deduced putative
proteins was consistent with the typical seven transmem-
brane (7TM) structure of a GPCR (Abbracchio et al, 2003).
Alignment of rat, mouse and human proteins showed almost
complete overlapping of TM3, TM6 and TM7 and conserva-
tion of a typical amino-acid motif in TM6 (H-X-X-R) that is
present in several GPCRs including all known P2Y and CysLT
receptors and is believed (at least for nucleotide receptors) to
be essential for ligand binding (Erb et al, 1995; Jiang et al,
1997; Jacobson et al, 2002) (Figure 1B).
In line with previous expression data (Blasius et al, 1998),
both human and rat GPR17 were highly present in the brain
(the present study) and in other organs typically undergoing
ischemic damage (kidney and heart) with very low expres-
sion in the liver and lung (Figure 1C). hGPR17 mRNA was
also found in some of the cell lines tested (see Figure 1
legend).
Functional characterization in heterologous systems
unveils the dual pharmacology of GPR17
In search for the natural ligand of GPR17, the cDNAs from
human and rat GPR17 were cloned into the expression vector
pcDNA3.1 and transfected in 1321N1, COS-7 and HEK-293
cells. As GPCR stimulation results in increased binding of
GTP to G-proteins (which can, in turn, be quantified by
measuring [
35
S]GTPgS binding to purified membranes;
Marteau et al, 2003), GPR17 activation was determined by
testing the increase of [
35
S]GTPgS binding by exogenously
added agonists in transfected cells (Kotani et al,2001;
Marteau et al, 2003; Fumagalli et al, 2004). Optimal
[
35
S]GTPgS binding conditions were determined in prelimin-
ary experiments (see Supplementary Materials and methods,
Supplementary Results and Supplementary Figure 7). In
1321N1 cells, which do not express any P2Y or CysLT
receptors (Communi et al, 2001; GE Rovati and MP
Abbracchio, unpublished data, 2005), hGPR17 expression
(Figure 2B) induced the appearance of concentration-depen-
dent responses to LTD
4
and LTC
4
(with LTC
4
bLTD
4
) and to
UDP, UDP-glucose and UDP-galactose (with UDP-
galactose ¼ UDP4UDP-glucose) (Figure 2A). ATP, ADP,
2-methyl-thio-ADP, UTP, a,b-methylene ATP and guanosine
had no effect (data not shown). Thus, the agonist-response
profile of GPR17 is different from that of CysLT
1
and CysLT
2
receptors (Brink et al, 2003; Capra, 2004), and, for nucleo-
tides, is intermediate between P2Y
6
and P2Y
14
receptors
(Abbracchio et al, 2003; Burnstock and Knight, 2004).
Interestingly, half-maximal response concentrations (EC
50
)
for agonist stimulation were in agreement with the character-
istics of known CysLT and P2Y receptors (Brink et al, 2003;
Burnstock and Knight, 2004; Capra, 2004), that is, in the
nanomolar (nM) and micromolar (mM) range for CysLTs and
nucleotides, respectively (Figure 2 and Supplementary Table
I). Expression of rGPR17 in 1321N1 cells (Figure 2D) also
induced responses to nM LTD
4
and LTC
4
and to mM UDP and
UDP-glucose (Figure 2C). However, at the rat receptor, UDP-
glucose was more potent than UDP, and UDP-galactose had
no effect; moreover, the relative potency of CysLTs was
inverted (Figure 2 and Supplementary Table I). The ligand
specificity of hGPR17 was also confirmed in additional cell
lines (i.e., COS-7 and HEK-293 cells). Transfection in COS-7
cells (which do not constitutively respond to CysLTs; Capra
et al, 2005) induced responses to LTD
4
and LTC
4
(Figure 2E).
COS-7 cells do express some P2Y receptors (Herold et al,
1997), so the ‘purinergic’ component of GPR17 could not be
studied in these cells. Transfection of hGPR17 in HEK-293
(which do not express P2Y receptors interfering with our
analysis; see Supplementary Discussion) (Figure 2F) or in
CHO cells (not shown) also induced responses to UDP, UDP-
glucose and UDP-galactose, with EC
50
values similar to those
observed in 1321N1 cells. In all cell systems, no response was
ever observed in cells transfected with corresponding empty
vectors (Supplementary Figure 1).
The ability of some known purinergic and leukotriene
antagonists to counteract agonist-induced [
35
S]GTPgS bind-
ing in 1321N1 cells expressing the human or rat receptor was
also assessed. Both cangrelor (formerly AR-C69931MX), a
P2Y
12
/P2Y
13
antagonist (Ingall et al, 1999; Marteau et al,
An orphan dualistic GPCR involved in ischemia
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2003; Fumagalli et al, 2004), and the P2Y
1
receptor antagonist
MRS2179 (Jacobson et al, 2002) concentration-dependently
inhibited [
35
S]GTPgS binding stimulated by 50 mM UDP-glu-
cose in cells expressing hGPR17, with half-maximal inhibi-
tion (IC
50
) in the nM range (Figure 3A and Supplementary
Table I). These values are comparable with the IC
50
of
cangrelor (Ingall et al, 1999; Marteau et al, 2003) and
MRS2179 (Nandanan et al, 1999) at human P2Y
12
/P2Y
13
and P2Y
1
receptors, respectively. These antagonists were
considerably more potent in inhibiting the effects induced
by 50 mM UDP-glucose on rGPR17 (Figure 3B and
Supplementary Table I), with IC
50
values in the picomolar
(pM) range. Moreover, MRS2179 was more potent than
cangrelor (Figure 3B). For the latter, affinity at rGPR17 is
1000-fold higher than that at rP2Y
13
(Fumagalli et al, 2004).
Such species differences in agonist/antagonist-response pro-
files will have to be taken into account when using rodents to
study the pathophysiological roles of GPR17. Conversely, the
CysLT
1
antagonists montelukast and pranlukast (Brink et al,
2003; Capra et al, 2006) concentration-dependently inhibited
activation of human (Figure 3C) and rat receptors (Figure 3D)
by 100 nM LTD
4
, with nM IC
50
values and similar potencies
(Supplementary Table I). Montelukast and pranlukast have
similar affinity at hCysLT
1
receptors (Capra et al, 2006).
Antagonists had no effect in cells transfected with the
pcDNA3.1 empty vector (see flat curves in Figure 3A–D).
A
B
C
Brain
1087 bp -
Liver
Kidney
bSMC
Lung
Heart
PBMC
Huvec
hGPR17
Brain
Liver
Kidney
Lung
Heart
1079 bp -
(β-Actin)
(β-Actin)
rGPR17
U937
Hep-G2
HeLa
SK-N-BE
MCF7
10 20 30 40 50 60 70 80 90
....|....|....|....|....|....|._________________________...|....|.._______________________
hGPR17 MNGLEVAPPGLITNFSLATAEQCGQETPLENMLFASFYLLDFILALVGNTLALWLFIRDHKSGTPANVFLMHLAVADLSCVLVLPTRLVY
mGPR17 MNGLEAALPSLTDNSSLAYSEQCGQETPLENMLFACFYLLDFILAFVGNALALWLFIWDHKSGTPANVFLMHLAVADLSCVLVLPTRLVY
rGPR17 MDGLETALPSLTDNASLAYSEQCGQETPLENMLFACFYLLDFILAFVGNALALWLFIWDHKSGTPANVFLMHLAVADLSCVLVLPTRLVY
TM1 TM2
100 110 120 130 140 150 160 170 180
....|....|....|______________________..|....|....|....|...____________________.|....|....|
hGPR17 HFSGNHWPFGEIACRLTGFLFYLNMYASIYFLTCISADRFLAIVHPVKSLKLRRPLYAHLACAFLWVVVAVAMAPLLVSPQTVQTNHTVV
mGPR17 HFSGNHWPFGEIPCRLTGFLFYLNMYASIYFLTCISADRFLAIVHPVKSLKLRRPLYAHLACAFLWIVVAVAMAPLLVSPQTVQTNHTVV
rGPR17 HFSGNHWPFGEIPCRLTGFLFYLNMYASIYFLTCISADRFLAIVHPVKSLKLRRPLYAHLACAFLWIVVAVAMAPLLVSPQTVQTNHTVV
TM3 TM4
190 200 210 220 230 240 250 260 270
....|....|....|..___________________...|....|....|..______________________|....|....|....|
hGPR17 CLQLYREKASHHALVSLAVAFTFPFITTVTCYLLIIRSLRQGLRVEKRLKTKAVRMIAIVLAIFLVCFVPYHVNRSVYVLHYRSHGASCA
mGPR17 CLQLYREKASHHALASLAVAFTFPFITTVTCYLLIIRSLRQGPRIEKHLKNKAVRMIAMVLAIFLICFVPYHIHRSVYVLHYRGGGTSCA
rGPR17 CLQLYREKASHHALASLAVAFTFPFITTVTCYLLIIRSLRQGPRIEKHLKNKAVRMIAMVLAIFLICFVPYHIHRSVYVLHYRGGGTSCS
TM5 TM6
280 290 300 310 320 330 340
....|...._____________________....|....|....|....|....|....|....|....|
hGPR17 TQRILALANRITSCLTSLNGALDPIMYFFVAEKFRHALCNLLCGKRLKGPPPSFEGKTNESSLSAKSEL*
mGPR17 AQRALALGNRITSCLTSLNGALDPVMYFFVAEKFRHALCNLLCSKRLTGPPPSFEGKTNESSLSARSEL*
rGPR17 AQRALALGNRITSCLTSLNGALDPVMYFFVAEKFRHALCNLLCSKRLTGPPPSFEGKTNESSLSARSEL*
TM7
CysLT1
P2Y
1
P2Y
2
P2Y
4
P2Y
11
P2Y
12
P2Y
13
P2Y
14
P2Y
6
CysLT2
GPR17
0.2
䊱䊱
Figure 1 Sequence analysis of GPR17 and expression of the human and rat receptors. (A) Phylogenetic tree showing the relationships between
GPR17 and P2Yand CysLT receptors. (B) Alignment of human, mouse and rat amino-acid GPR17 sequences highlighting the seven TM domains
and the conserved H-X-X-R motif in TM6. (C) RT–PCR amplification of the human (1087 bp) or rat (1079 bp) cDNA sequences in brain, kidney,
heart and in human umbilical vein endothelial cells (HUVEC) and breast adenocarcinoma MCF7 cells. No signal was found in human
peripheral blood mononuclear cells (PBMC), bronchial smooth muscle cells (bSMC), myeloid U937, hepatocellular carcinoma Hep-G2, cervix
carcinoma (HeLa) and neuroblastoma SK-N-BE cells. Parallel expression of the housekeeping gene beta-actin is shown.
An orphan dualistic GPCR involved in ischemia
P Ciana et al
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Two distinct binding sites (one for nucleotides and the other
for CysLTs) seem to be present on GPR17 (Supplementary
Figures 2 and 3 and Supplementary Results).
As both P2Y (Communi et al, 2001; Abbracchio et al, 2003;
Fumagalli et al, 2003) and CysLT receptors (Brink et al, 2003;
Capra et al, 2006) may couple to G-proteins of the Gi
subfamily, 1321N1 cells expressing hGPR17 were incubated
with the Gi-protein inhibitor pertussis toxin (PTX) before
membrane preparation and [
35
S]GTPgS binding. PTX strongly
inhibited [
35
S]GTPgS binding stimulated by UDP, UDP-galac-
tose, UDP-glucose and LTD
4
, thus establishing an essential
role for Gi proteins in GPR17 responses (Figure 4A). As Gi
proteins inhibit adenylyl cyclase and cAMP formation
(Milligan and Kostenis, 2006), we next evaluated the possible
coupling of GPR17 to cAMP inhibition and the effect of PTX
on this coupling. In 1321N1 cells expressing hGPR17, UDP,
UDP-glucose, UDP-galactose and LTD
4
concentration-depen-
dently inhibited the cAMP formation elicited by 10 mM for-
skolin (FK) (Figure 4B). These effects were detected at mM
concentrations of nucleotides and nM concentrations of
LTD
4
, in line with the [
35
S]GTPgS binding data. No inhibition
of FK-stimulated cAMP levels was obtained in cells trans-
fected in parallel with the empty plasmid (Figure 4C), con-
firming that agonist responses are specifically due to GPR17.
In line with Figure 4A data, pretreatment of GPR17-expres-
sing cells with PTX fully obliterated the agonists effects
on FK-induced cAMP formation (Figure 4D). Finally, as
P2Y
12,13,14
and CysLT
1
receptors can also couple to phospho-
lipase C and increase intracellular calcium ([Ca
2 þ
]
i
) likely
via Gi-protein bg subunits (Communi et al, 2001; Abbracchio
et al, 2003; Fumagalli et al, 2003; Hardy et al, 2004; Capra
et al, 2005, 2006), responses of GPR17 were also investigated
by single-cell calcium imaging. Approximately 30% of
1321N1 cells expressing hGPR17 responded to UDP-glucose,
RT: + – + –
-
1087 bp
–11 –10 –9 –8 –7 –6 –5 –4
Agonist concentration log[M]
[
35
S]GTPγS binding
(% over basal value)
A
g
onist concentration lo
g
[M]
–
4
[
3
5
S]GTPγS binding
(% over basal value)
A
g
onist concentration lo
g
[M]
[
35
S]GTPγS binding
(% over basal value)
Empty
vector
hGPR17-
pcDNA3.1
UDP = 1.14± 0.2 µM
UDP-glucose = 12± 1.1 µM
UDP-galactose = 1.1± 0.09 µM
LTD
4
= 7.2± 0.3 nM
LTC
4
= 0.33 ± 0.011 nM
UDP = 4.6± 0.6 µM
UDP-glucose = 530± 24 nM
LTD
4
= 5.9± 0.4 nM
LTC
4
= 65± 4.2 nM
LTD
4
= 4.4± 0.4 nM
LTC
4
= 14.8± 0.9 nM
–11 –10 –9 –8 –7 –6 –5 –10 –9 –8 –7 –6 –5
75
100
125
150
175
75
100
125
150
175
75
100
125
150
175
RT: + – + –
- 1079 bp
rGPR17-
pcDNA3.1
Empty
vector
UDP = 1.06± 0.3 µM
UDP-glucose = 9.5± 1.1 µM
UDP-galactose = 729± 42 nM
A
C
EF
B
D
hGPR17, 1321N1 cells
rGPR17, 1321N1 cells
hGPR17, COS-7 cells hGPR17, HEK-293 cells
–12 –11 –10 –9 –8 –7 –6 –5 –4 –3
75
100
125
150
175
[
35
S]GTPγS binding
(% over basal value)
Agonist concentration log[M]
Figure 2 GPR17 agonist specificity in [
35
S]GTPgS binding. (A, B) Agonist-response curves to CysLTs and nucleotides in 1321N1 cells
expressing hGPR17 (A), as shown by the presence of a specific 1087 bp amplification product (B). No products were detected in the absence of
retrotranscription (RT) (indicated as –RT), nor in cells transfected with the empty vector. (C, D), same as (A) and (B) for rGPR17 (RT–PCR
product of 1079 bp). (E) Responses to CysLTs in COS-7 cells expressing hGPR17. (F) Responses to nucleotides in HEK-293 cells expressing
hGPR17. Each point is the mean7s.d. of 4–9 independent experiments run in triplicate. For each agonist, EC
50
values are also reported.
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UDP, UDP-galactose or LTD
4
with increases of [Ca
2 þ
]
i
(Figure
5A–D). No responses were detected in cells transfected with
the empty vector (Figure 5E–H). In approximately 50% of
COS-7 cells expressing hGPR17, LTD
4
induced a [Ca
2 þ
]
i
response (Figure 5J) similar to that observed in cells expres-
sing hCysLT
1
receptor, here utilized as a positive control
(Figure 5I). These responses are independent of extracellular
calcium and due to calcium release from intracellular stores,
as expected for a GPCR (Supplementary Figure 4 and
Supplementary Results). Taken together, these results demon-
strate that GPR17 is a dualistic receptor specifically respond-
ing to two unrelated families of inflammatory molecules,
nucleotides and CysLTs. This novel dualistic receptor seems
different from that studied by Mellor et al (2001), as it was
not found in a human mast cell line (HMC-1 cells) nor in
mast cells isolated from cord blood mononuclear cells (see
Supplementary Figure 5 and Supplementary Results). GPR17
is coupled to Gi proteins and, in a similar way to many GPCRs
(Milligan and Kostenis, 2006), leading to both adenylyl
cyclase inhibition and [Ca
2 þ
]
i
increases.
Neuronal GPR17 expression in brain is markedly
increased after ischemia
To shed light on GPR17 pathophysiological roles, we next
developed an anti-GPR17 antibody for immunohistochemical
studies. Rabbits were immunized with a peptide derived from
the C-terminal region of the human receptor, which is highly
similar (94% identical) to the corresponding region of the rat
receptor. Sera from immunized rabbits specifically recog-
nized both human and rat GPR17 and did not crossreact
with other receptors of the P2Y family (e.g., P2Y
1
and P2Y
13
;
data not shown). In line with the pharmacological data
(Figure 2), positive immunoreactivity to antisera was
observed in 1321N1 (Figure 6B) or COS-7 cells (Figure 6E)
expressing rGPR17, but not in corresponding control cultures
transfected with the empty vector (Figure 6A and D). In both
cell systems, labeling was abolished by preabsorption of
antisera with the peptide used for immunization (Figure 6C
and F), confirming specificity of immunostaining. Based on
expression data (Figure 1), we next utilized this antibody to
study the cellular localization of GPR17 in rat cortical slices.
Confocal microscopy showed positive immunoreactivity
(green fluorescence) in the soma of a consistent number of
cortical cells (Figure 6G). No colocalization with glial fibril-
lary acidic protein (GFAP)-positive cells (red fluorescence
in Figure 6G) was found (see also Supplementary Results),
suggesting that GPR17 is not expressed by astrocytes.
Conversely, several GPR17-expressing cells also positively
stained for neuronal markers, such as SMI-311 or b-III tubulin
(yellow fluorescence in Figure 6H and I, respectively), which,
however, did not systematically colocalize with GPR17 (sug-
gesting that receptor expression is restricted to specific
neurons; see also Supplementary Results). As CysLTs and
nucleotides accumulate to a greater extent in ischemic brain
(Ohtsuki et al, 1995; Ciceri et al, 2001; Burnstock and Knight,
2004), based on the present data demonstrating the in vitro
activation of GPR17 by both families of endogenous ligands
(Figure 2), we next analyzed the role of GPR17 in an
established model of ischemic damage (the permanent mono-
lateral middle cerebral artery occlusion in the rat, MCAo).
A
Cangrelor = 0.7 ± 0.02 nM
MRS2179 = 508 ± 29 nM
hGPR17
Cangrelor = 22 ± 1.5 pM
MRS2179 = 0.18 ± 0.02 pM
rGPR17
75
100
125
–12.5 –10.0 –7.5 –5.0 –2.5
150
90
100
110
120
130
140
–15.0 –12.5 –10.0 –7.5
150
Antagonist concentration log[M]
[
35
S]GTPγS binding
(% over basal value)
[
35
S]GTPγS binding
(% over basal value)
[
35
S]GTPγS binding
(% over basal value)
[
35
S]GTPγS binding
(% over basal value)
B
Antagonist concentration log[M]
D
Montelukast = 196 ± 13 nM
Pranlukast = 31 ± 2.4 nM
rGPR17
–11 –10 –9 –8 –7 –6 –5 –4
90
100
110
120
130
140
150
Antagonist concentration log[M]
C
Montelukast = 60 ± 4.3 nM
Pranlukast = 10.5 ± 1.2 nM
hGPR17
–11 –10 –9 –8 –7 –6 –5 –4
75
100
125
150
Antagonist concentration log[M]
Figure 3 Effect of P2Y and CysLT
1
receptor antagonists on the activation of recombinant GPR17 in [
35
S]GTPgS binding. (A) Antagonism of
UDP-glucose stimulation of [
35
S]GTPgS binding by the indicated P2Y antagonists in 1321N1 cells expressing hGPR17. (B) Same as in (A), in
1321N1 cells expressing rGPR17. (C) Antagonism of LTD
4
stimulation of [
35
S]GTPgS binding by indicated CysLT
1
antagonists in 1321N1 cells
expressing hGPR17. (D) Same as in (C), in 1321N1 cells expressing rGPR17. Flat concentration–response curves in (A–D) refer to cells
transfected with the pcDNA3.1 empty vector. Each point is expressed as percentage of basal [
35
S]GTPgS-specific binding set to 100% and is the
mean7s.d. of 4–7 independent experiments run in triplicate. For each antagonist, IC
50
values are reported.
An orphan dualistic GPCR involved in ischemia
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& 2006 European Molecular Biology Organization The EMBO Journal VOL 25
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2006 4619

Forty-eight hours after ischemia, in the cortex of ischemic
rats, GPR17-immunoreactivity was markedly increased both
within (Figure 6J) and at the borders (Figure 6K) of the
ischemic infarct (gray area in the brain section drawing of
Figure 6), in comparison with corresponding cortical areas of
the unlesioned contralateral hemisphere (Figure 6L and M).
Within the ischemic lesion, GPR17 was highly expressed in
cells resembling pyramidal neurons (see inset of Figure 6J),
as also confirmed by costaining with SMI-311 (Figure 6N) (see
also Supplementary Results).
In vivo knockdown of dualistic GPR17 prevents
evolution of ischemic brain damage
Based on increased GPR17 expression after MCAo (Figure 6),
we next hypothesized that GPR17 activation during ischemia
may contribute to injury development. If this were true,
agents such as montelukast or cangrelor, which effectively
antagonize GPR17 in vitro (Figure 3), should contrast the
in vivo actions of GPR17 and attenuate brain damage evolution.
Moreover, we anticipated that agents interfering with either
one or both the signaling components of a dualistic receptor
50
75
100
125
150
175
– PTX
+ PTX
UDP
10 µM 50 µM
UDP-galactose
10 µM 50 µM
UDP-glucose
10 µM 50 µM
LTD4
10 nM 100 nM
[
35
S]GTPγS-specific binding
(% basal value)
A
D
50
60
70
80
90
100
1 µM
10 µM
50 µM
UDP
1 µM
10 µM
50 µ
M
1
µM
10
µ
M
50 µM
1 nM
10 nM
50 nM
UDP-
glucose
UDP-
galactose
LTD
4
FK
cAMP (% versus forskolin)
100
125
150
50
75
FK
UDP 50 µM
UDP-glucose 50 µM
UDP-galactose 50 µM
LTD
4
100 nM
cAMP (% versus forskolin)
BC
50
60
70
80
90
100
110
120
1 µM
50 µM
UDP
1 µM
50 µM
1 µM
50 µM
1 nM
10 nM
UDP-
g
lucose
UDP-
g
alactose
LTD
4
cAMP (% versus forskolin)
FK
Figure 4 A key role for Gi proteins and adenylyl cyclase in GPR17 function. (A) Pre-incubation of 1321N1 cells expressing the human receptor
with the Gi-protein inhibitor PTX (black columns) significantly reduced stimulation of [
35
S]GTPgS binding by the indicated agonists with
respect to untreated cells (white columns) (Po0.001). Data are the mean of three experiments run in triplicate. (B, C) Effect of uracil
nucleotides and LTD
4
on FK-stimulated cAMP accumulation. 1321N1 cells transfected with either hGPR17 (B) or with corresponding empty
plasmid (C) were treated with the indicated agonists in the presence of 10 mM FK. cAMP levels (means7s.e.m.; n ¼ 6) were quantified and
expressed as percentage of cAMP in the presence of FK alone set to 100% (white bar in graphs). A concentration-dependent inhibition of FK-
stimulated cAMP production was detected only in hGPR17-expressing cells. (D) Preincubation of 1321N1 cells expressing hGPR17 with PTX
under the same conditions reported in (A) before agonist treatment fully obliterated the effects of uracil nucleotides and LTD
4
on FK-stimulated
cAMP formation. Basal and FK-stimulated cAMP levels were 11.571.2 and 89.576.5 pmol/ml, respectively (values refer to 10 mg of protein/
sample and a 10 min incubation of intact cells with either medium alone or medium þ FK).
An orphan dualistic GPCR involved in ischemia
P Ciana et al
The EMBO Journal VOL 25
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2006 & 2006 European Molecular Biology Organization4620

should be able to very robustly modulate its in vivo activity.
Magnetic resonance imaging (MRI) of developing damage
showed that, after MCAo, brain infarct volume in lesioned
hemispheres increased dramatically between 2 and 48 h with
respect to the contralateral unlesioned side (see C1, C2 and
C3 in Figure 7). In vivo treatment of ischemic animals with
either montelukast or cangrelor markedly prevented increase
of damage at 24 and 48 h with respect to 2 h (Figure 7A and
B), suggesting that activation of GPR17 during ischemia
indeed contributes to injury development. However, as mon-
telukast and cangrelor are also antagonists at CysLT
1
and
P2Y
12,13
receptors, respectively, and some of these receptors
are expressed in the brain (Ingall et al, 1999; Brink et al, 2003;
Marteau et al, 2003; Fumagalli et al, 2004), to prove the
specific involvement of GPR17 in ischemia and to test the
efficacy of selective in vivo receptor knockdown, we utilized
an antisense oligonucleotide strategy (Stein, 2001), which has
been proven to very efficiently downregulate other GPCRs in
F
340/380
F
340/380
F
340/380
COS-7 cells Transfected with
hCysLT
1
-pcDNA3.1 Transfected with hGPR17-pcDNA3.1
Transfected
with pcDNA3.1
IJ K
Time
(
s
)
Time
(
s
)
Time
(
s
)
100 nM LTD
4
0 100 200 300 400
0.6
0.7
0.8
0.9
50 nM LTD
4
100 nM LTD
4
60 120 180 420 480 0
0.7
0.8
0.9
1.0
1.1
1.2
1.3
100 nM LTD
4
300 200 100 0
0.6
0.8
0.7
0.9
1.0
1.1
1.2
F
340/380
F
340/380
F
340/380
F
340/380
1321N1 cells transfected with hGPR17-pcDNA3.1
100 nM LTD
4
0 100 200
0.6
0.7
0.8
0.9
1.0
1.1
1.2
Time (s)
AB
Time (s)Time (s)
100 200
50 µM UDP
0
0.7
0.8
0.9
1.0
100 µM UDP-glucose
0 50 100 150
0.7
0.8
0.9
1.0
CD
Time (s)
0.9
0.8
50 µM UDP-galactose
1000
0.75
0.85
F
340/380
F
340/380
F
340/380
F
340/380
0
100 µM UDP-glucose
100 200 300
0.7
0.8
0.9
1.0
1.1
1.2
1 mM UDP-galactose
100 200 3000
0.7
0.8
0.9
1.0
100 nM LTD
4
1321N1 cells transfected with pcDNA3.1
EFGH
Time (s) Time (s) Time (s) Time (s)
100 200 3000
0.7
0.8
0.9
1.0
1.1
1.2
100 µM UDP
100 2000
0.7
0.8
0.9
1.0
1.1
1.2
Figure 5 Single-cell calcium imaging in cells expressing hGPR17. Each trace shows response recorded from one single cell. (A–C)
Approximately 30% of 1321N1 cells expressing hGPR17 showed responses to uracil nucleotides (mean calcium response to UDP-glucose:
DF
340/380
¼ 0.2370.06, mean7s.e.m., n ¼ 15; mean calcium response to UDP: DF
340/380
¼ 0.1270.01, n ¼ 11; mean calcium response to UDP-
galactose: DF
340/380
¼ 0.0570.003, n ¼ 3) or (D)toLTD
4
(DF
340/380
¼ 0.2670.05, mean7s.e.m., n ¼ 18). (E–H) The same agonists induced no
responses in cells transfected with the empty plasmid. (I) Approximately 45% of COS-7 cells transfected with hCysLT
1
receptor showed calcium
transients to LTD
4
(mean calcium response: DF
340/380
¼ 0.370.08, mean7s.e.m., n ¼ 17). (J) Similar responses were recorded from
approximately 50% of COS-7 cells expressing hGPR17 (mean calcium response: DF
340/380
¼ 0.1870.03, mean7s.e.m., n ¼ 22). (K)No
responses to LTD
4
were recorded in cells transfected with the empty plasmid.
An orphan dualistic GPCR involved in ischemia
P Ciana et al
& 2006 European Molecular Biology Organization The EMBO Journal VOL 25
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NO 19
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2006 4621

the brain (Tepper et al, 1997; Van Oekelen et al, 2003). Of
several antisense oligonucleotides designed on rGPR17
sequence, only oligo616 and, to a lesser extent, oligo241
reduced the in vitro expression of rGPR17 in HEK-293 cells
(Supplementary Figure 6), and were thus selected for the
in vivo MCAo study. In a similar way to montelukast and
cangrelor, intracerebroventricular (i.c.v.) injections of
oligo616 to ischemic rats 48 and 24 h before and 10 min
after MCAo markedly attenuated infarct size evolution
(Figure 7). A smaller protective effect was observed with
oligo241 (data not shown), which targeted a different
sequence on rGPR17. Bioinformatic analysis showed that
the two antisense oligonucleotides that effectively prevented
brain infarct evolution were not homologous to any other
GPCR (see Materials and methods). This, together with the
absence of activity of a ‘scrambled’ oligonucleotide both
in vivo (Figure 7) and in vitro (Supplementary Figure 6),
strongly indicates that GPR17 is indeed the specific target of
Figure 6 GPR17 immunoreactivity in transfected cells and in rat brain. (A–C) 1321N1 cells transfected with empty vector (A, control cells) or
rGPR17-pcDNA3.1 (B, transfected cells) were incubated with anti-GPR17 antisera in the absence or presence (C) of the peptide used for rabbit
immunization (neutralizing peptide). (D–F) Same as in (A–C) on COS-7 cells. (G–I) Double-labeling of rat cortical slices with anti-GPR17
antibody (green fluorescence) and astroglial (G, GFAP) or neuronal markers (H, SMI-311 or I, b-III tubulin) (red fluorescence). (J–N)At48h
after MCAo, rat brain slices were incubated with anti-GPR17 antisera followed by either horseradish peroxidase staining (J–M) or double-
staining with anti-SMI-311 (N). Micrographs were taken inside the ischemic area (J, gray area in left lesioned hemisphere) or at its borders (K),
or in the corresponding healthy brain areas of the contralateral unlesioned right hemisphere (L, M), as indicated by squares in the drawing of
coronal brain section. Inside the ischemic infarct, GPR17 immunoreactivity was found on pyramidal-like cells (higher magnification inset in J),
which were also positive for SMI-311 (N). Similar data were obtained in five experiments (24 brain sections in total). Scale bars 30 m m.
An orphan dualistic GPCR involved in ischemia
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oligo616 and oligo241. Importantly and rather surprisingly,
GPR17 knockdown by a single i.c.v. injection of oligo616
10 min after MCAo also resulted in comparable brain protec-
tion (Figure 7). This result might have important therapeutic
implications for human stroke, also in consideration of the
mechanism of action of antisense oligonucleotides, which, at
variance from antagonist receptor ligands, need time to exert
their effects. Thus, GPR17 may represent a novel target for
development of new relevant therapeutic approaches to
human stroke. Taken together, these data suggest that
GPR17 activation by CysLTs and nucleotides during ischemia
contributes to development of brain damage. In line with this
hypothesis, GPR17 knockdown by either pharmacological
agents or antisense technology results in robust protection
against brain damage, even when receptor inhibition is
accomplished after ischemia is induced.
Discussion
It is generally believed that each neurotransmitter/hormonal
receptor specifically and selectively responds to one single
endogenous ligand or a family of structurally related ligands
(Kenakin et al, 1992; Kenakin, 2004). Recently, two GPCRs
(the ALX and ChemR23 receptors) responding to lipid-
derived ligands have been reported to also recognize specific
peptides (Chiang et al, 2000; Arita et al, 2005). Mellor et al
(2001) were the first to postulate the existence of dualistic
receptors, by showing that both CysLT
1
and a yet unidentified
LTC
4
-preferring receptor (there referred to as the CysLT
3
receptor) mediated dual responses to both CysLTs and UDP.
Here, by heterologous expression of the previously orphan
receptor GPR17 in several cell lines (see Results and
Supplementary Discussion), we identify and fully character-
ize this receptor as a dualistic GPCR responsive to two
unrelated families of non-peptide small ligands (nucleotides
and CysLTs). Phylogenetically, GPR17 is located at an inter-
mediate position between ligand-specific receptor families
and is the closest receptor to a common ancestor that also
gave rise to the P2Y and CysLT families. In a similar way to
P2Y
12,13,14
and CysLT
1
receptors, GPR17 is coupled to Gi, and
its activation can inhibit cAMP formation and also stimulate
intracellular calcium release. The need for more selective,
organ- and cellular-specific responses to either extracellular
nucleotides or CysLTs may have driven the evolution of this
family towards the more specialized P2Yand CysLT receptors,
in parallel with a restriction of ligand selectivity. In line with
this hypothesis, both montelukast and pranlukast, two ‘spe-
cific’ CysLT
1
antagonists, also interfere with P2Y receptor
signaling (Mamedova et al, 2005). GPR17 does not seem to be
the elusive receptor described by Mellor and co-workers, as it
was not found in mast cells, suggesting that other dualistic
receptors remain to be characterized. GPR17 was instead
found to be highly expressed in organs that can typically
undergo ischemic injury (brain, heart and kidney) and may
Figure 7 Effect of montelukast (Mtk), cangrelor (Cang) and oligo616 on evolution of brain infarct size as determined by MRI at 2, 24 and 48 h
after MCAo. (A) Representative Tr(D) images of coronal brain sections from ischemic rats treated with either Mtk, Cang, oligo616 (616) or
scrambled oligonucleotide (Scr) in comparison with corresponding control animals treated with vehicle only (C1, C2, C3). Antisense
oligonucleotides were administered 48 and 24 h before and 10 min after MCAo (indicated as ‘pre’); in some experiments, animals only
received a single dose of antisense oligonucleotide (indicated as ‘post’, also see below). In left lesioned hemispheres, ischemia-associated brain
damage is shown by black areas. (B) Quantitative analysis of infarct size volume at 24 and 48 h after MCAo from rats receiving vehicle (C1,
n ¼ 5) or Mtk (2 mg/kg intravenously (i.v.), 10 min after MCAo; n ¼ 6); vehicle (C2, n ¼ 5) or Cang (4.5 mg/animal, i.c.v., 10 min after MCAo,
n ¼ 5), vehicle (C3, n ¼ 5) or either oligo616 or scrambled-oligo (400 ng/animal, i.c.v., 48 and 24 h before and 10 min after MCAo, n ¼ 5,
indicated as ‘pre’). Some animals received a single i.c.v. dose of 4.5 mg of either oligo616 (n ¼ 6) or scrambled-oligo (n ¼ 6) 10 min after
ischemia (indicated as ‘post’). Data are expressed as percentage variation of infarct volume at 24 and 48 h after MCAo compared to 2 h
considered as 100%.
y
Po0.05,
yy
Po0.01 versus 2 h; *Po0.05, **Po0.01 versus corresponding control animals;
#
Po0.05,
##
Po0.01 versus
corresponding Scr animals.
An orphan dualistic GPCR involved in ischemia
P Ciana et al
& 2006 European Molecular Biology Organization The EMBO Journal VOL 25
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thus represent a ‘primitive’, evolutionarily conserved, recep-
tor able to respond to gross tissue injuries (e.g., trauma and
ischemia) which do not require a very selective response. The
present data also add complexity to the already established
‘crosstalk’ between the purinergic and the leukotriene recep-
tor systems (see Introduction), suggesting GPR17 as an
additional means by which these two signaling systems
interact with each other.
The existence of dualistic receptors profoundly change the
current concept of receptor specificity, suggesting that the
same receptor may respond to either one or two families of
signaling molecules depending upon specific pathophysiolo-
gical conditions and/or ligand concentrations. In the case of
GPR17, not only are agonist-response profiles different from
those of both CysLTs (Brink et al, 2003; Capra et al, 2006) and
nucleotides (Abbracchio et al, 2003; Burnstock and Knight,
2004) (which suggests peculiar functions) but, most notably,
agonist EC
50
values are in the nM range for CysLTs and in mM
range for uracil nucleotides. Specifically for brain ischemia,
this might reflect the different modalities and times of accu-
mulation of these ligands after MCAo. In the same ischemia
model, levels of CysLTs in the lesioned cortex were sharply
increased 4 h after MCAo and rapidly declined afterwards
(Ciceri et al, 2001); extracellular concentrations of ATP were
instead constantly elevated starting from 20 min after MCAo
throughout a 220 min of microdialysis sampling, with a time
course similar to that of excitatory amino-acids (Melani et al,
2005). During this period, nucleotide release is likely to
originate from depolarized neurons or glial membrane chan-
nels (Melani et al, 2005). However, the local concentrations
of both ATP and other nucleotides in ischemic brain are likely
to further increase in the subsequent hours and days, as a
result of massive degradation of nucleic acids from damaged
and dying cells (Neary et al, 1996). Thus, at the site of injury,
neurons are likely exposed to high concentrations of nucleo-
tides for very prolonged periods of time. These data are also
consistent with the present results demonstrating that inhibi-
tion of GPR17 protects against brain damage even when
receptor knockdown is accomplished after the induction of
ischemia. Based on increased GPR17-expressing cells both
within and at the borders of the ischemic lesion 48 h after
MCAo, we speculate that GPR17 represents the common
molecular target of CysLTs and nucleotides that sensitizes
neurons to ischemic damage, thereby contributing to injury
propagation in the hours and days after ischemia. The
possibility of interfering with ischemia progression after
the ischemic insult has obvious relevant implications for
the development of innovative therapeutic approaches for
management of human stroke. Previous enthusiasm on anti-
stroke agents has been tempered by demonstration that the
efficacy of most neuroprotective agents diminishes quite
rapidly if they are given after the onset of ischemia (Huang
and McNamara, 2004). The present results might, thus,
generate novel neuroprotective strategies to counteract
damage evolution even after ischemia. More in general, we
anticipate that the existence of receptors responding to more
than one family of endogenous ligands will lead to the
development of novel dualistic pharmacological agents with
previously unexplored therapeutic potential. New chemical
entities targeting both components of dualistic receptors may
indeed prove extremely more effective in halting or prevent-
ing a variety of human diseases.
Materials and methods
Cell culture, transfection and treatments
Human astrocytoma cells (ADF cells), 1321N1, COS-7 and HEK-293
cells were cultured as described (Brambilla et al, 2000; Fumagalli
et al, 2004). For [
35
S]GTPgS, 10
6
1321N1, COS-7 or HEK-293 cells
were seeded on 75 cm
2
flasks and transfected by the calcium
phosphate precipitation method (Fumagalli et al, 2004). For calcium
imaging studies, 1321N1 and COS-7 cells were seeded on 2.4 cm
diameter glass coverslips (100  10
3
cells). In selected experiments,
cells were exposed to 100 ng/ml PTX (Sigma) for 18 h before
membrane preparation. For immunocytochemistry, 1321N1 and
COS-7 cells were seeded on 1.3-cm diameter glass coverslips
(1.5  10
4
cells) and transfected with FuGENE 6, according to the
manufacturer’s instructions.
For treatment of cultured cells with antisense oligonucleotides,
HEK-293 cells were utilized, based on their high transfection
efficiency. Briefly, 13 10
4
HEK-293 cells were seeded on 9 cm
2
dishes. On day 2 after plating, cells were transfected with pcDNA3.1
containing the construct encoding for rGPR17 together with the
neomycin resistance gene, here used as a reporter gene. The various
oligonucleotides described in Supplementary Figure 3 (all used at
0.3 mM final concentrations) were added to cells twice in a small
Fugene volume (125 ml): 16 and 40 h after transfection of rGPR17.
Twenty-four hours after the last Fugene addition, RNA was
extracted from cells as described below, and the transcripts for
both rGPR17 and the neomycin resistance gene determined as
specific RT–PCR amplification products of 1079 and 357 bp,
respectively.
Reagents
Culture media and sera were from Celbio. Reagents for RT–PCR,
cloning and transfection were from Invitrogen, with the exception
of FuGENE 6, which was from Roche Diagnostics. LTD
4
was
purchased from Cayman Chemical Co. (Ann Arbor, MI). Cangrelor
was kindly provided by The Medicines Company (Parsippany, NJ,
USA). Montelukast and pranlukast were a kind gift from MERCK &
Co (USA). The hCysLT
1
-pcDNA3.1 was a kind gift from Dr J Evans,
MERCK & Co (USA). Antisense oligonucleotides were selected
according to the general criteria for oligo design and synthesized by
MWG-Biotech AG. Thermodynamic criteria were set according to
Matveeva et al (2003) and care was taken to avoid internal loop,
palindrome of 6 or more base pairs, nucleotide repetition (more
than 3 base pairs), and where possible, an AGGG consensus
sequence shown to target RNase degradation was included (see
oligo616 and oligo241 in Supplementary Figure 3) (Smith et al,
2000). Oligo antisense sequence was mapped on GPR17 RNA
secondary structure predicted using GeneBee service (http://
www.genebee.msu.su/services/rna2_reduced.html) to choose oligo
mapping in the loop part of the hairpin; the scrambled oligonucleo-
tide was randomly generated on the basis of oligo616. We chose to
use unmodified oligonucleotides to avoid possible toxicity, while
the stability issue was faced by a multiple delivery experimental
design (see Figure 7 legend and text). Each oligo antisense sequence
was challenged with rat GenBank using BLAST (http://
www.ncbi.nlm.nih.gov/BLAST/) to exclude the presence of multi-
ple target sequences in the rat genome. All other reagents were from
Sigma-Aldrich.
Total RNA isolation and PCR analysis
Total RNA was extracted using the TRIZOL
s
Reagent (Invitrogen)
according to the manufacturer’s instructions. Retrotranscription to
cDNA and PCR reactions were carried out as described previously
(Fumagalli et al, 2004). The following primers were used to detect
the expression of human and rat GPR17, respectively:
Fw 5
0
-GACTCCAGCCAAAGCATGAA-3
0
and
Rw 5
0
-GGGTCTGCTGAGTCCTAAACA-3
0
;
Fw 5
0
-TAGACTTCTGCCTTCAGC-3
0
and
Rw 5
0
-TGACAGGACCTCCCCGAG-3
0
.
Cloning of human and rat GPR17
By using specific oligonucleotide PCR primers external to the open
reading frame (ORF) of the previously reported human receptor
sequence (GenBank accession no. U33447), we amplified a 1087 bp
product from human astrocytoma cells (ADF cells) and cloned it
into a pcDNA3.1 expression vector using the pcDNA3.1/V5-
An orphan dualistic GPCR involved in ischemia
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2006 & 2006 European Molecular Biology Organization4624

His
r
TOPO
s
TA Expression Kit (Invitrogen, Milan, Italy). With the
same strategy, we cloned the putative 1020 bp ORF of the rat
sequence (included in clone CH230-239E15, GenBank accession
no. AC112062) from rat brain. The sequence of the new cloned rat
receptor has been submitted to GenBank (accession no. DQ777767).
Constructs were verified by sequencing with the Applied Biosys-
tems Terminator cycle sequencing kit. A partial sequence of the
mouse ortholog of GPR17 is reported in GenBank (AY255543), and
the complete sequence, 98% identical to the rat receptor, was found
in a BAC clone (AC131761). Phylogenetic trees were generated with
the program Mega 2.1.
[
35
S]GTPcS binding assay
Control and transfected cells were homogenized in 5 mM Tris–HCl
and 2 mM EDTA (pH 7.4) and centrifuged at 48 000 g for 15 min at
41C. The resulting pellets (plasma membranes) were washed in
50 mM Tris–HCl and 10 mM MgCl
2
(pH 7.4) and stored at 801C
until used. Nucleotide-stimulated [
35
S]GTPgS binding in mem-
branes of cells expressing the human or rat receptor was performed
as described previously (Kotani et al, 2001; Marteau et al, 2003;
Fumagalli et al, 2004). For more information, see Supplementary
Materials and methods.
cAMP assay
Control and hGPR17-transfected 1321N1 cells were treated for
10 min with purinergic or leukotriene agonists in serum-free
medium containing the phosphodiesterase inhibitor Ro201724
(20 mM) in the absence or presence of 10 mM FK. Aliquots of cells
were pretreated with PTX before agonist treatments. Cells were then
harvested, lysed and assayed for cAMP accumulation using the
cAMP enzyme immunoassay system kit (Sigma-Aldrich), following
the manufacturer’s instructions.
Functional calcium imaging assay
Intracellular calcium concentrations ([Ca
2 þ
]
i
) were measured as
described previously (Fumagalli et al, 2003). At 48 h after
transfection, 1321N1, COS-7 and HEK-293 cells were loaded with
2 mM Fura-2 pentacetoxy methylester in Krebs–Ringer solution,
washed and transferred to the recording chamber of an inverted
microscope (Axiovert 100; Zeiss, NY) equipped with a calcium
imaging unit. In some experiments, agonists were applied in
calcium-free medium. Polychrome IV (TILL Photonics, Germany)
was used as the light source. Fura-2 and EGFP fluorescence images
were collected with a PCO Super VGA SensiCam (Axon Instruments,
Forest City, CA) and analyzed with the Axon Imaging Workbench
2.2 software (Axon Instruments). Images were acquired at 1–4
340/380 ratios/s.
Generation of an antibody against GPR17
Polyclonal antibodies recognizing human and rat GPR17 receptor
were raised in rabbits using a synthetic peptide corresponding to the
C-terminal region of the human receptor (amino acids (cg)SFEGKT-
NESSLSAKSEL) and 94.12% identical to the rat sequence (Figure 1).
The peptide was coupled to keyhole-limpet hemocyanin and
injected intradermally in rabbits using 200 mg of peptide/treatment.
Immunocytochemistry
Cells were fixed with 4% paraformaldehyde in 0.1 M phosphate
buffer, detergent permeabilized and incubated with anti-GPR17
polyclonal antibody (1:200) and anti-a-tubulin monoclonal anti-
body (1:5000; Sigma-Aldrich) followed by anti-rabbit-FITC-and
anti-mouse-TRITC-conjugated secondary antibodies (Jackson
Immunoresearch Laboratories, West Grove, PA, USA) (Verderio
et al, 1999). For negative controls, anti-GPR17 antibody and
neutralizing peptide were preincubated overnight at 41C before
addition to cells.
Histology and horseradish peroxidase immunohistochemistry
After dewaxing and rehydration, paraffin-embedded sections were
permeabilized in 10 mM sodium citrate and 0.05% Tween 20 (pH 6)
for 20 min at 901C and incubated in 0.01 M phosphate-buffered
saline pH 7.4 (10% normal goat serum, 0.1% Triton X-100).
Sections were then incubated with primary anti-GPR17 antibody
(1:200; see above) and stained for horseradish peroxidase
immunohistochemistry as described previously (Sironi et al, 2003).
Fluorescence immunohistochemistry
Sections treated and permeabilized as described above (Sironi et al,
2003) were double stained with anti-GPR17 polyclonal antibody
and anti-GFAP (1:1000; Sigma-Aldrich) or anti-SMI-311 (1:300;
Sternberger Monoclonal Inc.) or anti-b-III tubulin (1:1000; Prome-
ga) monoclonal antibodies at 41C overnight and then incubated
with anti-rabbit-FITC- and anti-mouse-TRITC-conjugated secondary
antibodies (Verderio et al, 1999).
Confocal microscopy
Labeled cultures or brain sections were examined with a Radiance
2100 confocal laser scanning microscope (Bio-Rad, Hemel Hemp-
stead, UK) mounted on a light microscope (Eclipse TE2000-S;
Nikon, Tokyo, Japan). Images were acquired using LaserSharp 5
software. For quantification of GPR17-expressing cells, see Supple-
mentary Materials and methods.
Induction of focal brain ischemia in the rat
Male Sprague–Dawley rats (Charles River) underwent permanent
MCAo as described previously (Tamura et al, 1981; Sironi et al,
2003). Procedures involving animals and their care at the
Department of Pharmacological Sciences of the University of Milan
respected the Institution’s guidelines, which comply with the
national and international rules and policies. Drug treatments were
as follows: montelukast (2 mg/kg, i.v., single bolus of 200 mlin
physiological solution) and cangrelor (4.5 mg/animal, i.c.v., 5 mlin
physiological solution) were administered 10 min after MCAo.
Cangrelor was used here to simply test the involvement of GPR17
in brain ischemia, and, being a very polar molecule likely to very
poorly permeate the blood–brain barrier, it was administered i.c.v.
Oligo616 and scrambled-oligo (400 ng in 5 ml of physiological
solution) were administered i.c.v. three times to each rat 48 and
24 h before and 10 min after MCAo. Some animals received a single
i.c.v. (4.5 mg of either oligo616 or scrambled-oligo) after ischemia
induction. Control groups received corresponding vehicle.
MRI analysis
MRI measurements were taken 2, 24 and 48 h after MCAo using a
4.7 T, vertical superwidebore magnet of a Bruker AMX3 spectro-
meter with microimaging accessory. Animal preparation, image
acquisition, trace of the diffusion tensor map computation,
ischemic volume determination and progression of the ischemic
damage over time were as described (Guerrini et al, 2002).
Statistical analysis
For [
35
S]GTPgS binding data, analysis and graphic presentation was
performed by the nonlinear multipurpose curve-fitting computer
program Graph-Pad Prism (GraphPad). For cAMP experiments, data
are mean7s.e.m. of six replicated from three independent experi-
ments and statistical analysis was performed by either Student’s
t-test or one-way analysis of variance (ANOVA) (multiple comparison
test).
For calcium imaging, data were normalized to the mean F
340/380
increase recorded in control cells. Data are presented as mean7
s.e.m. of 4–18 experiments run in triplicate. Statistical analysis was
performed by either Student’s t-test or one-way ANOVA (multiple
comparison test). Significance refers to results where Po0.05 was
obtained.
For NMR studies, data on progression of ischemic damage over
time were evaluated by ANOVA for repeated measures. For each
animal, the ischemic area at 2 h after ischemia induction was set to
100%, and the extension of the ischemic area at all other time
points was proportionally calculated, thus providing an internal
control of ischemia development. Then, variations of ischemic
volumes between animals and groups were compared. Data are
expressed as mean values7s.em. P-values o0.05 were considered
statistically significant.
Supplementary data
Supplementary data are available at The EMBO Journal Online
(http://www.embojournal.org).
Acknowledgements
We thank the Molecular Pharmacology Laboratory directed by
Professor Adriana Maggi at the Department of Pharmacological
An orphan dualistic GPCR involved in ischemia
P Ciana et al
& 2006 European Molecular Biology Organization The EMBO Journal VOL 25
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NO 19
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2006 4625

Sciences, University of Milan for the excellent support, and
Professor Marco Cattaneo, Professor Adriana Maggi, Dr Stefania
Ceruti and Dr Alessia Mazzola, University of Milan, for useful
discussion. We also thank Professor L Macchia (Department of
Allergology and Clinical Immunology, University of Bari, Italy) for
the kind gift of HMC-1 cells, Professor Joshua A Boyce (Department
of Medicine, Harvard Medical School, Boston, MA, USA) for the
kind gift of human mast cells from cord blood mononuclear cells
and Mrs Kerstin Griessmeier (University of Bonn, Germany) for
skilled technical assistance. This work was partially supported by
the Italian Ministry of Education (Project of National Research
Interest PRIN-COFIN 2002 and 2004, FIRB RBAUO19ZEN and
RBNE03YA3L to MPA, FIRB RBAU01AJTT001 to Professor Rodolfo
Paoletti, University of Milan), by the Italian CARIPLO Foundation
(2004 Projects ‘Promuovere la ricerca scientifica e tecnologica in tema
di salute e scienze della vita’ to GER, CVand MPA, project coordinated
by Professor Francesco Clementi, University of Milan, Italy) and by
EC FP6 funding to GER (LSHM-CT-2004-005033; this publication
reflects only the author’s views. The Commission is not liable for
any use that may be made of the information provided herein,
Cangrelor was a kind gift of The Medicines Company, Parsippany,
NJ, USA. PC, MLT, CV, GR, CM and MPA are inventors in the Italian
Patent Application #2004A002007 on ‘Modulatori del recettore GPR17
e loro impieghi terapeutici’ submitted on October 21, 2004.
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