Vol. 187, No. 2, 1992 September
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BIOCHEMICAL
16, 1992
Pages
CLONING AND FUNCTIONAL CHARACTERIZATION RECEPTOR Frederick Libertl, Czernilofsky', Helmut
OF A HUMAN Al
919-926
ADENOSINE
Jacqueline Van Sande', Anne Lefort', Jacques E. Dumont', Gilbert Vassart', A. Ensinger and Klaus D. Mendla3
Armin
'Institut de Recherche Interdisciplinaire and Service de Genetique Universite Libre de Bruxelles, Medicale, Faculte de Medecine, Campus Erasme, 808 route de Lennik, 1070 Bruxelles, Belgium 2Research
and Development,
Bender
and CO. GesmbH, Vienna,
Boehringer Research, 6507 Ingelheim/Rhein,
'Department Biochemical 173, Bingerstr.
Ingelheim Germany
Austria KG,
Received July 29, 1992 A human brain hippocampus cDNA library was screened by hybridization with a dog Al adenosine receptor cDNA probe. Sequencing of the resulting clones identified a 978 residue open reading frame encoding a 326 amino acid polypeptide showing 95.7% similarity with the dog Al adenosine receptor. Individual clones of stably transfected CHO cells expressing the human Al receptor were obtained and tested for their response to the Al agonist CPA [NG-cyclopentyladenosine] in the presence of forskolin. One clone was further characterized with respect to membrane binding of various adenosine agonists and antagonists. The rank order of affinities observed was typical of an Al adenosine receptor. A Kd 2.28 nM value of was determined using [3H]DPCPX [dipropylcyclopentyl-xanthine], an Al selective antagonist. 0 1992 ACadrmlC
Press,
Inc.
Adenosine
receptors
categories,
Al
adenosine
agonists
effect
recently
cyclase
"orphan" cloning Here
receptor
on the
while
A2
cDNAs,
receptor,
adenosine
cDNA clone,
receptors
and also
the
: Al
stimulate
isolation stable
919
of
expression
All
profile
of
receptors
inhibit
it
t21.
We have
mammalian cells This
from other
main
opposite
RDC7 and RDC8 [3], respectively.
two
on their
in different
receptors the
into
pharmacological
[l]
by expression
dog receptor
we present
classified
CAMP accumulation
adenosine of
been
and antagonists
characterized
and A2 [4,5] the
based
on intracellular
adenylyl two
and A2,
have
as an Al led the
species
a human Al of
the
Copyright 0 IY92 rights of reproduction
[4]
way to
[6,7,8,9]. adenosine recombinant
0006-291 X/92 $4.00 by Academic Press, Inc. in any form reserved.
Vol.
187,
No.
receptor its
in
coupling
human
2, 1992
Al
CHO to receptor
BIOCHEMICAL
(Chinese
AND
Hamster
a Gi protein. expressed
BIOPHYSICAL
Ovary)
RESEARCH
cells,
and
The pharmacological in
one
clonal
COMMUNICATIONS
evidence
behavior CHO
cell
for of
the
line
is
presented. MATERIALS
AND METHODS
Recombinant DNA. A human brain hippocampus cDNA library (Stratagene, La Jolla, CA, USA) was screened using a 2.3 kb cDNA fragment of the dog Al adenosine receptor containing the complete coding sequence [31. The same library was also screened with 5' and 3' sub-fragments of the same cDNA probe in order to select a full-length human cDNA clone. Isolation and sequencing of the cross-hybridizing clones were carried out according to standard methodologies [lo]. A recombinant clone containing the complete coding sequence was sequenced on both strands according to Sanger et al. Sequence alignment was performed as described [12]. [Ill. A 1.5 kb PstI-XbaI restriction fragment of the human Al adenosine receptor cDNA was cloned into the corresponding sites of the pSVL (Pharmacia, Milwaukee, WI, USA) eukaryotic expression vector. CHO cell transfection and cloning. CHO cells were co-transfected with the pSVL construct described above and the plasmid pSV2NEO using a modified calcium phosphate precipitation technique [13]. Neomycin resistant cell lines were selected in the presence of 0.4 mg/ml G418 and the resulting mixed population was subjected to limiting dilution to produce individual clonal cell lines. CHO cells were under standard grown culture conditions. Clones expressing the recombinant receptor were selected by measuring the decrease of CAMP accumulation induced by the addition of CPA to forskolin-stimulated CHO cells (see below). Bioassav. About 5.104 cells of each clone were seeded in glass tubes and further incubated for 24 h under standard culture conditions [141. Control cells (transfected with pSV2Neo alone) were treated similarly. The cells were pre-incubated in serum-free medium for 30 minutes in presence of 2u/ml adenosine deaminase. Incubation with 1 uM forskolin and increasing concentrations of CPA was performed in the presence of 2u/ml adenosine deaminase and 1 mM of the phosphodiesterase inhibitor Ro 20-1724 (gift from Hoffman-La Roche, Nutley, NJ, USA) for 20 minutes. The incubation was stopped by pouring off the medium and adding boiling water. CAMP levels were quantified by radioimmunoassay as described previously [15,16]. Membrane preparations. Cells from 4 plates (24.5 cm x 24.5 cm) grown to less than 50% confluency were harvested after rinsing the monolayer twice with ice-cold Ca+'- free phosphate buffered saline. All subsequent steps were performed on ice. Cells were scraped with a rubber policeman into a total volume of 30 ml of 25 mM sodium phosphate (pH 7.4) and 5 mM MgCl,. The suspended cells were lysed by sonication for 30 set (Branson sonifier) and centrifuged at 30,000 x g for 15 min at 4"~. The pellet was resuspended using a Dounce homogenizer and centrifuged again. The resulting crude membrane preparation was suspended in 50 ml of binding buffer (20 920
Vol.
187,
No.
2, 1992
mM HEPES-buffer were frozen concentrations
BIOCHEMICAL
in
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
pH 7.4, 10 mM MgCl, and 100 mM NaCl) and aliquots liquid nitrogen until used. Membrane protein were assayed according to the method of Peterson
[171.
Bindinq studies. Binding studies were conducted with [3H]DPCPX (120 Ci/mmol) (NEN-DuPont, Dreieich, Germany), [3H]CGS-21680 (47,2 Ci/mmol) (NEN-DuPont) and [3H]NECA Ci/mmol) (Amersham, (24 Braunschweig, Germany) as the radioligands. For competition binding experiments the following substances were tested : Caffeine, Theophylline, KFM 19 (Boehringer Ingelheim, Ingelheim, Germany); (-)PIA, (t)PIA, NECA (Boehringer Mannheim, Mannheim, Germany); CGS21680 (RBI, Cologne, Germany). Competition studies were performed with 0.5 nM [3H]DPCPX and 50 ug membrane protein in 1 ml binding buffer at 25"~ for 60 min. The non-specific binding was determined in the presence of 10 uM (-)PIA. Incubations were stopped by rapid filtration through Inotech GlO filters (Berthold, Wildbad, Germany) using an Inotech cell harvester and 48 or 96 well plates. The radioactivity was either measured in a liquid scintillation counter (Beckman, Munich, Germany) after adding 3 ml of Quickszint 2000 (Zinsser, Frankfurt, Germany) or using a two-dimensional autoradiograph (Berthold, Wildbad, Germany). The binding curves were evaluated for IC50values, Ki values, or dissociation constants (Kd) by using the software 'coupled mass equilibria' according to Rominger and Albert LIEI. RESULTS AND DISCUSSION A human hippocampus with
the
dog Al
adenosine
cross-hybridized
with
both
cDNA and was purified bp was identified
displayed of
dog Al
a very
the
dog adenosine
glycosylation could
acceptor
explain
the
iI91 I are present The definite a human Al analysis expected,
receptor site
in the
and binding recombinant
proof
human homologue
receptor properties receptor
frame
the
clone stable
expression 921
95.7%
This
already
of
978
coding clone
extension
devoid described
Two potential the
loop, bovine
Nwhich
receptor
(fig.1). we had isolated
has been provided of
this
(fig.1).
[3,4].
same
of
showed an overall
second extracellular
glycosylation that
of the
between
identity
subfamily
One clone
sub-fragments
(extracellular)
in the
observed
adenosine
of overlapping
a characteristic
sites,
[3,4].
An open reading
receptor
N-terminal
was screened
sub-fragment
Sequence comparison
adenosine
any N-glycosylation
for
and a 3'
94.2% residue
short
cDNA library
cDNA probe
sequencing
(fig.1).
including
similarity,
a 5'
from the
and the
receptor
to homogeneity.
in Ml3 derivatives region
Lambda ZAP II
by the
encoded functional
CHO transfectants. in
CHO cells
led
As to
an
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1 GAGCTCTGCCAGCTTTGGTGACCTTGGGTGCTTGGGTGcTTGccTcGTGccccTTGGTGcccGTcTGc ~
60
TGATGTGCCCAGCCTGTGCCC~CC~G~CG~CC~CCATCTCAGCTTTCCAGGCCGCCTAC DOG
A
E"
ISAFQAIAY
ATCGGCATCGAGGTGCTCATCGCCCTGGTCTCTGTGCCCGCGTGCTGGTGATCTGG I E " L IALVSVPGNVL v G
7%'
I
w
GCGGTGAAGGTGAACCAGGCGCTGCGGGATGCCACCTTCTGCTTC~TC~TG~CG~TG~CG RZAVKVNQALRDATFC 2F GTGGCTGATGTGGCCGTGGGTGCCCTGGTCATCCCCCTCGCCATCC~CATC~CATTGGG
22"
IPLAILINIG
E"
~"AD"A"GAL" CCACAGACCTyAC~TCCACACCTGCCTCATGGTTGCCTGTCCGGTCCTCATCCTCACCCAG HTC3LMVACPVL1L~Q 8 T
REP
180 32
360 92
AGCTCCATCCTGGCCCTGCTGGCAATTGCGGTGCGGTGGACCGCTACCTCCGGGTC~GATCCCT EMd&SSILALLAIAVD RYLRVKIP CTCCGGTACAAGATGGTGGTGACCCCCCGGAGGGCGGCGGTGGCCATAGCCGGCTGCTGG ~LRYKMVVTPRRAAVA I A G C T 4
W
480 132
ATCCTCTCCTTCGTGGTGGGACTGACCCCTATGTTTGGCTGG~C~TCTGAGTGCGGTG EzILSFVVGLTP;FGWN;L;;;
540 152
GAGCGGGCCTGGGCAGCCAACGGCAGCATGGGGGAGCCCGTGATCEAGTGCGAGTTCGAG E~~~RAWAANGS~GEFV~KC~F~
600 172
AAGGTCATCAGCATGGAGTACATGGTCTACTTCAACTTCTTTGTGTGGGTGCTCCCCCCG EO14&KVIS5MEYMVYFNFFVWVLPP CTTCTCCTCATGGTCCTCATCTACCTGGAGGTCTTCTACCT~TCCGC~GCAGCTC~C 1YLEVFYLIR;QL;
=Y%iLLLMVL
AAGAAGGTGTCGGCCTCCTCCGGCGACCCGCAGAAGTACTTC GDPQKYYGKELKI K " s A s s
B?EK
780 232
GCCRAGTCGCTGGCCCTCATCCTCTTC~TC~TT~CCTCAGC~G~TG~CTTTGCACATC EzAKS6LAL I L F L S L
H
I
CTCAACTGCATCACCCTCTTCTGCCCGTCCTGCCACEAGCCCAGCATCCTTACCTACATT TLFCPSCHKPSILTYI R 7 M GCCATCTTCCTCACGCACGGCAACTCGGCCATGAACCCCATTGTCTATGCCTTCCGCATC SAMNPIVYAFRI EYzAIFLTHGN
900 272
BTE!LNC1
960 292
CAGAAGTTCCGCGTCACCTTCCTTAAGATTTGGATTTGG~TGACCATTTCCGCTGCCAGCCTGCA E~QKFRVTFLKIWND HFRCQP? CCTCCCATT~AC~AG~AT~TC~CA~~~AG~GG~CTGAT~ACTAGACCCGCCTTCCGCTC l
HOP
p ; P A i * CCACCAGCCCACATCCAGTGGGGTCTCAGTCCAGTCCA~T~~T~~~~~~~C~~~~~~C~~A~~G~ TCTCCCTGAGCCTGCCCCAGCTGGGCTGTTGGGGGCGAG ATACCCACAGAGTGTGGTCCCTCCACTAGGAGTTAACTACCCTACACCTCTGGGCCCTGC AGGAGGC
840 252
1020 312
:;!z" 1140 1200 1260
1267
Fig.1. Nucleotide and deduced amino acid sequence of the human ~1 adenosine receptor. The non conserved amino acids between the dog and the human Al receptors are indicated. The putative transmembrane domains and the potential N-linked glycosylation sites in the second extracellular loop are underlined.
inhibition the
of
addition
resistance
forskolin-stimulated of
alone
same conditions Highly
keep a high
selective
distinguish
A Kd qf
about
expressing
derivative
(fig.2).
CHO cells level
of CAMP following
expressing
of intracellular
the
Neomycin
CAMP under
the
(fig.2).
clearly cells
CPA
accumulation
radioligands
between
2 nM was found the
L3H]DPCPX,
Al
using
recombinant a
have
been
and A2 binding
a membrane preparation
receptor
typical
of
that 211. CHO
and a dipropylxanthine
Al-adenosine 922
developped sites [l, 20,
receptor
Ligand
Vol.
No.
187,
BIOCHEMICAL
2,1992
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
.
LI : 0 -6 C -!3 -I log CPA concentration
0
Fiq.2. in a
A&F-
A-
(M)
Inhibition selected
by CPA of forskolin-stimulated CAMP accumulation CHO cell line expressing stably the human Al adenosine receptor. Results are expressed as percentage of maximal stimulation of CAMP accumulation observed with 1 gM forskolin alone.
[ZlI(Table using
1).
the
The rank is
No significant
A2 specific order
of potency
comparable
preparation
to from
that
is
shown in
in the after did
of
addition not
increased
3.
the
The two
apparent
of
receptor
Antagonists :
(-)PIA
: DPCPX > NECA >
GTP-analogue,
DPCPX as radioligand
affinity
of
to a single contrast, the
the
addition
(Table
receptor
low-affinity
antagonist
density
(nM)
shown)
and agonists
adenosine
[22].
antagonist
of
TABLE Kd
not
2).
states
affinity receptor
be detected
(Data
a non-hydrolysable
GppNHp, using
Al
Agonists
GppNHp. By way of the
an
(Table
could
antagonists
cortex
of GppNHp was shifted of
affect the
(-)PIA
figure
absence
for
> Caffeine.
of
[20]
tested
observed
>> CGS-21680
The influence on the binding
the
monkey cerebral
> (t)PIA
binding
[3H]CGS-21680
for
> KFM 19 >> Theophylline Adenosine
specific
ligand
1).
state of
GppNHp
[3H]DPCPX This
observation
1 Bmax
(pmol/mg)
['HIDPCPX
2.28
T!Z 0.72
1.92
+
0.52
(n=4)
[3H]DPCPX
2.13
? 0.16
3.78
2
0.35
(n=3)
+ GPPNHP binding constant of ['HldipropylcyclopentylTable 1. Equilibrium xanthine (DPCPX) and receptor density of membranes prepared from a selected CHO cell line expressing the human Al adenosine receptor (mean t S.D.). Effects of the addition of GppNHp (100 PM) are The number (n) of experiments performed is mentioned. indicated. 923
but
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
TABLE 2 Ki ANTAGONISTS
preparations effects
in
together
with
effective
the
rat
coupling
to
cyclase,
with
the
the
2 f k + -t
3,322 779 213 146 114
binding The
[231.
expression
functional
Considering similarity
for
brain
ectopic its
5,398 4,069 1.2 0.9
profile of the human Al adenosine CHO cell line.
reported
from
+ f 8.3 * 2.6 i
56,391 3,088 948 487 248
Table 2. Pharmacological in a stably transfected
previously
57,736 10,955
(n=4-5)
CGS21680 (+)PIA Adenosine NECA (-)PIA
has been
[3H1DPCPX
(n=4)
Caffeine Theophylline KFMlY DPCPX AGONISTS
(nM)
of
analysis Gi protein(s)
together
all
receptor,
pharmacological
of
are
indicative in
the
of
CHO cells. (sequence
activity
we conclude
these
receptor,
characteristics
biological
profile),
in membrane
recombinant
present its
[3H]XAC
demonstration
(fig.2)
the
dog
the
of
receptor
that
on adenylyl the
receptor
% BINDING
0 0.01
1 L-)PIA
100
10,000
lXllOl/l
concentration
Fic.3. Displacement of ["HIDPCPX binding to human Al adenosine receptor expressed in CHO cells by (-)PIA. Without (*) and in presence Shown is a representative (+) Of 100 PM GppNHp. experiment, each point has been done in duplicate. 924
an
Vol.
187,
we
No.
2, 1992
have
cloned
receptor
is
that Cloning
of Al the
receptor further
subfamily.
of
the
human Al cell
development
of
approached to
in adenosine when
as
the
has
of been
in
receptor and
in
is
its
for
coupling be
more
studies
are
activated tissue
by
ambient
the
types
the
normal
activation
cell
at
could
further
for
receptor
dissection,
Also,
reported
mice in vivo
this
modulation
Al
tool
G protein
permanent
various
and production of
the
lines.
adenosine
an invaluable
interaction,
CHO cells
expressed
of transgenic
provide
pharmacology
cell
if
The observation
endogenous
lines
pathway
the
Al
the
receptor
such
these
of
adenosine
receptor
determine
environment.
thyroid
using
COMMUNICATIONS
[1,2].
the
intracellular
RESEARCH
previously
applications of ligand
BIOPHYSICAL
counterpart
described
expressing
'constitutively'
receptor
human
been
level,
required
AND
has
subsequent
simply
the
Other
molecular and
BIOCHEMICAL
A2 [5]
adenosine and
in
the
[24].
ACKNOWLEDGMENTS We are grateful to C. Massart and Y. Mauquois for technical assistance. Supported by Boehringer Ingelheim, the Ministere de la Politique Scientifique (Interuniversity pole of attraction), the Fonds de la Recherche Scientifique Medicale (FRSM) and the Association Recherche Biomedicale et Diagnostic. The scientific responsibility is assumed by the authors. F.L. is Charge de Recherches of the Belgian FNRS. REFERENCES 1. Bruns, R.F., 29, 331-346. 2. Van Calker, Neurochem.
Lu,
G.H.
and Pugsley,
D., Muller, 33, 999-1005.
T.A.
(1986)
Mol.
B.
(1979)
M. and Hamprecht,
Pharmacol. J.
3. Libert, F., Parmentier, M., Lefort, A., Dinsart, C., Van Sande, J ., Maenhaut, C., Simons, M.J., Dumont, J.E. and Vassart, G. (1989) Science 244, 569-572. 4.
Libert, F., Gerard, C., 1677-1682.
5. Maenhaut, Parmentier, G. (1990)
Schiffmann, S.N., Lefort, Dumont, J.E. and Vassart,
A., G.
Parmentier, M., (1991) EMBO J. 10,
C., Van Sande, J., Libert, F., Abramowicz, M., M., Vanderhaeghen, J.J., Dumont, J.E. and Vassart, Biochem. Biophys. Res. Commun. 173, 1169-1178.
6. Mahan, L.C., MC Vittie, Monsma, F.J., Gerfen, Pharmacol. 40, l-7.
L.D., Smyk-Randall, C.R. and Sibley, D.R.
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M.E., (1992)
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18.Rominger, -420.
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lg.Stiles,
and Rivkees,
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J., Ludgate, M., Libert, G. and Parmentier, M. 171, 1044-1050.
15.Brooker, (1979)
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Linden, J., Robera, A.S., D'Angelo, (1992) FEBS Lett. 297, 107-111.
D.G. T., D.R.
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20.Jarvis, M.A. -893.
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G., Kufner-Muhl, (1991) Nucleosides
23.Freissmuth, M., 275, 651-656. 24.Ledent, EMBO J.
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G. and Parmentier,
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AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BIOCHEMICAL
16, 1992
Pages
CLONING AND FUNCTIONAL CHARACTERIZATION RECEPTOR Frederick Libertl, Czernilofsky', Helmut
OF A HUMAN Al
919-926
ADENOSINE
Jacqueline Van Sande', Anne Lefort', Jacques E. Dumont', Gilbert Vassart', A. Ensinger and Klaus D. Mendla3
Armin
'Institut de Recherche Interdisciplinaire and Service de Genetique Universite Libre de Bruxelles, Medicale, Faculte de Medecine, Campus Erasme, 808 route de Lennik, 1070 Bruxelles, Belgium 2Research
and Development,
Bender
and CO. GesmbH, Vienna,
Boehringer Research, 6507 Ingelheim/Rhein,
'Department Biochemical 173, Bingerstr.
Ingelheim Germany
Austria KG,
Received July 29, 1992 A human brain hippocampus cDNA library was screened by hybridization with a dog Al adenosine receptor cDNA probe. Sequencing of the resulting clones identified a 978 residue open reading frame encoding a 326 amino acid polypeptide showing 95.7% similarity with the dog Al adenosine receptor. Individual clones of stably transfected CHO cells expressing the human Al receptor were obtained and tested for their response to the Al agonist CPA [NG-cyclopentyladenosine] in the presence of forskolin. One clone was further characterized with respect to membrane binding of various adenosine agonists and antagonists. The rank order of affinities observed was typical of an Al adenosine receptor. A Kd 2.28 nM value of was determined using [3H]DPCPX [dipropylcyclopentyl-xanthine], an Al selective antagonist. 0 1992 ACadrmlC
Press,
Inc.
Adenosine
receptors
categories,
Al
adenosine
agonists
effect
recently
cyclase
"orphan" cloning Here
receptor
on the
while
A2
cDNAs,
receptor,
adenosine
cDNA clone,
receptors
and also
the
: Al
stimulate
isolation stable
919
of
expression
All
profile
of
receptors
inhibit
it
t21.
We have
mammalian cells This
from other
main
opposite
RDC7 and RDC8 [3], respectively.
two
on their
in different
receptors the
into
pharmacological
[l]
by expression
dog receptor
we present
classified
CAMP accumulation
adenosine of
been
and antagonists
characterized
and A2 [4,5] the
based
on intracellular
adenylyl two
and A2,
have
as an Al led the
species
a human Al of
the
Copyright 0 IY92 rights of reproduction
[4]
way to
[6,7,8,9]. adenosine recombinant
0006-291 X/92 $4.00 by Academic Press, Inc. in any form reserved.
Vol.
187,
No.
receptor its
in
coupling
human
2, 1992
Al
CHO to receptor
BIOCHEMICAL
(Chinese
AND
Hamster
a Gi protein. expressed
BIOPHYSICAL
Ovary)
RESEARCH
cells,
and
The pharmacological in
one
clonal
COMMUNICATIONS
evidence
behavior CHO
cell
for of
the
line
is
presented. MATERIALS
AND METHODS
Recombinant DNA. A human brain hippocampus cDNA library (Stratagene, La Jolla, CA, USA) was screened using a 2.3 kb cDNA fragment of the dog Al adenosine receptor containing the complete coding sequence [31. The same library was also screened with 5' and 3' sub-fragments of the same cDNA probe in order to select a full-length human cDNA clone. Isolation and sequencing of the cross-hybridizing clones were carried out according to standard methodologies [lo]. A recombinant clone containing the complete coding sequence was sequenced on both strands according to Sanger et al. Sequence alignment was performed as described [12]. [Ill. A 1.5 kb PstI-XbaI restriction fragment of the human Al adenosine receptor cDNA was cloned into the corresponding sites of the pSVL (Pharmacia, Milwaukee, WI, USA) eukaryotic expression vector. CHO cell transfection and cloning. CHO cells were co-transfected with the pSVL construct described above and the plasmid pSV2NEO using a modified calcium phosphate precipitation technique [13]. Neomycin resistant cell lines were selected in the presence of 0.4 mg/ml G418 and the resulting mixed population was subjected to limiting dilution to produce individual clonal cell lines. CHO cells were under standard grown culture conditions. Clones expressing the recombinant receptor were selected by measuring the decrease of CAMP accumulation induced by the addition of CPA to forskolin-stimulated CHO cells (see below). Bioassav. About 5.104 cells of each clone were seeded in glass tubes and further incubated for 24 h under standard culture conditions [141. Control cells (transfected with pSV2Neo alone) were treated similarly. The cells were pre-incubated in serum-free medium for 30 minutes in presence of 2u/ml adenosine deaminase. Incubation with 1 uM forskolin and increasing concentrations of CPA was performed in the presence of 2u/ml adenosine deaminase and 1 mM of the phosphodiesterase inhibitor Ro 20-1724 (gift from Hoffman-La Roche, Nutley, NJ, USA) for 20 minutes. The incubation was stopped by pouring off the medium and adding boiling water. CAMP levels were quantified by radioimmunoassay as described previously [15,16]. Membrane preparations. Cells from 4 plates (24.5 cm x 24.5 cm) grown to less than 50% confluency were harvested after rinsing the monolayer twice with ice-cold Ca+'- free phosphate buffered saline. All subsequent steps were performed on ice. Cells were scraped with a rubber policeman into a total volume of 30 ml of 25 mM sodium phosphate (pH 7.4) and 5 mM MgCl,. The suspended cells were lysed by sonication for 30 set (Branson sonifier) and centrifuged at 30,000 x g for 15 min at 4"~. The pellet was resuspended using a Dounce homogenizer and centrifuged again. The resulting crude membrane preparation was suspended in 50 ml of binding buffer (20 920
Vol.
187,
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2, 1992
mM HEPES-buffer were frozen concentrations
BIOCHEMICAL
in
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
pH 7.4, 10 mM MgCl, and 100 mM NaCl) and aliquots liquid nitrogen until used. Membrane protein were assayed according to the method of Peterson
[171.
Bindinq studies. Binding studies were conducted with [3H]DPCPX (120 Ci/mmol) (NEN-DuPont, Dreieich, Germany), [3H]CGS-21680 (47,2 Ci/mmol) (NEN-DuPont) and [3H]NECA Ci/mmol) (Amersham, (24 Braunschweig, Germany) as the radioligands. For competition binding experiments the following substances were tested : Caffeine, Theophylline, KFM 19 (Boehringer Ingelheim, Ingelheim, Germany); (-)PIA, (t)PIA, NECA (Boehringer Mannheim, Mannheim, Germany); CGS21680 (RBI, Cologne, Germany). Competition studies were performed with 0.5 nM [3H]DPCPX and 50 ug membrane protein in 1 ml binding buffer at 25"~ for 60 min. The non-specific binding was determined in the presence of 10 uM (-)PIA. Incubations were stopped by rapid filtration through Inotech GlO filters (Berthold, Wildbad, Germany) using an Inotech cell harvester and 48 or 96 well plates. The radioactivity was either measured in a liquid scintillation counter (Beckman, Munich, Germany) after adding 3 ml of Quickszint 2000 (Zinsser, Frankfurt, Germany) or using a two-dimensional autoradiograph (Berthold, Wildbad, Germany). The binding curves were evaluated for IC50values, Ki values, or dissociation constants (Kd) by using the software 'coupled mass equilibria' according to Rominger and Albert LIEI. RESULTS AND DISCUSSION A human hippocampus with
the
dog Al
adenosine
cross-hybridized
with
both
cDNA and was purified bp was identified
displayed of
dog Al
a very
the
dog adenosine
glycosylation could
acceptor
explain
the
iI91 I are present The definite a human Al analysis expected,
receptor site
in the
and binding recombinant
proof
human homologue
receptor properties receptor
frame
the
clone stable
expression 921
95.7%
This
already
of
978
coding clone
extension
devoid described
Two potential the
loop, bovine
Nwhich
receptor
(fig.1). we had isolated
has been provided of
this
(fig.1).
[3,4].
same
of
showed an overall
second extracellular
glycosylation that
of the
between
identity
subfamily
One clone
sub-fragments
(extracellular)
in the
observed
adenosine
of overlapping
a characteristic
sites,
[3,4].
An open reading
receptor
N-terminal
was screened
sub-fragment
Sequence comparison
adenosine
any N-glycosylation
for
and a 3'
94.2% residue
short
cDNA library
cDNA probe
sequencing
(fig.1).
including
similarity,
a 5'
from the
and the
receptor
to homogeneity.
in Ml3 derivatives region
Lambda ZAP II
by the
encoded functional
CHO transfectants. in
CHO cells
led
As to
an
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1 GAGCTCTGCCAGCTTTGGTGACCTTGGGTGCTTGGGTGcTTGccTcGTGccccTTGGTGcccGTcTGc ~
60
TGATGTGCCCAGCCTGTGCCC~CC~G~CG~CC~CCATCTCAGCTTTCCAGGCCGCCTAC DOG
A
E"
ISAFQAIAY
ATCGGCATCGAGGTGCTCATCGCCCTGGTCTCTGTGCCCGCGTGCTGGTGATCTGG I E " L IALVSVPGNVL v G
7%'
I
w
GCGGTGAAGGTGAACCAGGCGCTGCGGGATGCCACCTTCTGCTTC~TC~TG~CG~TG~CG RZAVKVNQALRDATFC 2F GTGGCTGATGTGGCCGTGGGTGCCCTGGTCATCCCCCTCGCCATCC~CATC~CATTGGG
22"
IPLAILINIG
E"
~"AD"A"GAL" CCACAGACCTyAC~TCCACACCTGCCTCATGGTTGCCTGTCCGGTCCTCATCCTCACCCAG HTC3LMVACPVL1L~Q 8 T
REP
180 32
360 92
AGCTCCATCCTGGCCCTGCTGGCAATTGCGGTGCGGTGGACCGCTACCTCCGGGTC~GATCCCT EMd&SSILALLAIAVD RYLRVKIP CTCCGGTACAAGATGGTGGTGACCCCCCGGAGGGCGGCGGTGGCCATAGCCGGCTGCTGG ~LRYKMVVTPRRAAVA I A G C T 4
W
480 132
ATCCTCTCCTTCGTGGTGGGACTGACCCCTATGTTTGGCTGG~C~TCTGAGTGCGGTG EzILSFVVGLTP;FGWN;L;;;
540 152
GAGCGGGCCTGGGCAGCCAACGGCAGCATGGGGGAGCCCGTGATCEAGTGCGAGTTCGAG E~~~RAWAANGS~GEFV~KC~F~
600 172
AAGGTCATCAGCATGGAGTACATGGTCTACTTCAACTTCTTTGTGTGGGTGCTCCCCCCG EO14&KVIS5MEYMVYFNFFVWVLPP CTTCTCCTCATGGTCCTCATCTACCTGGAGGTCTTCTACCT~TCCGC~GCAGCTC~C 1YLEVFYLIR;QL;
=Y%iLLLMVL
AAGAAGGTGTCGGCCTCCTCCGGCGACCCGCAGAAGTACTTC GDPQKYYGKELKI K " s A s s
B?EK
780 232
GCCRAGTCGCTGGCCCTCATCCTCTTC~TC~TT~CCTCAGC~G~TG~CTTTGCACATC EzAKS6LAL I L F L S L
H
I
CTCAACTGCATCACCCTCTTCTGCCCGTCCTGCCACEAGCCCAGCATCCTTACCTACATT TLFCPSCHKPSILTYI R 7 M GCCATCTTCCTCACGCACGGCAACTCGGCCATGAACCCCATTGTCTATGCCTTCCGCATC SAMNPIVYAFRI EYzAIFLTHGN
900 272
BTE!LNC1
960 292
CAGAAGTTCCGCGTCACCTTCCTTAAGATTTGGATTTGG~TGACCATTTCCGCTGCCAGCCTGCA E~QKFRVTFLKIWND HFRCQP? CCTCCCATT~AC~AG~AT~TC~CA~~~AG~GG~CTGAT~ACTAGACCCGCCTTCCGCTC l
HOP
p ; P A i * CCACCAGCCCACATCCAGTGGGGTCTCAGTCCAGTCCA~T~~T~~~~~~~C~~~~~~C~~A~~G~ TCTCCCTGAGCCTGCCCCAGCTGGGCTGTTGGGGGCGAG ATACCCACAGAGTGTGGTCCCTCCACTAGGAGTTAACTACCCTACACCTCTGGGCCCTGC AGGAGGC
840 252
1020 312
:;!z" 1140 1200 1260
1267
Fig.1. Nucleotide and deduced amino acid sequence of the human ~1 adenosine receptor. The non conserved amino acids between the dog and the human Al receptors are indicated. The putative transmembrane domains and the potential N-linked glycosylation sites in the second extracellular loop are underlined.
inhibition the
of
addition
resistance
forskolin-stimulated of
alone
same conditions Highly
keep a high
selective
distinguish
A Kd qf
about
expressing
derivative
(fig.2).
CHO cells level
of CAMP following
expressing
of intracellular
the
Neomycin
CAMP under
the
(fig.2).
clearly cells
CPA
accumulation
radioligands
between
2 nM was found the
L3H]DPCPX,
Al
using
recombinant a
have
been
and A2 binding
a membrane preparation
receptor
typical
of
that 211. CHO
and a dipropylxanthine
Al-adenosine 922
developped sites [l, 20,
receptor
Ligand
Vol.
No.
187,
BIOCHEMICAL
2,1992
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
.
LI : 0 -6 C -!3 -I log CPA concentration
0
Fiq.2. in a
A&F-
A-
(M)
Inhibition selected
by CPA of forskolin-stimulated CAMP accumulation CHO cell line expressing stably the human Al adenosine receptor. Results are expressed as percentage of maximal stimulation of CAMP accumulation observed with 1 gM forskolin alone.
[ZlI(Table using
1).
the
The rank is
No significant
A2 specific order
of potency
comparable
preparation
to from
that
is
shown in
in the after did
of
addition not
increased
3.
the
The two
apparent
of
receptor
Antagonists :
(-)PIA
: DPCPX > NECA >
GTP-analogue,
DPCPX as radioligand
affinity
of
to a single contrast, the
the
addition
(Table
receptor
low-affinity
antagonist
density
(nM)
shown)
and agonists
adenosine
[22].
antagonist
of
TABLE Kd
not
2).
states
affinity receptor
be detected
(Data
a non-hydrolysable
GppNHp, using
Al
Agonists
GppNHp. By way of the
an
(Table
could
antagonists
cortex
of GppNHp was shifted of
affect the
(-)PIA
figure
absence
for
> Caffeine.
of
[20]
tested
observed
>> CGS-21680
The influence on the binding
the
monkey cerebral
> (t)PIA
binding
[3H]CGS-21680
for
> KFM 19 >> Theophylline Adenosine
specific
ligand
1).
state of
GppNHp
[3H]DPCPX This
observation
1 Bmax
(pmol/mg)
['HIDPCPX
2.28
T!Z 0.72
1.92
+
0.52
(n=4)
[3H]DPCPX
2.13
? 0.16
3.78
2
0.35
(n=3)
+ GPPNHP binding constant of ['HldipropylcyclopentylTable 1. Equilibrium xanthine (DPCPX) and receptor density of membranes prepared from a selected CHO cell line expressing the human Al adenosine receptor (mean t S.D.). Effects of the addition of GppNHp (100 PM) are The number (n) of experiments performed is mentioned. indicated. 923
but
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
TABLE 2 Ki ANTAGONISTS
preparations effects
in
together
with
effective
the
rat
coupling
to
cyclase,
with
the
the
2 f k + -t
3,322 779 213 146 114
binding The
[231.
expression
functional
Considering similarity
for
brain
ectopic its
5,398 4,069 1.2 0.9
profile of the human Al adenosine CHO cell line.
reported
from
+ f 8.3 * 2.6 i
56,391 3,088 948 487 248
Table 2. Pharmacological in a stably transfected
previously
57,736 10,955
(n=4-5)
CGS21680 (+)PIA Adenosine NECA (-)PIA
has been
[3H1DPCPX
(n=4)
Caffeine Theophylline KFMlY DPCPX AGONISTS
(nM)
of
analysis Gi protein(s)
together
all
receptor,
pharmacological
of
are
indicative in
the
of
CHO cells. (sequence
activity
we conclude
these
receptor,
characteristics
biological
profile),
in membrane
recombinant
present its
[3H]XAC
demonstration
(fig.2)
the
dog
the
of
receptor
that
on adenylyl the
receptor
% BINDING
0 0.01
1 L-)PIA
100
10,000
lXllOl/l
concentration
Fic.3. Displacement of ["HIDPCPX binding to human Al adenosine receptor expressed in CHO cells by (-)PIA. Without (*) and in presence Shown is a representative (+) Of 100 PM GppNHp. experiment, each point has been done in duplicate. 924
an
Vol.
187,
we
No.
2, 1992
have
cloned
receptor
is
that Cloning
of Al the
receptor further
subfamily.
of
the
human Al cell
development
of
approached to
in adenosine when
as
the
has
of been
in
receptor and
in
is
its
for
coupling be
more
studies
are
activated tissue
by
ambient
the
types
the
normal
activation
cell
at
could
further
for
receptor
dissection,
Also,
reported
mice in vivo
this
modulation
Al
tool
G protein
permanent
various
and production of
the
lines.
adenosine
an invaluable
interaction,
CHO cells
expressed
of transgenic
provide
pharmacology
cell
if
The observation
endogenous
lines
pathway
the
Al
the
receptor
such
these
of
adenosine
receptor
determine
environment.
thyroid
using
COMMUNICATIONS
[1,2].
the
intracellular
RESEARCH
previously
applications of ligand
BIOPHYSICAL
counterpart
described
expressing
'constitutively'
receptor
human
been
level,
required
AND
has
subsequent
simply
the
Other
molecular and
BIOCHEMICAL
A2 [5]
adenosine and
in
the
[24].
ACKNOWLEDGMENTS We are grateful to C. Massart and Y. Mauquois for technical assistance. Supported by Boehringer Ingelheim, the Ministere de la Politique Scientifique (Interuniversity pole of attraction), the Fonds de la Recherche Scientifique Medicale (FRSM) and the Association Recherche Biomedicale et Diagnostic. The scientific responsibility is assumed by the authors. F.L. is Charge de Recherches of the Belgian FNRS. REFERENCES 1. Bruns, R.F., 29, 331-346. 2. Van Calker, Neurochem.
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Libert, F., Gerard, C., 1677-1682.
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