(Received for publication, June 2, 1995)
From the
To provide new insights into the regions of the human A adenosine receptor (A
AR) involved in ligand binding,
a series of chimeric human A
and rat A
adenosine receptors (A
/A
) were studied.
Binding studies were initially performed on acutely transfected COS
cells using fixed doses of the A
AR agonist
[
H]CGS-21680, the A
AR agonist
[
H]2-chloro-N
-cyclopentyladenosine
(CCPA), and the A
AR antagonist
[
H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX).
When the region of the A
AR from the amino terminus to the
end of transmembrane (TM) 1 was replaced by the corresponding region of
the A
AR (A
TM1/A
),
[
H]CGS-21680 and [
H]CCPA
binding was detectable. When an A
TM1-2/A
construct was studied, [
H]CGS-21680 binding
was lost and [
H] DPCPX binding appeared.
Saturation studies using [
H]CCPA revealed that
the A
TM1/A
construct had low affinity.
However, with the subsequent addition of A
AR TMs 2-4
receptor affinity improved markedly. Saturation studies using
[
H]DPCPX also revealed that the TMs 1-4 of
the A
AR conferred wild-type receptor affinity. When the
ligand binding properties of A
TM1-4/A
,
A
TM1-6/A
, and wild type A
AR
constructs were directly compared, no differences were found using 10
different compounds. When truncated A
ARs that extended from
the amino terminus to shortly after TM4 were examined, no binding was
detectable suggesting that the amino half of the receptor alone is not
sufficient for ligand binding. Collectively, these data suggest that
the important determinants for A
AR agonist and antagonist
binding and ligand specificity are present in TMs 1-4.
The nucleoside adenosine exerts its potent biological effects
via specific receptors that belong to the superfamily of G
protein-coupled receptors(1) . To date, four adenosine receptor
subtypes have been
cloned(2, 3, 4, 5, 6, 7, 8, 9, 10) .
These include the A and A
receptors that
inhibit adenylyl cyclase and the A
and A
receptors that stimulate adenylyl
cyclase(1, 6) . Each adenosine receptor subtype has
unique ligand binding properties and distinct patterns of tissue
expression(11, 12) .
Recently, there has been
considerable interest in A adenosine receptors
(A
ARs) (
)for several reasons. In the brain,
A
ARs are widely distributed (5, 7, 13) and are believed to tonically
inhibit neuronal activity(13) . A
ARs may influence
the pathogenesis of seizure disorders (14) and protect neurons
against excitotoxic damage (13) . In the heart, A
AR
activation influences the cardiac conduction system and may protect
against ischemic damage(15, 16, 17) .
The
AAR was initially cloned from the thyroid gland of dogs (2, 4) and later isolated from several other
species(5, 7, 18, 19, 20, 21) .
In each species, the A
AR cDNA encodes a protein of 326
amino acids that contains seven transmembrane spanning regions.
A
ARs have distinct ligand binding properties as compared to
other adenosine receptor
subtypes(12, 22, 23) . A
ARs have
high affinity for N
-substituted adenosine
analogues and have high affinity for C
-substituted xanthine
antagonists (11, 12) . Several highly selective
A
AR compounds are available including the agonist
2-chloro-N
-cyclopentyladenosine (CCPA) and the
antagonist 8-cyclopentyl-1,3-dipropylxanthine
(DPCPX)(24, 25) .
When compared to
AARs, the amino terminus of the A
ARs is longer
than that of A
ARs, while the carboxyl terminus of the
A
ARs is shorter than that of the
A
AR(5, 7, 8, 18, 19, 20, 21) .
At the amino acid level, A
and A
ARs are
generally 50% identical over all and 60% identical within the
transmembrane
domains(5, 7, 8, 18, 19, 20, 21) .
In contrast to A
ARs, A
ARs have high affinity
for 5`-substituted adenosine agonists and low affinity for N
-substituted compounds(11, 12) .
Highly selective A
AR agonists are available and include
the compound CGS-21680(26) .
Presently our understanding of
how ligands bind to AARs is at early stages. Similar to how
other G protein-coupled receptors bind small molecule
ligands(27) , it has been postulated that ligand-binding sites
of A
ARs are within transmembrane
regions(28, 29, 30) . Using computer
modeling, Ijzerman and colleagues (28, 29, 30) have suggested that the
adenosine ribose group interacts with transmembrane (TM) domain 5,
whereas the N
position interacts with TM7.
Site-directed mutagenesis also has been applied in efforts to
identify sites involved in ligand binding. Olah and co-workers (31) have suggested that His and His
are important for ligand binding. Townsend-Nicholson and Schofeld (32) and Tucker et al.(33) have reported that
Thr
may interact with the 5`-ribose position. Studies of
chimeric A
/A
adenosine receptors by Olah and
co-workers (34) have suggested the presence of a region in TM5
that influences the affinity for 5`-substituted compounds. Using
chimeric A
/A
receptors it has also been
suggested that the second extracellular loop may influence ligand
binding(35) . Most recently, Tucker and colleagues (33) have suggested that the A
AR 270 amino acid
interacts with the N
position of adenosine or the
C
position of xanthines.
Although the above reports have
provided important insights into adenosine-receptor interactions, we
still do not have a comprehensive understanding of how adenosine
receptors bind receptor subtype-selective ligands. For other G
protein-coupled receptors, chimeric receptor studies have provided
important insights about the transmembrane domains that are involved in
ligand binding(36, 37, 38, 39) . By
taking advantage of the distinct ligand binding properties of A and A
ARs and the availability of highly selective
A
and A
AR radioligands, we have used a broad
series of A
/A
AR chimeric receptors to identify
receptor regions involved in A
AR agonist and antagonist
binding. We now provide evidence that the first four transmembrane
domains of the human A
AR bind to and confer specificity for
A
AR-selective ligands.
To generate
A/A
chimeric receptors, oligonucleotide primer
pairs (primers A and B) were designed to generate a 5` fragment of the
A
AR. Another set of oligonucleotide primer pairs (primers C
and D) were made to generate a 3` fragment of the A
AR
receptor. To generate A
/A
chimeric constructs,
oligonucleotide primer pairs (A and B) were constructed to generate a
5` fragment of the A
AR, and oligonucleotide primer pairs
were designed to generate a 3` fragment of the A
AR receptor
(C and D). Receptor fragments were generated using >1 µg of DNA
as the substrate for PCR reactions, and PCR reactions were performed
using the Gene Amp Kit (Perkin-Elmer) reagents. PCR was generally
performed using 30 cycles at 94 °C for 1 min, 55 °C for 1 min,
and 72 °C for 2 min. PCR products were then separated on a 1%
agarose gel and eluted using NA45 paper (Schleicher and Schuell).
Receptor fragments (A-B and C-D) were then combined in third PCR
reaction to generate a full-length, chimeric receptor in a fusion
reaction using flanking primers (A and D). To generate truncated
A
ARs, a 5` fragment of the A
AR was generated
using the A
AR A primer and a D primer that included a stop
codon.
Flanking PCR primers contained HindIII (A primers) or XbaI (D primers) restriction endonuclease sites at the ends. After fusion reactions, PCR products were digested with HindIII and XbaI and were subcloned into the mammalian expression vector pcDNA1, (In Vitrogen, San Diego, CA). Chimeric receptors were then sequenced using Sequenase Version 2 (Amersham Corp.).
For binding reactions, the
radioligands used were [H]CCPA (DuPont NEN;
specific activity 33 Ci/mmol), [
H]DPCPX (DuPont
NEN; specific activity 100 Ci/mmol), and
[
H]CGS-21680 (DuPont NEN; specific activity 30
Ci/mmol). Generally, 50 µl cells (75-150 µg of protein)
were added to the radioligand in a final volume of 150 µl in
Multiscreen plates (GF/B 1.2-µm glass fiber filters; Millipore;
Bedford, MA). Reactions were incubated at 21 °C for 1 h with
shaking. Bound radioactivity was separated from free by vacuum
filtration and determined by liquid scintillation counting. All
determinations were performed in quadruplicate. Protein concentration
was measured using bicinconinic acid (Pierce) with bovine serum albumin
standards.
To define the regions of the AAR involved in
agonist and antagonist binding, a series of chimeric A
and
A
adenosine receptors were generated. These receptor
subtypes were chosen because each receptor subtype has distinct ligand
binding characteristics (11, 12, 22) and
highly selective radioligands for each receptor subtype are
available(24, 25, 26) .
We first generated
a series of chimeric A/A
receptors in which 5`
regions of the A
AR were replaced by progressively larger
5` regions of the A
AR (Fig. 1). Receptor splicing
took place at extra- or intracellular loops, and transmembrane domains
were transferred intact in the proper reading frame. To identify
receptor regions involved in ligand binding, chimeric constructs were
initially screened using fixed concentrations of the A
AR
agonist [
H]CGS-21680 (20 nM), the
A
AR agonist [
H]CCPA (5 nM),
or the A
AR antagonist [
H]DPCPX (5
nM) in four to six separate studies. COS 6M and COS 7 cells
were used for these studies. All subsequent studies involved COS 7
cells. At the above concentrations of radioligands, there was no
specific binding to non-transfected or sham transfected COS 6M or COS 7
cells.
Figure 1:
Schematic representation of
chimeric A/A
, A
/A
constructs and truncated A
ARs. Beside each receptor
is a representation of binding studies. cDNAs were acutely expressed in
COS cells. Binding was assessed 48 h later by radioreceptor assay using
the A
agonist [
H]CCPA (5
nM), the A
antagonist
[
H]DPCPX (DPX, 5 nM]), and
the A
agonist [
H]CGS-21680 (20
nM]). Specific binding is shown; -, no binding;
+ specific binding present at >50 fmol/mg of protein. Data
shown are representative of four to six separate studies/construct. All
samples were tested in quadruplicate in each separate
study.
Using [H]CGS-21680, we found that when
the region extending from the amino terminus to after the end of TM1 of
the A
AR receptor was replaced by that of the
A
AR (CR1), low level binding (25-50 fmol/mg of
protein) was present (Fig. 1). When both TMs 1 and 2 of the
A
AR were replaced by the corresponding A
AR
regions (CR2), [
H]CGS-21680 binding was no longer
detectable showing that TMs 1 and 2 of the A
AR are
important for [
H]CGS-21680 binding.
Using
[H]CCPA, no binding was detected on cells that
expressed the wild-type A
AR. However, with the
substitution of A
AR TM1 by that of the A
AR
(CR1), low level [
H]CCPA specific binding was
apparent (25-55 fmol/mg of protein). With the successive addition
of TMs 2 and 3 (CR 2 and CR3), low level binding continued to be
detected (30-75 fmol/mg of protein). With the subsequent
additions of TMs 4, 5, 6, and 7 (CRs 4-7),
[
H]CCPA binding was present at considerably
higher levels (75-250 fmol/mg of protein) than that observed with
the CR1, CR2, or CR3.
Next, [H]DPCPX was used
to examine antagonist binding to the chimeric receptor constructs (Fig. 1). When CR1 was examined, no specific binding was
detectable. With the successive addition of A
AR TMs 2 and 3
(CRs 2 and 3), specific low level binding was detectable (50-100
fmol/mg of protein). With the subsequent additions of TMs 4, 5, 6, and
7 (CRs 4-7), the amount of [
H]DPCPX binding
increased markedly (300-1500 fmol/mg of protein).
Because the
above studies suggested that TMs 1 and 2 were important for ligand
binding, we next tested another series of chimeric receptors in which
TMs 1 and 2 of the AAR were replaced by corresponding
domains of the A
AR. When A
AR TM1 alone was
replaced (CR8), [
H]CGS-21680 (50-75 fmol/mg
of protein) and [
H]DPCPX (75-125 fmol/mg of
protein) binding was apparent, but [
H]CCPA
binding was not observed. When both TMs 1 and 2 of the A
AR
were replaced by TMs 1 and 2 of the A
AR (CR9), binding was
not detected by any of the radioligands used.
Saturation studies
were next performed to determine if the differences in binding levels
observed using fixed doses of radioligands reflected differences in
chimeric receptor affinity (K) or
differences in receptor expression (B
). Although
the CR1 construct bound [
H]CCPA, ligand affinity
was low (Table 2; p < 0.01, ANOVA). With the addition
of A
AR TMs 2 and 3 (CRs 2 and 3), receptor affinity
increased, but remained less than that observed for the wild-type
A
AR (p < 0.05, ANOVA). With A
AR TM4
(CR4), the affinity for [
H]CCPA increased further
and approximated that of the wild-type A
AR (p >
0.05, ANOVA).
Next, saturation studies were performed using
[H]DPCPX (Table 3). When TMs 1-2 of
the A
AR were present (CR2), K
values reflected very low affinity (p < 0.01 versus wild type A
AR, ANOVA). However, when
A
AR TM3 was added (CR3), the affinity for
[
H]DPCPX increased, yet remained less than that
observed for the wild-type A
AR (p < 0.05,
ANOVA). With the addition of A
AR TM4 (CR4), receptor
affinity increased 7-fold and was actually greater than that observed
for the wild-type A
AR. Receptor expression also increased
with the addition of A
AR TM4. With the progressive
substitution of A
AR TMs 5, 6, and 7 (CRs 5-7),
chimeric constructs had high affinity and high levels of expression.
Because the CR4 construct was expressed at high levels, and
AAR TMs 1-4 contain regions that confer high affinity
for [
H]CCPA and [
H]DPCPX,
we next tested if A
AR TMs 1-4 alone were sufficient
for ligand binding. Two different truncated A
AR receptors
(T1 and T2) that contained TMs 1-4 and portions of the second
extracellular loop were generated and examined in parallel with the
wild-type A
AR. Although we could detect
[
H]DPCPX binding to the wild-type A
AR
after acute expression, we could not detect specific binding to either
of the truncated receptors. Thus, although TMs 1-4 contain
determinants for A
AR agonist and antagonist binding, TMs
5-7 may also be required for ligand binding.
After showing
that TMs 1-4 of the AAR alone are not sufficient for
ligand binding, we next tested if A
AR TMs 1-4 were
sufficient for conferring specificity for A
-selective
ligands. Competition studies were therefore performed on chimeric
constructs using 10 compounds selected for their ability to distinguish
different adenosine receptor subtypes(11, 12) . These
compounds included A
- and A
-selective agonists
and antagonists as well as non-selective adenosine agonists and
antagonists (see Fig. 2and Table 4). Because CR4, CR6,
and the wild-type A
AR had comparable affinity for
[
H]DPCPX and were expressed at high levels,
competition studies were performed using these three constructs. Each
construct was tested two or more times in side-by-side experiments
using each drug.
Figure 2:
Competition of adenosinergic compounds for
[H]DPCPX [2 nM] binding to the
wild-type A
AR and the A
TM1-4/A
(CR4) and A
TM1-6/A
(CR6)
constructs. Binding is expressed as a percentage of total binding. The
data shown are representative of three separate studies.
, DPCPX;
, CPA;
, NECA;
, CGS-21680. The abbreviations used
are as in Table 3.
Competition studies revealed the expected rank
order of potency of an AAR for each construct. We found
that CR4, CR6, and the wild-type A
AR each had low affinity
for A
-selective compounds (CGS-21680, CSC, and DMPA) and
had high affinity for A
-selective compounds (CPA,
CGS-15943, and DPCPX). The K
values for
each construct also were similar to values previously reported for
A
ARs(12) . For the compounds CPA, DPCPX, NECA, and
CGS-21680, which were tested in three or four separate side-by-side
studies, no significant differences among K
values were found (p > 0.05; ANOVA). Thus, TMs
1-4 of the A
AR are sufficient to confer specificity
for A
-selective compounds.
We next attempted to
determine the relative importance of the individual TM domains in the
amino half of the AAR in conferring specificity for
A
-selective ligands. Due to the low level of expression of
the CR1, CR2, and CR3 constructs, rigorous competition studies were
difficult to perform. Because saturation studies revealed that the
addition of TMs 2 and 3 resulted in increased affinity for
[
H]CCPA or [
H]DPCPX, we
tested the relative importance of these TM domains. Chimeric receptors
in which TM 2 or TM 3 of the A
AR was replaced by the
corresponding TM domains of the A
AR were therefore
generated (CR10 and CR11). Although we could detect
[
H] DPCPX binding to the wild-type
A
AR after acute expression, we could not detect specific
binding to either of these TM-substituted A
ARs. These
observations therefore suggest that both TMs 2 and 3 contain residues
that are important for ligand binding.
Our observations provide new insights into the potential
sites of ligand-receptor interactions for the human AAR.
Using a series of chimeric A
/A
receptors, we
have found that the region of the A
AR from the amino
terminus to the end of the fourth transmembrane domain is important for
ligand binding and conferring specificity for A
-selective
agonists and antagonists.
Using the highly selective AAR
agonist [
H]CCPA, we found that by replacing the
region from the amino terminus to after the end of TM1 of the
A
AR with that of the A
AR (CR1), specific
binding appeared. Supporting the notion that TM1 of the A
AR
contains an agonist binding site, [
H]CCPA binding
was lost when TM1 of the A
AR was replaced by that of the
A
AR (CR8). Although the CR1 construct bound
[
H]CCPA, the affinity for this ligand was low (K
= 26.4 nMversus 0.7 nM for wild-type A
AR). However, with the
addition of A
AR TM2 (CR2), the affinity for
[
H]CCPA increased greatly (K
= 4.5 nM). When A
AR TMs 3 and 4
(CR4) were added, the affinity for [
H]CCPA
increased again (K
= 1.9
nM) and approximated that of the wild-type A
AR.
CRs 6 and 7 also had K
values close to
that of the wild-type A
AR. CR5, in contrast, had lower
affinity than the CRs 4, 6, and 7. Since this receptor was generated by
a splice in the third cytoplasmic loop, it is possible that a
conformational change in this construct resulted in reduced affinity
for [
H]CCPA.
Using
[H]DPCPX, we also found that TMs 1-4 of the
A
AR receptor were important for antagonist specificity.
When the region from the amino terminus to the end of TM2 of the
A
AR was replaced by that of the A
AR,
[
H]DPCPX binding became detectable although this
construct had low affinity for the antagonist (K
= 11.1 nMversus 1.5 nM for the wild-type receptor). With the addition of A
AR
TM3 (CR3), the affinity for [
H]DPCPX increased
3-fold (K
= 3.1). With the
subsequent addition of A
AR TM4 (CR4; K
= 0.5 nM), receptor affinity actually
exceeded that of the wild-type A
AR. These observations are
consistent with the findings of [
H]CCPA binding
studies and support a role for TMs 1-4 in binding
A
-selective ligands.
Competition studies also showed
that AAR TMs 1-4 conferred ligand specificity. Using
10 drugs that distinguish among the different adenosine receptor
subtypes, we failed to detect any major differences in the
pharmacologic profiles of CR4, CR6, and the wild-type A
AR.
To better define the contributions of the domains within TMs
1-4, we also attempted competition studies using CR 1-3
constructs. However, the low level of expression of these constructs
made rigorous competition studies difficult to perform. Studies using
fixed doses of radioligands, however, revealed the presence of
important binding sites for A-selective ligands in TMs 1
and 2. When TMs 1-2 of the A
AR were replaced by
those of the A
AR (CR2), [
H]CGS-21680
binding was lost suggesting that A
AR agonist specificity
and binding involves TMs 1-2. Furthermore, when TMs 1, 2, and 3
of the A
AR were replaced by those of the A
AR,
neither [
H]CCPA nor [
H]
DPCPX binding was observed.
To date, structure function studies of
AARs have largely focused on sites within TMs 5-7.
Studies of chimeric A
/A
adenosine receptor
studies have suggested that 5` substituted compounds interact with
TM5(34) . Site-directed mutagenesis studies have suggested that
His
in TM6 may play a role in binding
antagonists(31) . Within TM7, differences in the amino acid at
position 270 among cows (Ile) and dogs (Met) are believed to account
for differences in affinity for A
-selective drugs among
these species(33) . It has been suggested that this position
interacts with the N
position of adenosine and the
C
position of xanthines(33) . Two separate studies (32, 33) have also suggested that the amino acid at
position 277 interacts with the 5`-ribose position. When His
of bovine A
AR is converted to Ala
, poor
affinity for agonists and antagonists is demonstrated, although
expression of this construct is not known(31) .
Although the
aforementioned reports suggest the importance of sites in TMs 5-7
in ligand binding, it is unlikely that they will distinguish among
A- and A
-selective ligands. Comparison of the
primary amino acids sequence among A
and A
ARs
reveals that the corresponding amino acids of the A
AR at
positions 256, 270, 277, and 278 are identical in
A
ARs(8) . Furthermore, our competition studies
show that TMs 1-4 are sufficient to confer the ligand binding
characteristics of an A
AR.
We do, however, agree with
the concept that the carboxyl half of the receptor is important for
ligand binding. When we tested two AARs truncated after
TM4, we did not detect either [
H]DPCPX or
[
H]CCPA binding. While it is possible that these
constructs did not express, the removal of binding sites in TMs
5-7 may have resulted in the loss of binding.
Based on studies
of other small molecule receptors(27) , we anticipate that
adenosine- and xanthine-binding sites will reside within the
transmembrane domains of AARs. However, because our
constructs contained parts of extra- and intracellular loops in
addition to TM domains, it is possible that non-transmembrane regions
may influence ligand binding. Furthermore, chimeric
A
/A
receptors have suggested that the second
extracellular loop between TMs 4 and 5 may influence receptor binding (35) . Thus, future studies involving smaller receptor
substitutions will be needed to examine the relative contributions of
transmembrane and non-transmembrane domains on ligand binding
specificity.
Currently, the orientation of adenosine with different
receptor regions is not known. Site-directed mutagenesis studies of
amino acid 270 have suggested that this site interacts with the N position of agonists and the C
position of xanthine antagonists(33) . It also has been
suggested that the Thr
position in TM 7 interacts with
the 5`-ribose of NECA(33) . Considering the expected close
proximity of these sites in TM7 (28) and the size and
confirmation of adenosine analogs(28, 44) , it is
unlikely that both the N
and the 5`-ribose
positions will interact with this region. Pharmacology studies show
that the N
-adenosine and C
-xanthine
positions are important for conferring receptor subtype ligand binding
specificity. However, because the 270 and 277 amino acids are the same
in A
and A
ARs, it is unlikely that these sites
are the primary determinants of the N
or C
binding.
In contrast to previously proposed models of
adenosine-receptor
interactions(28, 29, 30, 33) , based
on our findings we hypothesize that the N and
C
positions interact with sites within the first four
transmembrane domains of the A
AR and that the 5`-ribose
position interacts with TM7. If the 5`-ribose position is oriented
toward TM7, as previously suggested (33) , this would also
allow the ribose group to interact with TMs 1 and 2, if TMs 1 and 7 are
juxtaposed. The observation that [
H]CCPA binds to
the CR1 construct, whereas [
H]DPCPX does not,
supports this hypothesis since CCPA contains a ribose group whereas
DPCPX does not. If the ribose group binds to a TM7/TM1-2 region,
the N
group could then interact with TMs
2-4. Because the C
position xanthines are believed to
interact at sites to the N
adenosine
position(12) , we would also expect xanthines to interact with
this region. The N
or N
positions of adenosine and the N
or N
positions of xanthines, which are important for
ligand binding but are not important for receptor subtype
selectivity(12) , could then interact with sites in TMs
5-6.
Overall, our findings suggest that specificity for
AAR subtype-selective ligands is conferred by first four
TMs of the A
AR. With the background information provided by
these studies, we anticipate that individual transmembrane and single
residue substitutions in the amino half of the A
AR will
lead to the identification of the sites that confer specificity for
receptor subtype-specific ligands.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank]and X68485[GenBank].