From the
The A
Adenosine is a ubiquitous mediator (reviewed in Jacobson et
al. (1992a)) that regulates homeostasis in many organs and offers
protection under conditions of stress, such as ischemia. The
A
Structural studies of A
Until the present study, site-directed mutagenesis
of A
Chemical modification studies of A
A molecular model
of the rat A
Finally,
based on the results of site-directed mutagenesis, we have utilized
molecular modeling to predict the environment of the ligand binding
pocket. Molecular modeling serves to test the internal consistency of
the results through a unified model that is consonant with all of the
pharmacological observations. The approach to receptor modeling used in
the present study is based on homology with rhodopsin (Ballesteros and
Weinstein, 1995), for which low resolution electron density information
has been reported (Henderson et al., 1990). The homology in
sequence and structure between rhodopsin and other G-protein coupled
receptors has been explored (Baldwin, 1993). We have recently applied
the rhodopsin-based modeling approach to ligand binding at the P
For saturation and competition binding experiments, each tube
contained 100 µl of membrane suspension, 50 µl of radioligand,
and either 50 µl of buffer/competitor (50 mM Tris, pH 6.8,
10 mM MgCl
The A
To
provide more detail, we mutated several additional residues (Phe-180,
Cys-254, Phe-257, Tyr-271, Ile-274, and Ser-277), that were nearby in
the sequence and/or predicted by molecular modeling (IJzerman et
al., 1994) to be in proximity to the bound ligand. The F257A,
Y271A, and I274A mutant receptors did not bind either
[
Curiously, the S277A
mutant receptor was unable to bind significant amounts of
[
Although
substitution of either His residue with Ala prevented radioligand
binding, different results were obtained upon substitution with other
amino acids. Substitution of His-250 with Phe or Tyr preserved
radioligand binding, although the affinities of certain adenosine
agonists (5`-unsubstituted) were reduced by 10-50-fold
(). The affinities of CGS 21680, NECA, CGS 15943, and XAC
were nearly the same as those found with the wild type receptor.
However, substitution of His-278 with Gln, Phe, or Tyr abolished high
affinity binding of either [
Interestingly, there are two Met residues in the amino-terminal
segment of the human A
The complete understanding of ligand-receptor interactions
will lead to answers to questions, such as: (i) how are receptors
activated by agonist ligands and how is the binding site blocked by
antagonists? (ii) Which synthetic drug analogues are suitable to
control the function of the receptors? Even though mutagenesis studies
lead to only a limited understanding of detailed three-dimensional
structure, they are still a powerful tool for deriving an overall
hypothesis.
Modeling of G-protein coupled receptors (GPCRs) has
become an important tool in understanding drug-receptor interactions
and in the development of new ligands for these receptors. The first
widely accepted method was the homology modeling method by Hibert
et al.(1991). This method involved the alignment of the
receptor sequence with the sequence of bacteriorhodopsin and the
subsequent mapping of the sequence onto the structure of
bacteriorhodopsin that was determined by Henderson et
al.(1990). This procedure was based on the assumption that even
though GPCRs and bacteriorhodopsin, a proton pump in the outer membrane
of Halobacterium halobium, lacked any functional or sequence
homology, there would be considerable structural homology. This
structural homology was inferred by the extraordinary similarity in the
hydrophobicity plots, or Kyte-Doolittle plots, of the biogenic amine
subfamily of GPCRs and bacteriorhodopsin. Recently, a low resolution
electron density map of rhodopsin, a true member of the GPCR
superfamily, was published (Schertler et al., 1993). The low
sequence homology with bacteriorhodopsin, the structural differences
that must arise from the different placement of proline residues
(causing bends in the helices) in bacteriorhodopsin and GPCR sequences,
and the availability of an electron density map of a true member of the
GPCR superfamily prompted us (van Rhee et al. 1994) to adapt a
new method to build models of GPCRs (Ballesteros and Weinstein, 1995)
that is based on a computational approach rather than strict compliance
with the atomic coordinates of a distantly related protein, albeit with
higher resolution.
Although molecular models for the canine A
According to the present
mutagenesis results, substitution of either His-250 or His-278 with Ala
in human A
The H250A mutant receptor showed significant plasma membrane
expression (58%; ), but unlike H278A did not show any
detectable stimulation by 1 mM CGS 21680 of cyclic
AMP-production over control levels, using rolipram to inhibit
phosphodiesterases. These results suggest that the H250A mutant
receptor either has even lower affinity for CGS 21680 than H278A, or is
defective in G-protein coupling. Substitution of His-250 with Phe,
which does not readily form hydrogen bonds, diminished affinities of
certain ligands but did not preclude binding, suggesting that the
interaction between His-250 and ligands does not primarily involve
hydrogen bonding. Substitution of the imidazole group with other
aromatic side chains was particularly detrimental to the affinity of
R-PIA and DPMA, which have hydrophobic, aromatic
N
Phe-182 may also be involved in helix-helix packing,
according to our model in which TM5 is rotated in a fashion consistent
with rhodopsin in the model proposed by Baldwin(1993). Phe-182 is
located close to the adenosine C2 binding region in the model (viewed
as in Fig. 6A). If TM5 were rotated clockwise by several
degrees, then Phe-182 would be in even closer contact with the adenine
moiety.
A hydrogen bond between the
exocyclic NH and His-250 was proposed by IJzerman et
al.(1994). In our model the distance is too large for such a bond,
however, there is a possibility of H-bonding between the exocyclic NH
and Asn-253. It has been suggested that N
Substituting Asn-181 with Ser () indicated that this
amino acid selectively affects the affinity of
N
In the case of the
human A
Ser-281 (Ser-276
in the rat A
Substitution of Asn-253 with Ala, Gln, and
Ser prevented binding of [
The model predicts that Tyr-271 is at the interface between
TM7 and TM1, and when mutated to Ala, ligand affinity was greatly
decreased. Mutation of Tyr-271 to other aromatic residues (Phe or His)
or to Arg preserves binding of both agonists and antagonists (data not
shown).
Data are presented as means ± S.E. of two or three
independent experiments, each performed in duplicate. Each sample
contained 7-11 µg of membrane protein/tube.
Table shows mean ± S.E. of two independent
experiments, each performed in duplicate. [
Table shows mean ± S.E.
of two or three independent experiments, each performed in duplicate.
Agonist and antagonist binding affinities (K
Table shows mean ± S.E. of two or
three independent experiments, each performed in duplicate.
[
Table shows expression level as percentage of
Tag3-A
CGS
21680-stimulated cAMP production was examined in transiently
transfected COS-7 cells in the presence of 0.1 mM rolipram as
described under ``Experimental Procedures.'' In each case the
maximal stimulation was 3-4-fold over control (see Fig. 5). Table
shows mean ± S.E. of two or three independent experiments, each
performed in duplicate.
We thank Dr. Ad IJzerman (Leiden University) and Prof.
Gary Stiles, Dr. Mark Olah, and Dr. Timothy Palmer (Duke University)
for helpful discussions, and Dr. Marlene Jacobson (Merck) for providing
the human A
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
adenosine receptor is a member of the
G-protein coupled receptor family, and its activation stimulates cyclic
AMP production. To determine the residues which are involved in ligand
binding, several residues in transmembrane domains 5-7 were
individually replaced with alanine and other amino acids. The binding
properties of the resultant mutant receptors were determined in
transfected COS-7 cells. To study the expression levels in COS-7 cells,
mutant receptors were tagged at their amino terminus with a
hemagglutinin epitope, which allowed their immunological detection in
the plasma membrane by the monoclonal antibody 12CA5. The functional
properties of mutant receptors were determined by measuring stimulation
of adenylate cyclase. Specific binding of [
H]CGS
21680 (15 nM) and [
H]XAC (4
nM), an A
agonist and antagonist, respectively,
was absent in the following Ala mutants: F182A, H250A, N253A, I274A,
H278A, and S281A, although they were well expressed in the plasma
membrane. The hydroxy group of Ser-277 is required for high affinity
binding of agonists, but not antagonists. An N181S mutant lost affinity
for adenosine agonists substituted at N
or C-2, but not at
C-5`. The mutant receptors I274A, S277A, and H278A showed full
stimulation of adenylate cyclase at high concentrations of CGS 21680.
The functional agonist potencies at mutant receptors that lacked
radioligand binding were >30-fold less than those at the wild type
receptor. His-250 appears to be a required component of a hydrophobic
pocket, and H-bonding to this residue is not essential. On the other
hand, replacement of His-278 with other aromatic residues was not
tolerated in ligand binding. Thus, some of the residues targeted in
this study may be involved in the direct interaction with ligands in
the human A
adenosine receptor. A molecular model based on
the structure of rhodopsin, in which the 5`-NH in NECA is hydrogen
bonded to Ser-277 and His-278, was developed in order to visualize the
environment of the ligand binding site.
, A
, A
, and A
subtypes have been cloned (Mahan et al., 1991; Maenhaut
et al., 1990; Jacobson, 1995; Zhou et al., 1992). The
A
adenosine receptor activates adenylate cyclase (Hide
et al., 1992) via coupling to G
. Adenosine
regulates blood pressure by centrally (Barraco et al., 1994)
and peripherally mediated mechanisms (Olsson and Pearson, 1990).
Activation of A
receptors results in vasodilation, and
this effect has been examined as a potential hypotensive therapy using
selective A
agonists such as CGS 21680
(
)
(Jarvis et al., 1989). A
receptors
are also present in platelets, where they inhibit aggregation, and in
the liver. In the brain, A
receptors occur primarily in
the striatum, where they are involved in dopaminergic pathways
(Ferré et al., 1994) and elicit locomotor depression
(Nikodijevi&; et al., 1993). Consequently
therapeutic approaches, based on activating or inhibiting A
receptors, to diseases involving the dopaminergic system,
i.e. Parkinson's disease (Kanda et al., 1994),
schizophrenia (Martin et al., 1993), and Huntington's
disease (Nikodijevi&; et al., 1993), are under
investigation.
receptors have
been impeded by the lack of a selective high affinity A
antagonist. We have shown that an 8-phenylxanthine antagonist,
[
H]XAC, binds to A
receptors with
especially high affinity in the human (Ji et al., 1992) and
rabbit (Jacobson et al., 1992b) striatum (XAC is non-selective
in these species), and therefore this ligand was selected for use in
the present study for characterizing mutant human A
receptors.
receptors has not been reported, however, mutational
studies have provided insight into the ligand binding site of A
receptors. Olah et al.(1992) demonstrated the
involvement in ligand binding of two His residues in the sixth and
seventh transmembrane helical domains (TMs). In bovine A
receptors, His-250 (TM6) has been shown to be important for
antagonist binding, and His-278 (TM7) is important for both agonist and
antagonist binding. Mutagenesis studies of other adenosine receptor
subtypes have implicated a hexapeptide region in TM5 of rat A
receptors (Olah et al., 1994) and Ile-274 and Ser-277 in
TM7 of bovine A
receptors (Townsend-Nicholson and
Schofield, 1994; Tucker et al., 1994) in binding of adenosine
derivatives.
receptors indicated that, as in A
receptors, His
residues appear to be involved in ligand binding (Jacobson et
al., 1992b). Two conserved His residues are present in TM6 and
TM7. Using an agonist molecular probe, the bovine A
receptor was photoaffinity labeled (Barrington et al.,
1989). The site of this labeling was later shown using peptide mapping
to occur on TM5 (Piersen et al., 1994).
receptor (IJzerman et al., 1994) was
deduced from the primary sequence and by computer assisted homology
modeling, based on the electron density map of bacteriorhodopsin.
According to the model, TM5, TM6, and TM7 are most likely involved in
ligand binding. As with other receptors, the transmembrane helices tend
to be amphipathic, with the more hydrophilic sides postulated to be
facing the ligand binding cleft and in direct contact with the ligand.
In particular, residues Phe-182, His-250, Asn-253, His-278, and Ser-281
(numbering for human sequence) were predicted to be involved in ligand
binding. In the present study, these and other residues in the same
transmembrane region were selected as sites for the replacement of
single amino acids, to probe the influence of individual side chains on
molecular recognition by the human A
receptor.
(ATP) receptor (van Rhee et al., 1994).
Materials
Human A adenosine
receptor cDNA (pSVLA2a) was provided by Dr. Marlene A. Jacobson (Merck
Research Labs, West Point, PA). Taq polymerase for the
polymerase chain reaction (PCR) was purchased from Perkin Elmer
(Emeryville CA). All enzymes used in this study were obtained from New
England Biolabs (Beverly, MA). The agonists CGS 21680, NECA,
R-PIA, 2-chloroadenosine, and DPMA, and the antagonists XAC
and CGS 15943 were from RBI (Natick, MA). [
H]CGS
21680 (38.3 Ci/mmol) and [
H]XAC (118 Ci/mmol)
were obtained from DuPont NEN, and [
H]adenine (15
Ci/mmol) was purchased from American Research Chemicals Inc. (St.
Louis, MO). IB-MECA was prepared as described (Gallo-Rodriguez et
al., 1994). Fetal bovine serum and o-phenylenediamine
dihydrochloride were purchased from Sigma. The Sequenase kit, ATP, and
cyclic AMP were from U. S. Biochemical Corp. (Cleveland, OH). All
oligonucleotides used were synthesized by Bioserve Biotechnologies
(Laurel, MD). A monoclonal antibody (12CA5) against a hemagglutinin
epitope (HA) was purchased from Boehringer Mannheim Biochemicals
(Indianapolis, IN), and goat anti-mouse IgG (
-chain specific)
antibody conjugated with horseradish peroxidase was purchased from
Sigma. DEAE-dextran was obtained from Pharmacia Biotech Inc.
(Piscataway, NJ). Rolipram was a gift of Schering AG (Berlin, Germany).
Plasmid Construction and Site-directed
Mutagenesis
The coding region of pSVLA was
subcloned into the pcD cDNA expression vector (Okayama and Berg, 1983),
yielding pcDA
. All mutations were introduced into
pcDA
using standard PCR mutagenesis techniques (Higuchi,
1989). The correctness of all PCR-derived sequences was confirmed by
dideoxy sequencing of the mutant plasmids (Sanger et al.,
1977).
Epitope Tagging
A 9-amino acid sequence derived
from the influenza virus hemagglutinin protein (TAC CCC TAC GAC GTC CCC
GAC TAC GCC; peptide sequence: YPYDVPDYA) was inserted into three
different locations at the extracellular NH terminus of the
A
adenosine receptor gene (Fig. 3A).
Oligonucleotides containing the HA-tag sequence were designed and used
to generate PCR fragments, which were then used to replace the
homologous wild type pcDA
sequences.
Figure 3:
A, introduction of HA epitope tags into
the amino terminus of the human A adenosine receptor.
B, amount of absorbance detected in ELISA experiments with the
wild type and the various epitope-tagged A
receptors
expressed in COS-7 cells. ELISA measurements were carried out in
96-well plates as described under ``Experimental
Procedures.'' Data are presented as means ± S.E. of two or
three independent experiments, each performed in
triplicate.
Transient Expression of Mutant Receptors in COS-7
Cells
2 10
COS-7 cells were seeded into
100-mm culture dishes containing 10 ml of Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum. Cells
were transfected with plasmid DNA (4 µg of DNA/dish) by the
DEAE-dextran method (Cullen, 1987) about 24 h later, and grown for an
additional 72 h at 37 °C.
Membrane Preparation and Radioligand Binding
Assay
Cells were scraped into ice-cold lysis buffer (4 ml of 50
mM Tris, pH 6.8, at room temperature, containing 10
mM MgCl). Harvested cells were homogenized using a
Polytron homogenizer and then spun at 27,000
g for 15
min. Cell membranes (pellet) were resuspended in the same buffer.
) or 50 µl of 80 µM CADO in buffer (to define nonspecific binding) and finally 2
units/ml adenosine deaminase (Boehringer Mannheim). The mixtures were
incubated at 25 °C for 90 min, filtered, and washed three times
with
5 ml of ice-cold buffer each using a Brandel cell harvester.
Data analysis was performed by using the GraphPad program.
Cyclic AMP Determination
cAMP levels were
determined by measuring the conversion of [H]ATP
to cyclic [
H]AMP. One day after transfection,
cells were transferred from 100-mm dishes into 6 well dishes (about 3
10
cells/well) and incubated with culture media
containing 2 µCi/ml [
H]adenine. After 24 h,
the cultures were washed and incubated with 1 ml/well Hank's
balanced salt solution containing 0.1 mM rolipram for 15 min
at 37 °C. The cells were incubated with different concentrations of
the agonist CGS 21680 (in culture media) for 30 min at 37 °C. The
reaction was terminated by aspiration of the media and addition of 1 ml
of ice-cold 5% trichloroacetic acid containing 1 mM ATP and 1
mM cAMP. After 30 min incubation at 4 °C, cell lysates
were eluted through sequential chromatography on Dowex and alumina
columns (Enjalbert and Bockaert, 1983). Cyclic AMP formation is
expressed as fold-stimulation of conversion of
[
H]ATP into [
H]cyclic AMP
(Weiss et al., 1985).
ELISA
For indirect cellular ELISA measurements,
cells were transferred to 96-well dishes (4-5 10
cells/well) 1 day after transfection. About 48 h after splitting,
cells were fixed in 4% formaldehyde in phosphate-buffered saline for 30
min at room temperature. After washing with phosphate-buffered saline
three times and blocking with Dulbecco's modified Eagle's
medium (containing 10% fetal bovine serum), cells were incubated with
HA-specific monoclonal antibody (12CA5), 20 µg/ml, for 3 h at 37
°C. Plates were washed and incubated with a 1:2000 dilution of a
peroxidase-conjugated goat anti-mouse IgG antibody (Sigma) for 1 h, at
37 °C. H
O
and o-phenylenediamine
(2.5 mM in 0.1 M phosphate/citrate buffer, pH 5.0)
served as substrate and chromogen, respectively. The enzymatic reaction
was stopped after 30 min at room temperature with 1 M
H
SO
solution containing 0.05 M
Na
SO
, and the color development was measured
bichromatically in the BioKinetics reader (EL 312, Bio Tek Instruments,
Inc., Winooski, VT) at 490 and 630 nm (reference wavelength).
Western Blot Analysis
Whole cell or membrane
fractions (20 µg) were fractionated by denaturing
SDS-polyacrylamide gel electrophoresis (12%) and were assayed by
Western analysis (detailed procedure found in Bio-Rad product guide).
Anti-HA antibody (20 µg/ml) was incubated for 2 h, and the
secondary alkaline phosphatase-conjugated goat anti-mouse IgG was used
in 1:2,000 dilution.
Molecular Modeling
A sequence alignment of 40
G-protein coupled receptors was constructed to assist in locating
common amphipathic patterns in helical regions. The human A model was built and optimized using the InsightII/Discover
modeling package (BIOSYM Technologies, San Diego, CA, versions 2.2.0
and 2.90, respectively), employing the Amber force field, running on a
Silicon Graphics Indigo XZ4000 workstation (Silicon Graphics Inc.,
Mountain View CA), based on the methods described elsewhere (van Rhee
et al., 1994; Ballesteros and Weinstein, 1995). Briefly, TMs
were identified with the aid of Kyte-Doolittle hydrophobicity and Emini
surface probability parameters. Transmembrane helices were built from
the sequences and minimized individually. The minimized helices were
then grouped together to form a helical bundle that matches the overall
characteristics of the electron density map of rhodopsin (Schertler
et al., 1993) and the model of rhodopsin as proposed by
Baldwin(1993). The helical bundle was minimized in a stepwise process
gradually releasing tethering of the backbone. Initially, 500 steps
conjugate gradient were performed with the backbone of the helices
tethered with a force constant of 100 kcal/Å. In consecutive runs
(500 steps conjugate gradient each), the force constant was reduced to
50 kcal/Å, then 25 kcal/Å and, finally, without tethering.
NECA was then docked into the helical bundle with the express purpose
to explain as many experimental data as possible, regardless of the
conformation of the ligand. The NECA
A
complex was
then minimized using conjugate gradient until the root mean square
derivative was <0.1 kcal/mol/Å. The energy of the complex was
36.8 kcal/mol lower than the components.
receptor has been cloned initially from a
canine thyroid cDNA library (Maenhaut et al., 1990) and later
in other species from the brain (Jacobson, 1995). A sequence alignment
was carried out, and the fifth, sixth, and seventh transmembrane
helical domains thereof (TM5-TM7) are shown in Fig. 1A.
In these regions there is a high degree of sequence homology with rat,
canine, and guinea pig A
receptors and with other subtypes
of adenosine receptors.
Figure 1:
A, location of mutations carried out in
this study, illustrated through an alignment of TMs 5, 6, and 7 of
selected adenosine receptor subtypes. Individually mutated residues in
the A (present study), A
(Townsend-Nicholson
and Schofield, 1994; Tucker et al., 1994), and m3 muscarinic
receptors (Wess et al., 1991; Blüml et al.,
1994) and a hexapeptide mutated in a chimeric bovine A
/rat
A
construct (Olah et al., 1994) are shown in bold
type. Accession numbers are: hA2a (human) P29274; rA2a (rat) P30543;
cA2a (canine) P11617; gpA2a (guinea-pig) U04201; hA2b (human) P29275;
bA1 (bovine) P28190; cA1 (canine) P11616; hA1 (human) P30542; hA3
(human) P33765; rA3 (rat) P28647; m3 (rat) P08483;
2 (hamster)
P04274; and NK1 (human) P25103. B, structures of ligands used
in this study.
The 12 residues of the human A receptor, selected as targets for site-directed mutagenesis, are
shown in bold type (Fig. 1A). They include residues that
are conserved among all known adenosine receptor sequences (Phe-182,
Asn-253, Ile-274, His-278, and Ser-281), conserved among all A
and A
receptors (Phe-181 and His-250), conserved
among all A
and A
receptors (Ser-277), present
only in A
receptors (Phe-257), or not conserved (Tyr-271).
Cys-254 is conserved among nearly all adenosine receptors, and Phe-180
is conserved as Phe or Tyr. Each of these amino acid residues was
individually replaced with Ala and/or other amino acids (see below). In
addition, each mutant contained an epitope-tag sequence included at the
NH
terminus for immunological detection (see below), and
pharmacological properties were compared with the wild type receptor
similarly modified.
Ligand Binding Properties of Mutant Human
A
A modeling
study of AAdenosine Receptors
receptors hypothesized that the residues:
Phe-182 (TM5), His-250 (TM6), Asn-253 (TM6), His-278 (TM7), and Ser-281
(TM7) might be involved in direct ligand contact (IJzerman et
al., 1994). Radioligand binding studies showed that mutant
receptors, in which each of these residues was individually modified to
Ala, were unable to bind either the agonist
[
H]CGS 21680 () or the antagonist
[
H]XAC (). The five Ala mutant
receptors displayed less than 2% (<500 cpm) of the specific binding
of [
H]CGS 21680 (15 nM) observed with
the wild type receptor (typically 28,000 cpm/25 µg of protein).
Similarly, in these Ala mutants, the specific binding of 4 nM
[
H]XAC was greatly diminished (<800 cpm/25
µg of protein for mutants versus 8800 cpm for wild type).
Assuming that the Ala mutant receptors are properly expressed on the
cell surface (see ELISA results below), these results indicate that the
above five residues are important, either directly or indirectly, for
the high affinity binding of both agonist and antagonist ligands.
H]CGS 21680 (15 nM) or
[
H]XAC (4 nM). However, the F180A and
C254A mutant receptors showed ligand binding properties similar to
those of the wild type receptor (). Competition binding
studies showed that the F180A and C254A mutant receptors had the same
affinities for NECA, DPMA, CGS 15943, and XAC (structures in
Fig. 1B) as the wild type receptor. Therefore, the
aromatic group in Phe-180 and the -SH group in Cys-254 are not required
for binding of either agonists or antagonists.
H]CGS 21680 present at 15 nM
(), but showed normal affinity for
[
H]XAC ( and Fig. 2). Thus,
the hydroxy group present in the side chain of Ser-277 is required for
high affinity binding of agonists, but not antagonists.
[
H]XAC competition assays (I) showed
that the affinities of the S277A mutant receptor for the agonists CGS
21680, NECA, and CADO were decreased by about 2-3 orders of
magnitude. N
-Substituted agonists such as R-PIA
and DPMA showed somewhat less pronounced reductions in binding
affinities for the S277A mutant receptor (43- and 107-fold,
respectively) than 2- and 5`-substituted adenosine derivatives. It is
interesting that IB-MECA, which is both 5`- and
N
-substituted, also showed a less pronounced decrease in
affinity at the receptor (66-fold versus 400-fold reduction
for NECA). On the other hand, the S277A substitution slightly increased
the affinity for the antagonist CPX, and the affinity for the
antagonist CGS 15943 remained the same. The affinity of
[
H]XAC for the S277A mutant receptor was at least
as high as for the wild type receptor (). These results
indicate that the hydroxyl group of Ser-277 is not involved in binding
of antagonists, but is very important for the high affinity binding of
agonists, especially N
-unsubstituted adenosine agonists.
Figure 2:
Displacement of binding of the antagonist
radioligand [H]XAC from Tag3-A
wild
type (
) and S277A (
) mutant receptors expressed in COS-7
cells. Competitors used were CGS 21680 (A), 2-chloroadenosine
(B), and N
-phenylisopropyladenosine
(C). Competition binding studies were carried out using
membrane homogenates prepared from transfected COS-7 cells, as
described under ``Experimental Procedures.'' Data are
presented as means ± S.E. of two or three independent
experiments, each performed in duplicate.
All Ala mutant receptors except F180A and C254A were unable to bind
specifically with high affinity either [H]CGS
21680 () or [
H]XAC ().
To further probe the role of each residue in ligand-binding at the
human A
adenosine receptor, we introduced several
nonalanine mutations (). Substitution of Asn-181 with Ser
only slightly affected the affinity of all examined antagonists and the
agonist NECA (), while it led to a 14- and 9-fold reduction
in the affinity of the N
-substituted adenosine derivative
R-PIA and 2-chloroadenosine (CADO), respectively. Substitution
of Phe-182 with Tyr or Trp also resulted in 5-9-fold reductions
in affinities for R-PIA or NECA, while having little effect on
antagonist affinities. Substitution of Asn-253 with Ser or Gln almost
completely abolished [
H]CGS 21680 or
[
H]XAC binding. Substitution of Phe-257 with Arg
essentially retained binding affinity for [
H]CGS
21680 (K
= 23 nM,
B
= 2.4 pmol/mg protein). Substitution of
Ser-277 with Asn or Thr resulted in only minor changes (
2-fold) in
binding affinities of all ligands examined. Substitution of Ser-281
with Thr (present in A
receptors) led to an about 10-fold
increase in affinity for the agonists CADO and R-PIA, both of
which are more potent at A
versus A
receptors (Jacobson et al., 1992a).
H]CGS 21680 (15
nM) or [
H]XAC (4 nM).
HA-tag Construction
A strategy for the
immunological detection of mutant receptors that lacked binding of
either agonist or antagonist radioligands was required in order to
discern whether these results were due to a reduced expression of the
mutant receptors on the cell surface or to greatly decreased binding
affinities. Thus, prior to mutagenesis, the human A receptor was tagged at the extracellular NH
terminus
with a 9-amino acid HA epitope (see ``Experimental
Procedures''). The HA-tag was initially inserted after the first
Met residue in the amino terminus of the wild type A
receptor (``Experimental Procedures'' and
Fig. 3A). The tagged receptor (Tag1-A
)
showed the same affinity for [
H]CGS 21680 as the
wild type receptor (), but could not be detected by
immunological techniques (ELISA, Western blotting, or
immunocytochemistry) as shown in Fig. 3B.
adenosine receptor. The
introduction of the HA-tag following the first Met might affect the
initiation of protein translation, causing protein expression to start
at the second Met. This would result in the deletion of the first 3
amino acids (MPI) and the HA-tag not being translated. However, the
resulting mutant receptor may still be functional. We therefore
introduced an HA-tag after the second Met, resulting in
Tag2-A
. This mutant receptor showed the same affinity for
[
H]CGS 21680 and the same expression level
(B
) as the non-tagged receptor ().
Moreover, we could detect the Tag2-A
receptor by
immunological techniques (ELISA, but not Western blotting,
Fig. 3B). To test if the first Met is important for the
expression of Tag2-A
, we deleted the first Met to
construct the Tag3-A
receptor (Fig. 3A).
The Tag3-A
receptor showed the same
[
H]CGS 21680 binding properties ()
and gave a similar signal in ELISA assay as the Tag2 wild type receptor
(Fig. 3B). We therefore concluded that the second Met
may represent the physiological translation start site in the human
A
adenosine receptor. Based on these results, the Tag3
epitope-tag was introduced into all other mutant receptors
(). However, it was not possible to detect Tag3-A
receptor by Western blotting.
Expression Levels of Ala Mutant Receptors
To
estimate approximate levels of receptor protein present in the plasma
membrane, a standard curve was constructed from different batches of
transfected COS-7 cells expressing different levels of Tag3-A wild type receptors (Fig. 4). To decrease the density of
Tag3-A
, COS-7 cells were transfected with progressively
reduced amounts of receptor DNA (supplemented with pcD vector DNA to
maintain 4 µg of total DNA per dish). From the linear region of
this standard curve, an equation was obtained by linear regression, and
this equation (see ) was used to calculate the amount of
receptor protein for the Ala mutants. Experiments were done with
nonpermeabilized cells, therefore the ELISA assay detected only
receptors in the plasma membrane. To establish this point, control
experiments with nonpermeabilized COS-7 cells expressing a
COOH-terminally HA-tagged form of the rat m3 muscarinic receptor were
carried out. The resulting OD readings were similarly low as those
found with the non-tagged versions of the m3 muscarinic and A
adenosine receptors (data not shown). This finding, together with
the observation that the COOH-terminally tagged m3 muscarinic receptor
can be easily detected in permeabilized cells demonstrates that the
employed ELISA procedure does not interfere with the intactness of the
plasma membrane barrier.
Figure 4:
Correlation between the strength of the
absorbance signal determined by the ELISA method and the density
(B) of receptors. The receptor densities were
determined by [
H]CGS 21680 saturation binding
experiments using transfected COS-7 cells derived from the same plate
as those for the ELISA experiments. About 24 h after transfections,
aliquots of COS-7 cells were transferred into 96-well plates, and the
remaining cells were grown for saturation binding assays. ELISA studies
were carried out as described under ``Experimental
Procedures.''
All Ala mutant receptors including H278A
were present at relatively high levels in the plasma membrane, i.e. greater than 25% compared to the wild type receptor. However, this
calculation did not always match the results of saturation binding
experiments. For example, we determined receptor density for the S277A
mutant receptor by the ELISA method to be 25% of wild type, while by
saturation binding the mutant showed increased density (25.6 pmol/mg
versus 12.3 pmol/mg for wild type; ). This
discrepancy might have resulted from different transfection
efficiencies, since we did not carry out the experiments
simultaneously. Since the nine Ala mutant receptors were expressed at
significant levels in the plasma membrane, dramatically decreased
radioligand affinity, rather than insufficient expression, is the
likely source of the observed lack of radioligand binding.
Functional Assay
To determine whether Ala mutant
receptors that lacked high affinity radioligand binding were still
functional at high agonist concentrations, their ability to mediate
increases in intracellular cyclic AMP levels was studied. Untransfected
COS-7 cells showed a strong stimulation of cyclic AMP production
(4-fold; Fig. 5) at >100 µM CGS 21680, using 0.1
mM rolipram as a phosphodiesterase inhibitor (Hide et
al., 1992). Since at this concentration, CGS 21680 is nonselective
among adenosine receptor subtypes (Hide et al., 1992), perhaps
this response may occur through activation of another receptor, such as
the A subtype, since the concentration required is above
the range of A
receptors. COS-7 cells transfected with the
wild type Tag3-A
receptor showed a pronounced stimulation
of cyclic AMP production (3.2-fold) following CGS 21680 treatment, with
an EC
value of 3 nM. The fact that stimulation
occurs at concentrations below the K
value for CGS 21680 (21 nM) suggests that under these
conditions only a minor fraction of the receptors needs to be activated
for effective signal transduction.
Figure 5:
Stimulation of adenylyl cyclase in COS-7
cells transiently expressing Tag3-A wild type or mutant
A
adenosine receptors in the presence of 2 units/ml
adenosine deaminase and 0.1 mM rolipram. The following
receptors were studied: wild type (
), S277A (
), I274A
(
), H278A (
); untransfected COS-7 (
). Transfected
COS-7 cells were incubated for 30 min at 37 °C (for details, see
``Experimental Procedures'') with increasing concentrations
of CGS 21680. Data are presented as fold increase in cyclic AMP above
basal levels (400-600 cpm) in the absence of CGS 21680. Each
curve represents the fold-stimulation average of two or three
independent experiments, each carried out in duplicate. Alternately,
the conversion factor (f) was calculated using the following
equation, f = cAMP(cpm)/(cAMP(cpm) + ATP(cpm)).
The ATP fraction gave very similar values in all samples (598,000
± 4000 cpm). The maximum conversion factor was
0.7.
The receptors I274A, S277A, and
H278A, containing mutations in TM7, also showed dose-dependent
stimulation of cyclic AMP production following treatment with CGS 21680
(Fig. 5; ) with EC values in the
10
-10
M range. The
agonist efficacy in these mutant receptors was at least as much as for
the wild type receptor. With all other Ala mutations (), we
could not detect any stimulation over control up to 1 mM CGS
21680.
Construction of a Human A
A
human AAdenosine Receptor Model: Ligand Binding Hypothesis
receptor model was constructed, independent of the
mutagenesis results, using standard computational methods (see
``Experimental Procedures'') and based on rhodopsin as a
template. Docking of NECA to the human A
receptor model
was carried out manually, followed by energy minimization, in a fashion
that placed the ligand in the center of a region defined by the
essential residues, determined by mutagenesis, shown in green in
Fig. 6B.
(
)
The orientation of the
ligand was such that the adenine ring rested in a hydrophobic pocket
defined by His-250, Phe-182, and Phe-257 (TM5 and TM6), and the
N
-substituent would align with Asn-253 (H-bonding distance
to the exocyclic NH) and Met-270. The ribose ring pointed in the
direction of His-278, while the 5`-uronamide group was in proximity to
Ser-277 and Ile-274 (all TM7). Although a recent study (Olah et
al., 1994) found that replacement of a 6-amino acid cassette in
TM5 (see Fig. 1A) affected mostly binding of
5`-substituted adenosine derivatives, we were unable to place the
5`-uronamide of NECA in direct proximity to this region in our model
while maintaining other favorable interactions.
Figure 6:
Molecular model of the human
A receptor containing NECA bound in the proposed binding
site, viewed either (A) perpendicularly to the membrane from
the extracellular side or (B) as a side view of the residues
in TM5, TM6, and TM7 in proximity to the bound ligand. The model was
based on the structure of rhodopsin and minimized using the Discover
program (BIOSYM Technologies, San Diego CA, Version 2.90) employing
the Amber force field. Side chains in dark green were those
residues found in mutation experiments in this study to be essential
for ligand binding. Side chains in yellow were those residues
found in mutation experiments in this study to be non-essential for
ligand binding, thus Ala mutants at these positions were fully
functional in binding of both agonist and antagonist
radioligands.
Residues that are in
proximity of the ribose moiety in our model are Thr-88, Gln-89, Ser-90,
Ser-91, Ile-92, Ala-273, Ile-274, Ser-277, and His-278. Of these
residues, Thr-88, Gln-89, Ser-90, and Ala-273 seem to be interacting
with NECA primarily through their backbone atoms rather than through
their side chains. Thr-88 may, in addition, show hydrogen bonding to
both the 2`-O and 3`-O. The 5`-OH of adenosine, transformed into 5`-NH
in NECA, is most notably hydrogen bonded to Ser-277 and His-278 in our
model.
(IJzerman et al., 1992), rat A
(van Galen
et al., 1994), and rat A
adenosine receptor
(IJzerman et al., 1994) have been published, we thought it
worthwhile to construct a model of the human A
adenosine
receptor based on the electron density map of rhodopsin rather than on
the atomic coordinates of bacteriorhodopsin. Using rhodopsin as a
template allowed us to better interpret the mutation studies described
in this paper. We have derived a human A
receptor model
mainly through a computational approach, and docked in the binding site
the high affinity agonist ligand, NECA, in a fashion that is consistent
with all of the available pharmacological data. The docking of
antagonist ligands and other modified agonists will be the subject of
future studies. The region of ligand binding (TM5-TM7) is similar to
that predicted in the bacteriorhodopsin-based modeling study of
IJzerman et al.(1994), although novel specific interactions
are proposed.
Histidine Side Chains Involved in Ligand Binding
The His
residues of TM6 and TM7, which have been previously implicated through
mutagenesis in ligand binding to A receptors (Olah et
al., 1992), oriented according to both present and previous
adenosine receptor models in the direction of the binding cleft.
However, even for A
receptors we have had little
information to understand precisely the noncovalent bonding
interactions. It was proposed in the modeling study by IJzerman et
al.(1994) that His-278 coordinates both the 2`- and 3`-hydroxyl
groups of the ribose moiety, but in our model the 5`-hydroxyl group of
adenosine (or amide NH of NECA) is in hydrogen bonding proximity. It is
probably this residue that is referred to as ``the agonist
histidine'' in the literature (Jacobson et al., 1992b).
The homologous His residue has also been postulated to play a role in
ribose coordination in the A
receptor (van Galen et
al., 1994). More recently, Askalan and Richardson(1994) provided
support for the assumption that the ``agonist histidine''
directly interacts with the 2`-hydroxyl group of the ribose moiety. We
found it impossible to dock the ligand into the receptor in such a
fashion that His-278 interacts directly with the 2`-hydroxyl group
while maintaining all the other interactions. Analysis of various
mutants with several (radiolabeled) ribose-modified ligands could,
perhaps, further elucidate this matter.
receptors dramatically reduced affinity of both
agonist and antagonist radioligands. These mutant receptors were
expressed in the plasma membrane, at levels not qualitatively different
from that of the wild type receptors (). In a functional
assay CGS 21680 was able to stimulate cAMP production in the H278A
mutant receptor but with a
300-fold decreased potency. These
results are in contrast to a previous study of bovine A
receptors (Olah et al., 1992), in which mutation of the
corresponding His in TM7 to Leu similarly abolished binding, but
mutation of the His of TM6 only somewhat reduced affinity for XAC and
did not affect agonist binding. Even though His-278 is adjacent to
Ser-277 in TM7 (see below), the H278A mutant receptor did not bind
either [
H]CGS 21680 or
[
H]XAC, while S277A failed to bind only
[
H]CGS 21680, an agonist (). The fact
that His-278 is located in the putative ribose binding region and also
affects antagonist binding suggests that there is a possibility that
His-278 may be involved in other interactions as well as ligand
binding. This possibility is supported by the effects of the following
other mutations: substitution of His-278 with Gln, Phe, and Tyr also
prevented binding of either [
H]CGS 21680 or
[
H]XAC, suggesting that His-278 may be essential
for maintaining the conformation of A
adenosine receptors.
-substituents, and of CADO (). The affinities
of 5`-substituted adenosine analogues and various antagonists
() were nearly unaffected. It is possible that the
5`-uronamide group may form a better anchor than 5`-hydroxyl
derivatives by favorable interactions with TM7 (see below), thus
reducing dependence on the imidazole interactions. In summary, these
data suggest that mainly aromatic (hydrophobic) interactions in the
region of His-250 are important for the binding of both agonists and
antagonists.
Other Aromatic Residues Involved in Ligand
Binding
The molecular model predicts that there is a
hydrophobic/aromatic binding pocket, putatively the site of
coordination of the adenine moiety, defined by His-250 and Phe-257
(TM6) and Phe-182 (TM5). Mutation of residue Phe-182 or Phe-257 to Ala
resulted in loss of specific binding of either
[H]CGS 21680 or [
H]XAC,
indicating the importance of these side chains in ligand recognition.
Mutation of Phe-182 to Trp reduced agonist binding affinities
3-10-fold, whereas antagonist affinity was not affected.
Replacement of the same residue with Tyr, however, decreased agonist
affinity 8-10-fold, while antagonist affinity was decreased only
2-fold. The propensity for hydrogen bond formation, increasing as
residue 182 is changed from Phe to Trp to Tyr, inversely correlates
with binding affinity. This notion supports the aromatic interaction
hypothesis.
Aliphatic and Hydrophilic Side Chains Involved in Ligand
Binding
Met-270 (common to both human A and canine
A
adenosine receptors; Fig. 1A) was shown to
be important for species differences, i.e. the lower affinity
of N
-substituted adenosine derivatives at canine versus bovine A
receptors (Tucker et al., 1994). The
sulfur atom of this residue was positioned at 6.0 Å from the
N
atom in NECA in our model, thus consistent with direct
contact with the N
-substituent as an explanation for the
observed effect. Met-270 is conserved among species in the A
(Fig. 1A) and A
adenosine receptors,
and may be responsible for the generally low affinity of
N
-substituted derivatives at A
versus A
adenosine receptors.
- and
C-2-substituents occupy overlapping positions in the receptor-bound
conformations of adenosine agonists (IJzerman et al.(1994) and
references therein). The present results support this concept, since
N
- (R-PIA and DPMA, having aromatic bulk) and
C-2-modified (CADO, hydrophobic substituent) adenosine analogues (all
5`-CH
OH) behaved similarly. The side chain carbonyl of
Asn-253 is favorably located at 4.3 Å from the N
atom
in NECA according to the model. Mutation of this residue, even to Gln,
led to a total loss of specific [
H]CGS 21680 and
[
H]XAC binding, thus the structural requirements
for adenosine and xanthine derivatives at this site are highly
restrictive. Interaction of Asn-253 with the N
atom of
adenine, as suggested by IJzerman et al.(1994) for the rat
A
adenosine receptor, is less likely in our model.
-substituted agonists and CADO. N181S had slightly
decreased (only
2-fold decrease) affinity for CGS 21680, NECA, CGS
15943, and XAC, but 20-fold decreased affinity for R-PIA.
Residue Asn-181, although not hydrophobic, according to our model is
facing the lipid bilayer. If TM5 were rotated in the model (see above),
Asn-181 would then be located in the inter-helical contact region,
adjacent to Pro-139 of TM4. Thus, it is possible that the side chain of
this Asn residue is involved in hydrogen bonding to the backbone.
Disturbance of this hydrogen bond by mutation may affect overall
receptor structure, and indirectly ligand binding. Such an assumption
is consistent with the effects observed here.
adenosine receptor, previous studies have shown
that Thr-277 in TM7 is critical for the interaction with the ribose
ring, especially for binding of a subset of agonist ligands, i.e. those containing the 5`-uronamide modification, such as NECA
(Townsend-Nicholson and Schofield, 1994). This residue has also been
indicated to be involved in species-dependent ligand binding at A
receptors (Tucker et al., 1994). In the A
receptor the corresponding residue, Ser-277, conserved across
species (see Fig. 1A), was found in this study to
contain an hydroxyl group that is essential for agonist binding. The
S277A mutation only slightly changed the affinity for the antagonists,
indicating that the Ser hydroxyl group does not contribute
energetically to antagonist binding (). The affinity of
N
-substituted agonists was less affected by the S277A
substitution, perhaps as a result of additional stabilization of the
N
-substituent by neighboring hydrophobic residues. Hence,
the interaction of the ribose ring with Ser-277 may be relatively weak
compared to other interactions at the N
-region.
Furthermore, mutation of Ser-277 to either hydrogen bonding residues
Thr or Asn affected agonist affinity only moderately ().
These results corroborate a model where the ribose moiety, essential
for agonist activity, occupies a predominantly hydrophilic region of
the receptor that is not occupied by antagonists.
adenosine receptor) was implicated by
IJzerman et al.(1994) to be involved in binding the 5`-hydroxy
region of adenosine. The S281A mutant receptor indeed does not display
high affinity for [
H]CGS 21680. In our model the
side chain oxygen of Ser-281 is separated from the 5` nitrogen of NECA
by 8.2 Å, a distance too large to accommodate hydrogen bonds.
However, our model also suggests the possibility of a conjugated
hydrogen bond from Ser-281 through His-278 to the 5` region. Such a
conjugated hydrogen bond system would be disrupted by the mutation and
explain the observed loss of ligand affinity. Of further interest is
residue Ile-274. In the ribose-binding region, according to the model,
the side chain of Ile-274 is in close proximity of the terminal methyl
group of the 5`-uronamide of NECA (3.2 Å), a ligand region that
has strict steric requirements (Jacobson et al., 1992a). The
dramatically decreased affinity of the I274A mutant receptor for
[
H]CGS 21680 and [
H]XAC
might be related to the loss of hydrophobicity resulting from this
mutation. However, this receptor was well expressed (72% of wild type),
and showed good stimulation of cAMP-production with a 33-fold decreased
affinity for CGS 21680.
H]CGS 21680
(, footnote b), suggesting that Asn-253 is also essential
for ligand recognition. According to the present model Asn-253 is
within hydrogen bonding distance from the N
H of NECA.
Substitution of Ser-281 with Thr showed increased affinity for some
agonists, but S281A was unable to bind either
[
H]CGS 21680 or [
H]XAC in
spite of significant plasma membrane expression (36%; ).
Taken together, we conclude that Ser-277 and Ser-281 might be involved
in the interaction with the ribose ring of agonists.
Side Chains That Are Not Involved in Ligand
Binding
The molecular model predicts that Phe-180 and Cys-254
(shown in yellow in Fig. 6) are pointing away from the ligand
binding cleft into the lipid bilayer. If indeed located in the lipid
bilayer, mutation of these residues should have little effect on ligand
affinity, unless the receptor structure itself is corrupted. Indeed,
mutagenesis studies are consistent in that Ala substitutions at these
sites have no effect on ligand affinity. Other residues found to be
essential in ligand recognition were generally predicted by the model
to be facing in the direction of the putative ligand binding site
(shown in green in Fig. 6) or in the inter-helical contact
regions.
Comparison to Essential Side Chains in Other
Receptors
Residues Asn-181, His-250, and His-278 of A receptors align with Thr-234, Asn-507, and Tyr-533, respectively,
in the rat m3 muscarinic receptor (Fig. 1A), which have
been shown by mutagenesis to be involved in ligand binding (Wess et
al., 1991; Blüml et al., 1994). Residues Phe-182 and
Ser-281 correspond to serines 204 and 319 in the hamster
adrenergic receptor, which are also involved in ligand
recognition (Strader et al., 1989). His-250 is equivalent to
His-265 in the NK1 receptor. Mutation of this residue to alanine
influenced the selectivity of antagonists, but did not affect the
affinity of agonists (Fong et al., 1994).
Table:
Binding characteristics of wild type and mutant
human A adenosine receptors using an agonist radioligand
Table:
[H]XAC binding properties
of wild type, S277A, and other mutant human A
adenosine
receptors
H]XAC
saturation binding studies were carried out with membrane homogenates
prepared from transiently transfected COS-7 cells as described under
``Experimental Procedures.'' B
values
indicate the maximum number of binding sites/mg of membrane protein.
Table:
Ligand binding properties of wild type and
S277A human A receptors
values) were determined in [
H]XAC (2
nM) competition binding studies using membrane homogenates
prepared from transiently transfected COS-7 cells, as described under
``Experimental Procedures.'' K
values were calculated from IC
value using the
GraphPad program. About 15 µg of membrane protein/tube were used.
Table:
[H]CGS 21680 binding
properties of wild type and three epitope-tagged human A
receptor constructs
H]CGS 21680 saturation binding studies were
carried out with membrane homogenates prepared from transiently
transfected COS-7 cells as described under ``Experimental
Procedures.''
Table:
ELISA
detection of human A receptor mutants on the surface of
COS-7 cells
wild type (=100%).
Table:
CGS 21680-induced stimulation of cAMP
production mediated by mutant A adenosine receptors
-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adeno-sine;
ELISA, enzyme-linked immunosorbent assay; GPCR, G-protein coupled
receptor; HA, hemagglutinin; IB-MECA,
N
-(3-iodobenzyl)adenosine-5`-N-methyluronamide;
NECA, 5`-N-ethylcarboxamidoadenosine; PCR, polymerase chain
reaction; R-PIA,
R-N
-phenylisopropyladenosine; TM,
transmembrane helical domain; XAC,
8-[4-[[[[(2-amino-ethyl)amino]carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine.
plasmid. We thank Dr. Xiao-duo Ji, Dr. Neli
Melman, and Patricia Evans for assisting with binding experiments and
Carola Gallo-Rodriguez for preparing IB-MECA.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.