From the Molecular Recognition Section, Laboratory of
Bioorganic Chemistry, NIDDK, National Institutes of Health,
Bethesda, Maryland 20892 and the § Department of
Pharmacology, University of North Carolina School of Medicine,
Chapel Hill, North Carolina 27599
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ABSTRACT |
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The P2Y1 receptor is a
membrane-bound G protein-coupled receptor stimulated by adenine
nucleotides. Using alanine scanning mutagenesis, the role in receptor
activation of charged amino acids (Asp, Glu, Lys, and Arg) and
cysteines in the extracellular loops (EL) of the human P2Y1
receptor has been investigated. The mutant receptors were expressed in
COS-7 cells and measured for stimulation of phospholipase C induced by
the potent agonist 2-methylthioadenosine-5'-diphosphate (2-MeSADP). In
addition to single point mutations, all receptors carried the
hemagglutinin epitope at the N- terminus for detection of cell-surface
expression. The C124A and C202A mutations, located near the exofacial
end of transmembrane helix 3 and in EL2, respectively, ablated
phospholipase C stimulation by P2 receptors have been divided into two structurally distinct
families as follows: the P2X receptor class of ligand-gated ion
channels, which are primarily activated by ATP, and the P2Y receptor
class of G protein-coupled receptors
(GPCRs),1 which are activated
by both extracellular adenine and uridine nucleotides (1-3). As many
as 11 subtypes of P2Y receptors have been cloned, but only 5 of these
are from mammalian species and have been shown to be functionally
activated by extracellular nucleotides. Of these 11, several may be
either non-nucleotide receptors such as P2Y5,
P2Y7, P2Y9, and P2Y10 or species
homologues like p2y3 and P2Y6. The P2Y1 subtype
specifically recognizes adenine nucleotides, which act as either potent
agonists (e.g. 2-methylthio-ADP) or selective antagonists
(e.g. MRS 2179). The P2Y1 receptor, which is
coupled to activation of phospholipase C (PLC), is expressed in heart
muscle, skeletal muscle, and various smooth muscle cells and is
associated with the vasodilatory action of ATP (3). In platelets, it
serves as one of the receptors through which ADP induces aggregation,
an action attenuated by ATP and other triphosphate derivatives (4).
Previously we have investigated the determinants of ligand recognition
in P2Y1 receptors using site-directed mutagenesis of specific amino acid residues in the transmembrane helical domains (TMs)
(5), molecular modeling based on similarity to rhodopsin in sequence
and overall geometry (6), and the chemical synthesis of novel agonists
(7) and antagonists (8). Positively charged and other conserved
residues in TM3, TM6, and TM7, such as Arg128,
Arg310, and Ser314, were found to be critical
for the activation by nucleotides of human P2Y1 receptors.
A molecular model of the TM domains based on these findings has been
proposed (6).
Most of the interest in GPCRs to date has focused on the TM domains for
locating amino acids that are specifically involved in recognition of
small, non-peptide ligands, including nucleotides (5, 9, 10). However,
for peptide receptors residues within the extracellular loops (ELs) are
essential for ligand recognition (11). Currently, there is accumulating
evidence that residues within the ELs are also important in GPCRs that
recognize small molecules as ligands. In adenosine receptors, amino
acids in EL2 were shown to be involved in agonist and antagonist
binding (12) and high affinity binding of xanthine antagonists (13). In
the thyrotropin-releasing hormone receptor, amino acids in EL2 and EL3
were found to be important for ligand binding (14). In the A conserved disulfide bridge between Cys residues in the second
extracellular loop (EL2) and the exofacial end of TM3 has been shown to
be essential for various biogenic amine and other GPCRs. It is
generally thought that this disulfide bond is required to maintain
overall receptor geometry, although its role in transport to the cell
surface is contradictory (18, 19). An additional disulfide bridge
between a cysteine in the EL3 and the N-terminal domain was found in
the angiotensin receptor (20). The extracellular portions of the human
P2Y1 receptor carry four cysteines, and sequence comparison
has revealed that all of these cysteines are conserved among P2Y
receptors (21). Hence P2Y receptors might form two disulfide bridges
within the extracellular receptor portions.
Interestingly, the P2Y receptors are GPCRs that recognize small
molecules as ligands, but they exhibit the highest sequence identity
with peptide receptors such as somatostatin, platelet-activating factor, angiotensin II, and neuropeptide Y receptors (22). Because of
this paradox (binding of a small ligand and sequence homology with
peptide receptors), we have investigated the role of amino acids in the
ELs in human P2Y1 receptor activation.
The aim of this study was to identify amino acids that potentially may
be involved in either direct ligand contact or in conformational restriction of the ELs. We hypothesized that disruption of either of
the two potential disulfide bonds in the ELs or substitution of amino
acids potentially involved in ionic interactions (Asp, Glu, Lys, or
Arg) might have a marked effect on ligand recognition and thus
receptor-mediated activation of PLC.
Materials--
The expression construct coding for the human
P2Y1 receptor (pCDP2Y1) was prepared as
described previously (5). Vent DNA polymerase and all endonuclease
restriction enzymes used in this study were obtained from New England
Biolabs (Beverly, MA). The agonists 2-MeSATP and 2-MeSADP were from
Research Biochemicals (Natick, MA). The agonist
2-hexylthioadenosine-5'-monophosphate (HT-AMP) was synthesized as
described (7, 23) as the ammonium salt, which was more soluble in
aqueous medium than the triethylammonium salt.
myo-[3H]Inositol (15 Ci/mmol) was obtained
from American Radiolabeled Chemicals. Fetal bovine serum (FBS) was from
Life Technologies, Inc. o-Phenylenediamine dihydrochloride
was purchased from Sigma. The Sequenase Kit version 2.0 was from
Amersham Pharmacia Biotech. All oligonucleotides were synthesized by
Bioserve Biotechnologies (Laurel, MD). A monoclonal antibody (12CA5)
against a hemagglutinin epitope (HA) was purchased from Roche Molecular
Biochemicals, and goat anti-mouse IgG ( Plasmid Construction and Site-directed Mutagenesis--
All
mutations were introduced into pCDP2Y1 (5) using standard
polymerase chain reaction mutagenesis techniques (24). The accuracy of
all polymerase chain reaction-derived sequences was confirmed by
dideoxy sequencing of the mutant plasmids (25).
Epitope Tagging--
As described previously (5) a 9-amino acid
sequence derived from the influenza virus hemagglutinin (HA) protein
(TAC CCA TAC GAC GTG CCA GAC TAC GCG; peptide sequence: YPYDVPDYA) was inserted after the initiating methionine residue in the extracellular N
terminus of the human P2Y1 receptor gene. A hexahistidine
tag (26) was also included at the C terminus immediately after the ultimate leucine residue resulting in a construct suitable for potential affinity chromatography using a nickel column.
Transient Expression of Mutant Receptors in COS-7
Cells--
4 × 106 COS-7 cells were seeded into
150-mm culture dishes containing 25 ml of Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10% FBS, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 µmol/ml glutamine. Cells were transfected
approximately 24 h later with plasmid DNA (10 µg DNA/dish) using
the DEAE-dextran method (27) for 40 min, followed by treatment with 100 µM chloroquine for 2.5 h, and grown for an
additional 24 h at 37 °C and 5% CO2.
Inositol Phosphate Determination--
Assays were carried out
according to the general approach of Harden et al. (28).
About 24 h after transfection, the cells were split into 6-well
plates (Costar, ~0.75 × 106 cells/well) in DMEM
culture medium supplemented with 3 µCi/ml myo-[3H]inositol. After a 24-h labeling
period, cells were preincubated with 10 mM LiCl for 20 min
at room temperature. The mixtures were slightly swirled to ensure
uniformity. Following the addition of agonists, the cells were
incubated for 30 min at 37 °C, 5% CO2. The supernatant
was removed by aspiration, and 750 µl of cold 20 mM
formic acid was added to each well. After a 30-min incubation at
4 °C, cell extracts were neutralized with 250 µl of 60 mM NH4OH. The inositol monophosphate fraction
was then isolated by anion exchange chromatography (29). The contents
of each well were applied to a small anion exchange column (Bio-Rad
AG-1-X8) that had been pretreated with 15 ml of 0.1 M
formic acid, 3 M ammonium formate, followed by 15 ml of
water. The columns were then washed with 10 ml of water followed by 15 ml of a solution containing 5 mM sodium borate and 60 mM sodium formate. [3H]Inositol phosphates
were eluted with 4.5 ml of 0.1 M formic acid, 0.2 M ammonium formate and quantitated by liquid scintillation spectrometry (LKB Wallace 1215 Rackbeta scintillation counter).
Pharmacological parameters were analyzed using the KaleidaGraph program
(Abelbeck Software, version 3.01).
ELISA--
For cell surface ELISA measurements, cells were
transferred to 96-well dishes (4-5 × 104 cells per
well) 1 day after transfection. About 72 h after transfection, cells were fixed in 4% formaldehyde in phosphate-buffered saline (PBS)
for 30 min at room temperature. After washing three times with PBS and
blocking with DMEM (containing 10% FBS), cells were incubated with the
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. Hydrogen peroxide and o-phenylenediamine (each
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 H2SO4 solution containing 0.05 M Na2SO3, and the color development
was measured bichromatically in the BioKinetics reader (EL312, BioTek
Instruments, Inc., Winooski, VT) at 490 and 630 nm (base line). The
reading for cells transfected with expression constructs coding for the human P2Y1 wild-type receptor was approximately 0.5 OD
units, and approximately 0.2 OD units for cells transfected with vector DNA alone. The difference was normalized as 100% surface expression.
Western Blotting--
Wild-type and all cysteine mutant
receptors were detected with the polyhistidine monoclonal antibody (0.5 µg/ml, clone BMG-His-1, Roche Molecular Biochemicals) directed
against the hexa-His tag present at the C-terminal domain of all
receptors. Samples containing 50 µg of solubilized membrane protein
prepared from transfected COS-7 cells as described previously (30) were
resolved by SDS-polyacrylamide gel electrophoresis (4-20% miniplus
sepragel, Owl Separation Systems, Woburn, MA), electroblotted onto
nitrocellulose, and probed with the polyhistidine monoclonal antibody
as described (31). Immunoreactive proteins were detected by incubation
with horseradish peroxidase-conjugated sheep anti-mouse antibody
(1:2500, Amersham Pharmacia Biotech) and visualized using an enhanced
chemiluminescence system (Amersham Pharmacia Biotech).
Cysteine Residues--
Among P2Y receptors, four cysteines in the
extracellular domains are conserved (21). For the human
P2Y1 receptor these cysteines are Cys42 in the
N-terminal domain, Cys124 near the exofacial end of TM3,
Cys202 in EL2, and Cys296 in EL3. Each of these
cysteine residues was individually mutated to alanine, and the
corresponding mutants were tested for activation of PLC by three
different agonists (2-MeSATP, 2-MeSADP, and HT-AMP). The results are
summarized in Table I. The C202A mutant
receptor showed no activation up to 100 µM concentration
of all agonists used in this study. Analogously, the C124A mutant
receptor exhibited the same properties, with no increased stimulation
of PLC detectable at
All mutant receptors contained an HA epitope at the N terminus,
allowing cell-surface expression levels to be measured by ELISA (Table
I). Both C124A and C202A mutant receptors showed strongly diminished
surface expression. The C124A mutant receptor was detectable at
approximately 10% of the level of the wild-type receptor, whereas the
level of the C202A mutant receptor was not significantly different from
vector-transfected control. Western blot analyses of membrane fractions
prepared from transfected cells showed that these cysteine mutant
proteins were expressed in almost equal amounts compared with the
wild-type receptor (Fig. 1). These
results, together with the lack of agonist-promoted functional
activity, suggest that a disulfide bridge between Cys124
and Cys202 is critical for proper receptor trafficking of
the human P2Y1 receptor to the cell surface. These results
are in concordance with previous studies indicating that this conserved
disulfide bridge found in the vast majority of GPCRs is important for
proper receptor function (11, 32).
In contrast to the above cysteine mutant receptors, the C42A and C296A
mutant receptors were activated by 2-MeSADP; however, the 2-MeSADP
concentration-response curves were shifted by more than 1000-fold to
the right compared with the wild-type receptor. The same relative shift
in receptor activation was observed for the two other agonist ligands
(Table I), indicating a similar influence on the general recognition of
the agonist ligands. The double point mutant C42A/C296A exhibited a
similarly impaired response as the single alanine mutants, indicating
that the effect of this cysteine substitution is not additive (Fig.
2).
Although the expression levels of C42A, C296A, and C42A/C296A mutant
receptors were low, activation of PLC was clearly detectable. Control
experiments (inositol phospate assay and ELISA) in which the amount of
DNA used for transfection was varied showed that the wild-type
P2Y1 receptor was significantly stimulated, even at
expression rates as low as 10% of the normal expression level attained
using 10 µg of DNA. No significant shift in EC50 values was observed with a 10% expression rate of wild-type receptor (Fig.
3, A and B); thus,
the EC50 values observed for these three cysteine mutant
receptors likely reflect the intrinsic activity of these
constructs.
Extracellular Loop 1--
Only two positions in EL1 were selected
for investigation of their influence on agonist activation of
P2Y1 receptors as follows: Cys124 (see above)
and Lys125, which is functionally conserved within the P2Y
family. The K125A mutant showed no difference compared with the
wild-type receptor in agonist-promoted PLC activation. ELISA data for
this mutant receptor showed approximately 45% of wild-type receptor
expression, indicating that Lys125 does not influence
receptor activation. No further mutations were made in EL1, since this
loop is presumed to be located distal to the ATP-binding site (6).
Extracellular Loop 2--
Seven positively or negatively charged
amino acids and one cysteine residue (see above) are located in EL2.
All of these amino acids were mutated individually to alanine. Although
R195A, K196A, K198A, D208A, and R212A mutant receptors showed no
detectable changes in activation of PLC and were well expressed on the
cell surface (Table I), the D204A and E209A mutant receptors behaved much differently. The D204A mutant receptor was activated at
20-25-fold higher agonist concentrations compared with the wild-type
receptor. Furthermore, this shift was observed for all agonists,
independent of the length of the 5'-phosphate chain. To investigate
further the role of Asp204 in receptor activation, we
constructed D204E and D204N mutant receptors. Extension of the alkyl
carboxylate side chain at position 204 (D204E) further impaired
agonist-promoted activation of the receptor, resulting in a 55-65-fold
shift of the concentration-response curves. Surprisingly, the
relatively small change of a carboxylate to carboxamide in the D204N
mutant receptor caused an even greater change (200-270-fold shift in
EC50 values) in receptor activation (Fig.
4 and Table I).
In contrast, the E209A mutant receptor exhibited a dramatically
different activation pattern. This mutant receptor was activated by
>1000-fold higher concentrations of each agonist than the wild-type receptor (Fig. 5 and Table I). The nature
of the interaction of this carboxylate side chain with agonists was
further tested by additional mutant receptors E209D, E209Q, and E209R.
These mutant receptors were fully active and responded in a manner
indistinguishable from wild-type receptors (Table I).
Extracellular Loop 3--
Beside Cys296 (see above),
five charged amino acids were investigated in EL3. The R285A, D289A,
D300A, and R301A mutations had no effect on PLC activation, whereas the
R287A mutant receptor was activated at more than 1000-fold higher
agonist concentrations than wild-type receptors, and the profile of the
concentration-response curves was surprisingly similar to that of the
E209A mutant receptor (see Fig. 5). Thus, Arg287 was
targeted for a more detailed analysis. The R287K, R287Q, and R287E
mutant receptors were generated and tested for PLC activation. The
R287Q mutant receptor required >1,000-fold higher agonist concentrations for activation of PLC compared with the wild-type receptor, whereas the R287E mutant receptor was not significantly activated by agonist concentrations up to 100 µM. In
contrast to these findings, receptor activity could be partially to
fully retained by changing Arg287 to lysine. However, the
concentration-response curves for the nucleoside diphosphate and
triphosphate were shifted to the right (35- and 17-fold, respectively,
compared with wild-type receptors), whereas the concentration-response
curve for the nucleoside monophosphate was not shifted.
Since the E209A and R287A mutant receptors exhibited very similar
activation patterns when stimulated with 2-MeSADP (Fig. 5), we tested
the hypothesis that a direct interaction between these residues might
be required for stabilization of the EL. Molecular modeling studies of
the EL (33) suggested that Glu209 and Arg287
might form an ionic interaction. The double mutant E209R/R287E and the
double deletion mutant E209A/R287A were constructed and investigated.
The double mutant E209R/R287E was not activated by agonist
concentrations up to 100 µM, indicating that the amino acids at these positions were not interchangeable. The double mutant
E209A/R287A exhibited an additive shift in concentration-response curves for receptor activation (Fig. 5) compared with the single mutants, suggesting that Glu209 and Arg287
affect receptor activation independently.
In the present study we have identified both charged residues and
Cys residues within the ELs of the human P2Y1 receptor that are critical for the activation of the receptor. We have shown that the
highly conserved disulfide bridge between the EL2 and TM3 is required
for proper functioning of the receptor. Since the double cysteine
mutant receptor (C42A/C296A, see Fig. 2) showed PLC activation similar
to the single cysteine mutants (C42A and C296A), we deduced
the existence of an additional, critical disulfide bridge between the
EL3 and the N-terminal domain, which seems to be involved in receptor
activation as well as proper receptor trafficking to the cell surface.
A similarly situated disulfide bridge was found in the angiotensin
AT1 receptor (26, 27), and it was concluded that this
disulfide bridge functions to position properly extracellular amino
acids that are involved in the binding of AT II. Sequence comparisons
show that the angiotensin AT1 receptor is one of the most
closely related receptor to the family of P2Y receptors (21). This
novel disulfide bridge would covalently constrain the helical bundle in
a circular arrangement (33).
Moreover, each of the charged residues of EL2 and EL3 regions has
been mutated, leading to the observation that three of these residues
(Asp204, Glu209, and Arg287) appear
to be involved in receptor activation and/or ligand recognition. Unfortunately, due to the lack of a high affinity radioligand (34), we
could not distinguish between effects on agonist binding or on G
protein coupling efficiency. At present the function of Asp204 remains to be clarified, but this position appears
to be very sensitive to even minor structural changes.
Glu209 was substituted by different amino acids, and all
mutant receptors (except E209A) were fully active and responded in a
manner indistinguishable from wild-type receptors (Table I). This
indicates that this carboxylate group is more likely involved in
hydrogen bonding rather than an ionic interaction. The conformational
requirements of this hydrogen bonding would be largely flexible, since
deletion of one methylene group (E209D mutant receptor) or change of
the charge (E209R mutant receptor) had no further influence on receptor activation.
Arg287 was substituted by various amino acids. Removal
(R287Q mutant) or change (R287E mutant) of the charge of the amino acid side chain was not tolerated. Substitution of arginine by lysine (R287K
mutant) could partially to fully retain receptor activity, depending on
the length of the phosphate chain of the agonist used for receptor
stimulation. Hence, Arg287 appears to participate in a
direct ionic interaction with the phosphate group of an agonist, which
is crucial for receptor activation.
The possible existence of an ionic bridge between EL2 and EL3 via the
side chains of Glu209 and Arg287 could not be
clarified by mutagenesis. Both residues might have a combined function
in maintaining the overall conformation of the receptor and/or through
direct interaction with the ligand, as indicated by the different
response of the R287K mutant receptor to different agonists (Table I).
This issue is being explored in more detail through molecular modeling
and molecular dynamics simulations (33). An energetically sound
conformational hypothesis for the receptor has been calculated that
includes transmembrane (TM) domains (using the electron density map of
rhodopsin as a template), extracellular loops, and a truncated
N-terminal region. ATP may be docked in the receptor, both within the
previously defined TM cleft and within two other regions of the
receptor, termed meta-binding sites, defined by the extracellular
loops. The first meta-binding site is located outside of the TM bundle, between EL2 and EL3, and the second meta-binding site is positioned immediately underneath EL2 (33). In meta-binding site I, the side chain
of Glu209 (EL2) is within hydrogen bonding distance (2.8 Å) of the ribose O-3', and Arg287 (EL3) coordinates both
Our data have identified two or three stable, steric constraints on the
conformation of otherwise highly flexible peptide loops suggesting a
preferred three-dimensional arrangement of these ELs (33). These
constraints might be responsible for proper positioning of amino acids
involved in ligand recognition and/or receptor activation. Fig. 6
visually summarizes the currently available mutagenesis data for the
human P2Y1 receptor, obtained in the present and previous
studies (5).
Unlike the transmembrane domains in GPCRs, the length and amino acid
composition of the extracellular loops is highly variable. However, it
is possible that the position rather than the actual amino acid within
the extracellular loops, especially the second loop, is important for
ligand recognition. For the human P2Y1 receptor, two amino
acids, Asp204 and Glu209, were identified as
being critical for receptor activation. These amino acids are located
two and seven residues, respectively, beyond the Cys that forms the
critical disulfide bridge between EL2 and TM3 (Fig.
6 and 7).
Previous studies have shown the involvement of EL2 of other GPCRs in
ligand recognition (Fig. 7). The amino acid in position
Cys+22 was found to be
involved in ligand binding for the human Y1 neuropeptide Y receptor
(35), the thyrotropin-releasing hormone receptor of rat and mouse (14,
36), and subtype-specific agonist binding in bombesin receptors (37)
and cholecystokinin receptor types A and B (38). For the substance P
(NK-1) receptor from rat a six-amino acid portion of EL2, including the
Cys+2 position, was found to be labeled by a photoreactive analogue of
substance P (39). Almost the same region of the human NK-1 receptor was found to be responsible for a loss of agonist binding affinity, when
substituted with the corresponding sequence of the human NK-3 receptor
(40). A study of chimeric adenosine A1/A3
receptors identified an 11-amino acid portion of EL2, including the
critical cysteine, to be responsible for subtype specificity of ligand binding (13). Interestingly, in this chimeric receptor 8 amino acid
residues were altered, but the amino acid in position Cys+2 was
conserved. In the human A2A receptor, a glutamate residue three amino acids C-terminal to the cysteine in EL2 was found to be
involved in ligand binding (12). Interestingly, the position Cys+3 was
not conserved in the study of chimeric adenosine
A1/A3 receptors (13). For the AT1 angiotensin
receptor, the position Cys+3 was also found to interact with the
agonist (41). For the 100 µM 2-MeSADP. Surface enzyme-linked immunosorbent assay detection of both mutant receptors showed <10% expression, suggesting that a critical disulfide bridge between EL2 and the upper part of transmembrane 3, as found in many
other G protein-coupled receptors, is required for proper trafficking
of the P2Y1 receptor to the cell surface. In contrast, the
C42A and C296A mutant receptors (located in the N-terminal domain and
EL3) were activated by 2-MeSADP, but the EC50 values were
>1000-fold greater than for the wild-type receptor. The double mutant
receptor C42A/C296A exhibited no additive shift in the concentration-response curve for 2-MeSADP. These data suggest that
Cys42 and Cys296 form another disulfide bridge
in the extracellular region, which is critical for activation.
Replacement of charged amino acids produced only minor changes in
receptor activation, with two remarkable exceptions. The E209A mutant
receptor (EL2) exhibited a >1000-fold shift in EC50.
However, if Glu209 were substituted with amino acids
capable of hydrogen bonding (Asp, Gln, or Arg), the mutant receptors
responded like the wild-type receptor. Arg287 in EL3 was
impaired similarly to Glu209 when substituted by alanine.
Substitution of Arg287 by lysine, another positively
charged residue, failed to fully restore wild-type activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-adrenergic receptor, three amino acids at the
C-terminal end of EL2 were shown to be responsible for subtype-specific
antagonist binding (15). In angiotensin II type 2 receptors, amino acid residues in EL2 and EL3 contribute to angiotensin II binding (16), whereas in angiotensin II type 1 receptors, an additional residue in
EL1 is involved in this process (17).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain-specific) antibody
conjugated with horseradish peroxidase was purchased from Sigma.
DEAE-dextran was obtained from Amersham Pharmacia Biotech.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
100 µM agonist.
Receptor-stimulated PLC activation ([3H]inositol phosphate
accumulation) and surface expression assays (ELISA) of wild-type and
mutant human P2Y1 receptors
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Fig. 1.
Western blot analysis of receptor mutant
expression. Wild-type and all cysteine mutant receptors were
transiently expressed in COS-7 cells. Samples containing 50 µg of
solubilized membrane proteins prepared from whole transfected COS-7
cells were applied to each lane and resolved by SDS-polyacrylamide gel
electrophoresis. Proteins were detected with a polyhistidine monoclonal
antibody directed against the hexa-His tag at the C-terminal domain of
all receptors and a horseradish peroxidase-conjugated sheep anti-mouse
antibody. Lanes 1-7 correspond to control (transfected with
PCD-vector, lane 1), wild type (lane 2), C42A
(lane 3), C124A (lane 4), C202A (lane
5), C296A (lane 6), and C42A/C296A (lane 7)
mutant receptors. Lanes 2-7 show an additional band at
approximately 42 kDa, which is the size expected for the wild-type P2Y1
and mutant receptors. All bands are of similar intensity, indicating
equal expression levels for wild-type and mutant receptors.
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Fig. 2.
Concentration-response curves of
P2Y1 receptors with mutated cysteine residues in the
N-terminal domain and the third extracellular loop. Wild-type
human P2Y1 receptor (squares) or mutant
receptors in which Cys42 (C42A, circles) and/or
Cys296 (C296A, diamond, and C42A/C296A,
inverted triangle) were converted to alanine and
transiently expressed in COS-7 cells. [3H]Inositol
phosphate accumulation was measured following a 30-min incubation with
increasing concentrations of 2-MeSADP in the presence of 10 mM LiCl (see "Experimental Procedures" for details).
Maximal responses ranged from 2.5- to 4-fold increases in
[3H]inositol phosphate accumulation over basal.
Concentration-response curves represent the mean values ± S.E. of
2 to 6 replicate experiments.
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Fig. 3.
Stimulation of PLC in COS-7 cells transiently
expressing wild-type human P2Y1 receptors
(A, concentration-response curves; B,
surface expression of receptor). COS-7 cells were
transfected with varied amounts of plasmid DNA coding for human
P2Y1, supplemented with the PCD-PS vector DNA to keep the
amount of total DNA constant at 10 µg/dish. A,
concentration-response curves for activation of PLC by stimulation with
2-MeSADP. Transfections were done with the following amounts of
P2Y1 plasmid DNA: 10 µg (squares), 1 µg
(circles), 100 ng (triangle), 10 ng
(inverted triangle), and none
(diamonds). B shows receptor surface expression
of the same transfected cells as determined by ELISA (see
"Experimental Procedures" for details). Surface expression is
presented as percent expression relative to the expression observed in
cell transfection with 10 µg of plasmid coding for human
P2Y1.
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Fig. 4.
Concentration-response curves of
P2Y1 receptors with mutated Asp204 residues in
the second extracellular loop. Wild-type human P2Y1
receptor (squares) or mutant receptors in which
Asp204 was mutated to Ala (D204A, circles), Glu
(D204E, diamond), or to Asn (D204N,
triangle) were transiently expressed in COS-7 cells.
[3H]Inositol phosphate accumulation was measured
following a 30-min incubation with increasing concentrations of
2-MeSADP in the presence of 10 mM LiCl (see "Experimental
Procedures" for details). Maximal responses ranged from 2.5- to
4-fold increases in [3H]inositol phosphate accumulation
over basal. Concentration-response curves represent the mean
values ± S.E. of 2 to 6 replicate experiments.
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[in a new window]
Fig. 5.
Concentration-response curves of
P2Y1 receptors with mutated residues in the second
(Glu209) and third extracellular loop
(Arg287). Wild-type human P2Y1 receptor
(squares) or mutant receptors in which Glu209
(E209A, triangle) and/or Arg287 (R287A,
diamond, and E209A/R287A, circle) were mutated to
alanine and transiently expressed in COS-7 cells.
[3H]Inositol phosphate accumulation was measured
following a 30-min incubation with increasing concentrations of
2-MeSADP in the presence of 10 mM LiCl (see "Experimental
Procedures" for details). Maximal responses ranged from 2.5- to
4-fold increases in [3H]inositol phosphate accumulation
over basal. Concentration-response curves represent the mean
values ± S.E. of 2 to 6 replicate experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and
-phosphates of the triphosphate chain, consistent with the
insensitivity in potency of the 5'-monophosphate agonist, HT-AMP, to
mutation of Arg287. Additional experiments showed a
selective reduction of potency for 3'-NH2-ATP in activating
the E209R mutant receptor (33). This is consistent with the hypothesis
of direct contact between Glu209 in EL2 and the nucleotide
ligands, because the 3'-NH2 group is positively charged and
expected to have a repulsive interaction with the positively charged
arginine in the E209R mutant receptor.
1-adrenergic receptor, three amino
acids beyond the Cys were found to be responsible for a
subtype-specific antagonist binding profile (15).
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Fig. 6.
Hypothetical human P2Y1 receptor
topology. All information is given for receptor stimulation upon
application of 2-MeSADP. Shaded amino acids exhibited 5-fold
change in EC50 when mutated to alanine. Shaded
and underlined amino acids displayed a 5- to 20-fold shift
when mutated to alanine or other amino acids as indicated. Bold
circled amino acids showed >20-fold shift when mutated to alanine
or other amino acids as indicated. The included data about residues in
the transmembrane domain were taken from Jiang et al. (5).
Only one (Asn27) of 4 possible glycosylation sites is
indicated. The other possible glycosylation sites are
Asn11, Asn113, and Asn197.
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Fig. 7.
Alignment of extracellular loop 2 amino acid
sequences from various GPCRs. Residues in boldface were
identified by point mutations and shown to be important for receptor
function, and underlined sequences were important for
receptor function in chimeric receptors (see "Discussion" for
details). All sequences were aligned with respect to the cysteine
forming a disulfide bridge between EL2 and TM3. The numbers on the
left indicate the position of the starting amino acid in the
receptor sequence.
Although there exist various examples in the literature for the importance of position Cys+2, there are only a few reports that correlate with the importance of the glutamic acid at the Cys+7 position. The only report showing the identical position to be important for ligand recognition was found for the human Y1 neuropeptide Y receptor (35). Studies on thrombin receptors have found a glutamic acid at the Cys+6 position to be important for agonist specificity (42). The same group published a report on chimeric constructs of human and Xenopus thrombin receptors, showing that an exchange of the C-terminal portion of the EL2, including position Cys+7, results in a robust constitutive activity (43). Hence, it appears that this portion of the EL2 might be important for maintaining the receptor in a resting conformation. None of the positions that were found to be important in our study were found to be important in the human interleukin-8 type A receptor (44). Nevertheless, six amino acids in EL2 play an important role in agonist binding and/or subsequent Ca2+ mobilization.
The situation is different for EL3, because there is no conserved disulfide bridge in this loop that could be used as a reference point for a sequence alignment. A smaller number of studies reported amino acids in this loop to be important for proper receptor function. In most cases the reported amino acids were located in vicinity to the TMs. In various GPCRs, amino acids in the EL3 close to the top of TM6 (within 4 amino acids) are important for receptor function or subtype specificity (30-32, 35, 40, 45). This region would correspond to Arg287, which we found to be important for receptor function.
In conclusion, our data so far are consistent with the presence of two
critical disulfide bridges and involvement of charged residues in
ligand recognition. As suggested by mutagenesis and molecular modeling,
Glu209 and Arg287 serve bifunctional roles. In
the model Glu209 interacts with the 3'-OH group of the
ribose moiety, whereas Arg287 interacts with the
-phosphate of ATP (33). In the resting state of the receptor, these
residues are likely to form an ionic bridge between EL2 and TM6, thus
acting as a gate for ligand entry to the binding site within the
helical bundle. The function of Asp204 still remains to be
clarified, but since the shift in concentration-response curves was
consistent among mono-, di-, or triphosphates, this result most likely
excludes a direct involvement of this amino acid in possible
Mg2+ coordination.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ivar von Kügelgen for helpful discussions and Dr. Yong Chul Kim for synthesis of HT-AMP (Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health). Furthermore we would like to thank the Information system for G-protein-coupled receptors (http://swift.embl-heidelberg.de/7tm/) and the Gprotein-coupled receptor mutant data base (http://www-grap.fagmed.uit.no/GRAP/homepage.html) for valuable information.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Molecular Recognition Section, Bldg. 8A, Rm. B1A-17, NIDDK, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-9024; Fax: 301-480-8422; E-mail: kajacobs{at}helix.nih.gov.
2 The amino acids in positions Cys+2, Cys+3, and Cys+7 are located 2, 3, and 7 residues, respectively, beyond the cysteine.
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ABBREVIATIONS |
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The abbreviations used are: GPCR, G protein-coupled receptor; 2-MeSADP, 2-methylthioadenosine-5'-diphosphate; 2-MeSATP, 2-methylthioadenosine-5'-triphosphate; DMEM, Dulbecco's modified Eagle's medium; EL, extracellular loops; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; HA, hemagglutinin; HT-AMP, 2-(hexylthio)adenosine-5'-monophosphate; PBS, phosphate buffered saline; PCR, polymerase chain reaction; PLC, phospholipase C; TM, (helical) transmembrane domain; AT, angiotensin.
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