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Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 (Q.J., D.G., B.X.L., A.M.v. R., Y.-C.K., K.A.J.), and Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599 (R.A.N., J.R.S., T.K.H.)
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Summary |
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Summary
Introduction
Procedures
Results
Discussion
References
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We conducted a mutational analysis of residues potentially involved in
the adenine nucleotide binding pocket of the human P2Y1
receptor. Mutated receptors were expressed in COS-7 cells with an
epitope tag that permitted confirmation of expression in the plasma
membrane, and agonist-promoted inositol phosphate accumulation was
assessed as a measure of receptor activity. Residues in transmembrane
helical domains (TMs) 3, 5, 6, and 7 predicted by molecular modeling to
be involved in ligand recognition were replaced with alanine and, in
some cases, by other amino acids. The potent P2Y1 receptor
agonist 2-methylthio-ATP (2-MeSATP) had no activity in cells expressing
the R128A, R310A, and S314A mutant receptors, and a markedly reduced
potency of 2-MeSATP was observed with the K280A and Q307A mutants.
These results suggest that residues on the exofacial side of TM3 and
TM7 are critical determinants of the ATP binding pocket. In contrast,
there was no change in the potency or maximal effect of 2-MeSATP with
the S317A mutant receptor. Alanine replacement of F131, H132, Y136,
F226, or H277 resulted in mutant receptors that exhibited a 7-18-fold
reduction in potency compared with that observed with the wild-type
receptor. These residues thus seem to subserve a less important
modulatory role in ligand binding to the P2Y1 receptor.
Because changes in the potency of 2-methylthio-ADP and
2-(hexylthio)-AMP paralleled the changes in potency of 2-MeSATP at
these mutant receptors, the 
- and 
-phosphates of the adenine
nucleotides seem to be less important than the 
-phosphate in
ligand/P2Y1 receptor interactions. However, T221A and T222A
mutant receptors exhibited much larger reductions in triphosphate (89- and 33-fold versus wild-type receptors, respectively) than in
diphosphate or monophosphate potency. This result may be indicative of
a greater role of these TM5 residues in -phosphate recognition.
Taken together, the results suggest that the adenosine and
-phosphate moieties of ATP bind to critical residues in TM3 and TM7
on the exofacial side of the human P2Y1 receptor.
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Introduction |
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Summary
Introduction
Procedures
Results
Discussion
References
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Extracellular ATP acts via membrane-bound receptors as a neurotransmitter in the central and peripheral nervous systems and as a regulator of vascular and smooth muscle tone (1). Two pharmacologically distinct families of ATP receptors, the P2X receptor class of ligand-gated ion channels and the P2Y receptor class of GPCRs, have been described (2, 3). As many as seven subtypes have been cloned within each family.1
P2Y receptors are members of the superfamily of rhodopsin-like GPCRs. Many other members of this superfamily, including adenosine receptors, have been probed using site-directed mutagenesis (4-10). Among P2Y receptors, only the P2Y2 receptor (11), which binds both purine and pyrimidine nucleotides, has been studied using mutagenesis. The P2Y1 receptor subtype is a phospholipase C-activating ATP receptor present in heart, skeletal muscle, and various smooth muscles. Both P2Y1 (12) and P2Y2 (11, 13) receptors have been modeled using rhodopsin (14-16)2 or bacteriorhodopsin (17), respectively, as a template. There are substantial pharmacological differences between these two subtypes, and the overall percent identity of amino acids in the human sequences is only 38%.
In this study, we attempted to characterize the determinants of ligand recognition in the family of P2Y receptors by site-directed mutagenesis. Activation of phospholipase C by three nucleotide ligands was used as an indicator of receptor function. We explored the role of positively charged and conserved residues in TM3, TM6, and TM7 and found that residues3 R128(3.29), R310(7.39), and S314(7.43) are critical for the binding of nucleotides to human P2Y1 receptors.
A long-range goal of this investigation is the design of more selective pharmacological agents based on structural differences in receptors. A limitation in the study of P2 receptors is the lack of high affinity antagonist radioligands. Although no specific, high affinity agonists or antagonists are available for the cloned subtypes, we previously introduced agonist probes that reveal pharmacological heterogeneity within the two superfamilies (18-20). One such agonist used in this study is HT-AMP (20), which activates P2Y1 but not P2Y2 receptors with a potency greater than that of the endogenous agonist ATP. The availability of a potent AMP derivative allowed us to compare various agonists to probe the phosphate binding region of the receptor.
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Experimental Procedures |
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Summary
Introduction
Procedures
Results
Discussion
References
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Materials.
The human P2Y1 receptor
cDNA (pcDNA3P2Y1) was prepared as previously
described (21). KlenTaq polymerase for the PCR was purchased from
Clontech (Palo Alto, CA). 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 RBI (Natick, MA). The agonist
HT-AMP was synthesized as previously described (18, 20) as the ammonium
salt, which was more soluble in aqueous medium than the
triethylammonium salt. The antagonists suramin and PPADS were from
Tocris Cookson (St. Louis, MO). Reactive Blue 2 was from Aldrich
(Milwaukee, WI). myo-[3H]inositol
(15 Ci/mmol) was obtained from American Radiolabeled Chemicals (St.
Louis, MO). FBS and o-phenylenediamine dihydrochloride were
purchased from Sigma Chemical (St. Louis, MO). The Sequenase Kit, ATP,
and cAMP were from United States Biochemical (Cleveland, OH). All
oligonucleotides were synthesized by Bioserve Biotechnologies (Laurel,
MD). A monoclonal antibody (12CA5) against an HA epitope 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-LKB (Piscataway, NJ).
Plasmid construction and site-directed mutagenesis. The coding region of pCDNA3P2Y1 was subcloned into the pCD-PS expression vector (22), yielding pCDP2Y1. All mutations were introduced into pCDP2Y1 using standard PCR mutagenesis techniques (23). The accuracy of all PCR-derived sequences was confirmed by dideoxy sequencing of the mutant plasmids (24).
Epitope tagging. A 9-amino acid sequence derived from the influenza virus HA protein (TAC CCA TAC GAC GTG CCA GAC TAC GCG; peptide sequence, YPYDVPDYA) was inserted after the M residue at the extracellular amino terminus of the human P2Y1 ATP receptor gene (21). Oligonucleotides containing the HA-tag sequence were designed and used to generate a PCR fragment, which was then used to replace the corresponding wild-type P2Y1 sequence. A HexaHis tag (25) was also included at the carboxyl terminus immediately after the L residue, resulting in a construct suitable for potential affinity chromatography using a nickel column.
Transient expression of mutant receptors in COS-7 cells. COS-7 cells (2 × 106) were seeded onto 100-mm culture dishes containing 10 ml of DMEM supplemented with 10% FBS. Cells were transfected ~24 hr later with plasmid DNA (4 µg of DNA/dish) according to the DEAE-dextran method (26) and grown for an additional 72 hr at 37°.
Inositol phosphate determination. COS-7 cells were subcultured into 12 × 100-mm dishes (Falcon) at a density of ~1 × 106 cells/dish. After overnight incubation at 37° and 5% CO2, the cells were transfected with plasmids containing receptor-coding sequence using the DEAE-Dextran method for 40 min, followed by treatment with 100 µM chloroquine for 2.5 hr. Typically, two dishes of COS-7 cells were transfected for each mutant tested.
The assay was carried out according to the general approach of Harden et al. (27). At ~24 hr after transfection, the cells were split into six-well plates (~0.75 × 106 cells/well; Costar, Cambridge, MA) in DMEM culture medium (plus 100 units/ml of penicillin, 100 µg/ml streptomycin, 2 µmol/ml glutamate, and 10% fetal calf serum) supplemented with 3 µCi/ml of myo-[3H]inositol. After a 24-hr labeling period, cells were preincubated for 30 min at 37° with 10 mM LiCl. The mixtures were swirled to ensure uniformity before they were allowed to stand for 20 min at room temperature. 2-MeSATP was added, and the cells were incubated for 30 min at 37° and 5% CO2. The supernatant was removed by aspiration, and 750 µl of cold 20 mM formic acid was added to each well. Cell extracts were collected after a 30-min incubation at 4° and neutralized with 250 µl of 60 mM NH4OH. The inositol monophosphate fraction was then isolated by anion exchange chromatography (28). The contents of each well was applied to a small anion exchange column (AG-1-X8; BioRad, Hercules, CA) 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 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 quantified by liquid scintillation counting (LKB Wallace 1215 Rackbeta scintillation counter). Pharmacological parameters were analyzed using KaleidaGraph (version 3.01; Abelbeck/Synergy Software, Reading, PA). Statistical analysis was performed using the Alternate t test (InStat version 2.04; GraphPAD, San Diego, CA).ELISA. For indirect cellular ELISA measurements, cells were transferred to 96-well dishes (4-5 × 104 cells/well) at 1 day after transfection. At ~72 hr after transfection, cells were fixed in 4% formaldehyde in PBS for 30 min at room temperature. After washing three times with PBS and blocking with DMEM (containing 10% FBS), cells were incubated with HA specific monoclonal antibody (12CA5; 20 µg/ml) for 3 hr at 37°. Plates were washed and incubated with a 1:2000 dilution of a peroxidase-conjugated goat anti-mouse IgG antibody (Sigma) for 1 hr at 37°. 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 with a BioKinetics reader (EL 312; Bio Tek Instruments, Winooski, VT) at 490 and 630 nm (base-line).
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Results |
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Summary
Introduction
Procedures
Results
Discussion
References
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Sequence alignments for selected transmembrane regions of P2Y receptors and other G protein-coupled receptors are shown in Fig. 1. The residues of the human P2Y1 receptor selected as targets for site-directed mutagenesis are shown in bold; they include the positively charged residues R128, K280, and R310 in TM3, TM6, and TM7, respectively, which also appear in some but not all of the other P2Y sequences. K280 and R310 were suggested in our modeling study of the chick P2Y1 receptor (12)2 to be involved in the binding of the phosphate moiety of nucleotides. Mutated residues also include F226 and S317, which are highly conserved among GPCRs. Another mutated residue, H277, is conserved among six of the seven known P2Y receptor sequences and among three of the four adenosine receptor sequences. Other mutated residues include H132, T221, and Q307, which are unique to P2Y1 receptors within the P2Y class of GPCRs. In addition, other uncharged residues targeted for mutagenesis that have been predicted through molecular modeling to be in proximity to the ligand binding site are: F131 and Y136 in TM3, T222 in TM5, and Q307 and S314 in TM7. Each of these amino acid residues was individually replaced with alanine and/or other amino acids (see below). In addition, each mutant contained an epitope-tag sequence included at the amino terminus for immunological detection (see below) and a hexa-His tag at the carboxyl terminus, potentially for future purification using Ni-affinity chromatography (25). The pharmacological properties were compared with those of the wild-type receptor that was similarly modified.
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ELISA assay for receptors located at the cell surface. Mutant P2Y1 receptors were detected immunologically at the plasma membrane surface by virtue of the HA-tag and the 12CA5 monoclonal antibody. This assay is specific for receptor proteins in which the amino-terminal sequence is accessible to the extracellular medium (i.e., of the proper orientation in the plasma membrane). This ELISA procedure does not interfere with the intactness of the plasma membrane barrier (5). 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 HA-tagged P2Y1 wild-type receptors (see Experimental Procedures; Ref. 5).
Table 1 shows that the mutant receptors were expressed on the cell surface using ELISA (5). Expression levels for the various mutants (HA-tagged wild-type receptor = 100%) determined by this method (eight experiments) ranged from 50% to 260% (Table 1). The combination of ELISA and the functional assays discussed below indicates which residues are important, either directly for activation of the receptor or indirectly for the high affinity binding of agonists.
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Functional assay. Agonist-promoted production of inositol phosphates was measured to determine whether mutant receptors were functionally active (Table 2). COS-7 cells transiently expressing wild-type human P2Y1 receptors displayed a substantial increase of inositol phosphates in response to the potent agonist 2-MeSATP (EC50 = 6.75 nM), which was not observed in control cells (Fig. 2). At the highest concentration of the agonist tested (1 mM), there was a slight increase of inositol phosphates in control COS-7 cells, presumably from an endogenous nucleotide receptor. The difference in basal inositol phosphate levels of the various expressed receptors occurs due to release of endogenous ATP and concomitant differential activation of receptors that are sensitive to activation by low concentrations of nucleotide versus mutant receptors that are only activated by higher concentrations of nucleotide. The broad concentration-effect curves reflect both the noise introduced by a limited number of data points and the fact that the assays were for a length of time (30 min) during which agonist-induced desensitization of the inositol lipid signaling response occurred.
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- and -phosphates
with the receptor. The residues that were found most critical for
activation by 2-MeSATP (R128, R310, and S314) were also absolutely
critical for activation of the receptor by monophosphates and
diphosphates. In addition, the K280A mutant receptor had greatly diminished potency of monophosphates and diphosphates as well as the
triphosphates. The one instance of an alanine mutation with no effect
on triphosphate potency (S317A) yielded identical results for
monophosphates and diphosphates. However, subtle differences were noted
in the relative effects of a given mutation on potency of
monophosphates versus triphosphates. For example, the F131A mutation
was relatively more important in reducing the potency of HT-AMP
(15-fold) than of 2-MeSATP (6.5-fold). The potency shifts for agonists
at other mutant receptors versus wild-type receptors were less
pronounced for HT-AMP versus 2-MeSATP: H132A (6.7- versus 12-fold),
Y136A (4.8- versus 18-fold), T221A (5.1- versus 89-fold), and T222A
(3.4- versus 33-fold). Among these cases of divergence between HT-AMP
and 2-MeSATP, the differences between the two agonists were the
greatest at T221A and T222A mutant receptors. The change in potencies
of 2-MeSADP at T221A and T222A mutant receptors (13- and 8.8-fold,
respectively) versus wild-type receptors were intermediate between
those for monophosphates and triphosphates. The consistency of this
trend at T221A and T222A mutant receptors suggests a greater contribution of the -phosphate moiety of ATP in ligand recognition at this region of TM5.
The effects of ATP antagonists (29) at wild-type receptors and at six
mutant receptors at which 2-MeSATP was fully active (S317A, S314T,
H277A, Q307A, K280A, and R310K) were probed using the same functional
assay (Fig. 4). The weak, nonselective
antagonists suramin, PPADS, and Reactive Blue 2, at concentrations of
100 µM, were tested in the presence of a concentration of
2-MeSATP that caused ~50% stimulation of phospholipase C in each
case (Fig. 4A). Suramin and PPADS each partially (~20%) blocked the
agonist effects at this concentration; however, Reactive Blue 2 had no significant effect. Although IC50 values were not
determined, it seemed that both suramin and PPADS retained antagonist
properties at all of the mutant receptors examined (Fig. 4B).
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Discussion |
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Summary
Introduction
Procedures
Results
Discussion
References
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Stimulation of phospholipase C by mutant human P2Y1
receptors.
A mutational analysis of residues potentially involved
in the adenine nucleotide binding pocket of the human
P2Y1 receptor has been carried out. Single amino
acid residues in TMs 3, 5, 6, and 7 predicted by molecular modeling to
be involved in ligand recognition were targeted for replacement with
alanine and, in some cases, by other amino acids. Mutated receptors
were expressed in COS-7 cells with an epitope tag that permitted
confirmation of expression in the plasma membrane. A functional assay
(i.e., agonist-promoted inositol phosphate accumulation) was used as a
measure of receptor activity. We have attempted to supplement these
results by using the radioligand
[35S]-thio-2
-deoxy-ATP, as reported by
Barnard et al. (30). However, untransfected cells displayed
high levels of binding that was comparable to COS-7 cells transfected
with wild-type P2Y1 receptors but no comparable
activation of phospholipase C by 2-MeSATP (data not shown). Similar
conclusions were reached by Schachter et al. (31) concerning
the unsuitability of [35S]-thio-2-deoxy-ATP
as radioligand for P2Y1 receptors. Because we
already demonstrated the limitations of the binding of another radioligand, [35S]-thio-ADP binding (32),
for characterization of P2Y receptors, no suitable radioligand was
available.
-hydroxyl group of the ribose moiety of
bound ATP. Overall, these results suggest that residues on the
exofacial side of TM3, TM6, and TM7 are critical determinants of the
ATP binding pocket.
In contrast, there was no change in the potency or maximal effect of
2-MeSATP with the S317A mutant receptor. Thus, this residue is
insensitive in adenine nucleotide binding. Furthermore, alanine replacement of F131, H132, Y136, F226, or H277 resulted in mutant receptors that exhibited a 7-18-fold reduction in potency of 2-MeSATP compared with that observed with the wild-type receptor. These five
residues in TM3, TM5, and TM6 thus seem to subserve a less important
modulatory role in ligand binding to the P2Y1
receptor. The three residues in TM3 in this modulatory category would
be aligned along the same face of the helix as the critical residue R128 but are located further from the exofacial side by approximately one (F131 and H132) or two (Y136) helical turns.
Because changes in the potency of 2-MeSADP and HT-AMP paralleled the
changes in potency of 2-MeSATP at these mutant receptors, the - and
-phosphates of the adenine nucleotides seem to be less important
that the -phosphate in ligand/P2Y1 receptor
interactions. However, T221A and T222A mutant receptors exhibited much
larger reductions in the potency of 2-MeSATP (89- and 33-fold versus wild-type receptors, respectively) rather than HT-AMP and 2-MeSADP potency. This result may be indicative of a greater role of these TM5
residues in -phosphate recognition. The remaining residues in TMs 3, 5, 6, and 7 found to be either essential or modulatory for agonist
potency did not show any striking differences among monophosphate,
diphosphate, and triphosphate agonists. Thus, they would be expected to
be associated preferentially with the -phosphate and/or adenosine
moieties of ATP.
These findings are largely but not entirely consistent with our
P2Y1 receptor molecular model based on a
rhodopsin template (12), which predicted that [numbering translated by
homology from the chick P2Y1 receptor sequence]
H132(3.33), Y136(3.37), F226(5.47), H277(6.52), K280(6.55), and
R310(7.39), but not R128(3.29), are in proximity to the triphosphate
moiety of bound ATP. The model further predicted that S314 and S317 are
in proximity to the ribose moiety. However, only mutation of the former
residue, which is located closer to the exofacial side, affects ligand binding. Residues F131 and Q307 were predicted to be in proximity to
the adenine moiety (the latter in potentially hydrogen-bonding proximity from the exocyclic amine), and indeed mutation of these residues caused a substantial loss of agonist potency (particularly at
Q307).
PPADS and suramin retained potency as antagonists (1, 29) in the H277,
K280, Q307A, R310K, S314T, and S317A mutant receptors. Thus, the
noncritical (and possibly modulatory) residues H277, K280, and Q307A
seem to be involved especially in agonist recognition. The S317A
mutation seems to be inconsequential for agonist binding. The rescued
mutants R310K and S314T are functional for both agonists and
antagonists, although the potencies determined precisely for agonist
are ~1 (S314T) or several orders of magnitude (R310K) less than those
for wild-type receptors. It is not feasible with the present
experimental paradigms to test the ability of the nonactivatable R310K
and S314T mutants to recognize the antagonists PPADS and suramin.
Structural homology of human P2Y1 receptors to other
GPCRs.
The essential S314(7.43) corresponds to important
hydrogen-bonding residues in other GPCRs, such as tyrosine in
adrenergic and muscarinic acetylcholine receptors, and the essential
H278 in human A2A receptors. Although there are
numerous parallels between the P2Y1 receptors and
homologous sites in other GPCRs (4), there are some striking
differences. For example, S317(7.46) corresponds to a critical serine
residue in the hamster 2 receptor (33), yet it
apparently has no role in ligand recognition in the human
P2Y1 receptor. Residue F131(3.32) corresponds to
the essential D residue in biogenic amine receptors (counterion to ligand). For human P2Y1 receptors, this site is
less critical for ligand recognition than in biogenic amine receptors,
because its mutation to A resulted in only a 6.5-fold loss of 2-MeSATP potency.
Conclusions.
The present site-directed mutagenesis study
confirms that many of the residues of TM3, TM5, TM6, and TM7 predicted
in a modeling study of P2Y1 receptors to form the
nucleotide binding pocket (12) are indeed involved in ligand
recognition. Thus, it seems that positively charged residues near the
exofacial side of both TM3 and TM7 and, to a lesser extent TM6, are
important for recognition of ATP, presumably through interaction with
the negatively charged phosphate moiety. The potency of monophosphate
and diphosphate nucleotide analogues generally varied in parallel to
changes for the triphosphate analogue. Therefore, most
structure-function relationships for the receptor were not dependent on
interactions with the - and -phosphates. An exception was the
T221T222 sequence in TM5, for which mutation to A was more detrimental
to triphosphate than either monophosphate or diphosphate potency,
suggesting a greater role of this region in -phosphate binding.
-phosphate moieties of ATP seem to bind to human
P2Y1 receptors preferentially in a TM region
proximal to the exofacial side, with the most critical residues located
in TM3 and TM7. This study has revealed differences between
P2Y1 and P2Y2 receptors
(11), in which positively charged residues of TM6 and TM7 principally have been proposed to coordinate the phosphate group of uridine nucleotides. The characterization of structure function differences among P2Y receptor subtypes will likely aid in the design of highly potent and selective ligands.
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Acknowledgments |
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We thank Dr. Jürgen Wess for helpful discussions.
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Footnotes |
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Received February 26, 1997; Accepted May 27, 1997
2 The coordinates of the P2Y1 receptor model are available from the protein database (Brookhaven National Laboratory) using the URL http://www.pdb. bnl.gov/cgi-bin/browse under the ID code 1ddd.
3 Residue identifiers as described in van Rhee and Jacobson (4) specify the helix (X) and the position relative to a key conserved residue in that helix, which is designated (X0.50). For example, for the helices mutated in the present study, the key conserved residues are R149(3.50), P229(5.50), P275(6.50), and P321(7.50).
1 P2Y1-P2Y7 sequences have been cloned and named in the literature (3), however, several of these may be either receptors of a different family (e.g., P2Y5 and possibly P2Y7) or possibly species homologues (e.g., P2Y3 and P2Y6). The identity and relationship among these clones require further study.
Send reprint requests to: Dr. K. A. Jacobson, Chief, Molecular Recognition Section, Bldg. 8A, Room B1A-17, NIDDK, NIH, Bethesda, MD 20892. E-mail: kajacobs{at}helix.nih.gov
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Abbreviations |
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2-MeSATP, 2-methylthio-ATP;
2-MeSADP, 2-methylthio-ADP;
DMEM, Dulbecco's modified Eagle's medium;
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;
TM, (helical) transmembrane domain;
PPADS, pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid.
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References |
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Summary
Introduction
Procedures
Results
Discussion
References
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| 1. | Fredholm, B. B., M. P. Abbracchio, G. Burnstock, J. W. Daly, T. K. Harden, K. A. Jacobson, P. Leff, and M. Williams. Nomenclature and classification of purinoceptors: a report from the IUPHAR subcommittee. Pharmacol. Rev. 46:143-156 (1994)[Medline]. |
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| 5. |
Kim, J. H.,
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100-fold loss of potency was
observed with 2-MeSATP. Gray circles, residues conserved
between P2Y1 and P2Y2 receptors (R128, R310,
and S314 are also conserved).
