(Received for publication, March 11, 1997)
From the Division of Nephrology and Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2372
The prostaglandin EP3 receptor
binds Prostaglandin E2 in a ligand binding pocket formed in
part by seven transmembrane -helices. The present studies
demonstrate that the second extracellular loop of the receptor is
involved in prostanoid ligand recognition as well. Site-directed
mutagenesis of seven conserved residues clustered in the amino portion
of the second extracellular loop was performed. Receptors with single
amino acid substitutions at each of these positions were transiently
transfected into HEK293tsA201 cells, their ligand binding profiles
assessed, and each receptor was tested for its ability to decrease
intracellular cAMP levels. Substitution of Trp199 or
Thr202 with alanine resulted in receptors with increases in
affinity up to 128-fold for prostanoid compounds with a C1 methyl ester but wild type affinities for natural prostanoid ligands that have a
carboxylate moiety at the C1 position. In contrast, substitution of
Pro200 with serine caused a loss of selectivity up to
20-fold for naturally occurring prostanoid agonists as compared with
the wild type EP3 receptor: the PS200 receptor displayed a
decrease in affinity for E-ring compounds and an increase in affinity
for F- and D-ring compounds. The EC50 for inhibition of
cAMP remained unchanged for each receptor tested.
Prostaglandin E2 (PGE2)1 is a mediator of a variety of physiological functions. Evidence suggests that many of these effects are the results of PGE2 interacting with specific seven transmembrane G-protein-coupled receptors (GPCRs). Based upon their ligand binding selectivity and signaling pathway activation, these receptor subtypes are classified as EP1, EP2, EP3, and EP4 (1, 2).
Extensive structural information is available for GPCRs that bind small
ligands (biogenic amine neurotransmitters, nucleotides, and opsin), and
such studies have provided significant insights regarding the receptor
amino acid residues required for ligand recognition (for review, see
Ref. 3). Evidence supports the role of residues embedded in the
transmembrane as important in receptor-ligand interactions (4). In
contrast, the extracellular regions appear to be relatively unimportant
for binding of these small ligands, with the exception of a role of an
extracellular cysteine disulfide bridge demonstrated in several
receptors including the -adrenergic receptor (5) and the thromboxane
A2 receptor (6) as well as a portion of the second
extracellular loop of the adenosine A1 and A2a
receptors (7, 8).
Less is known regarding the structural determinants of EP receptor-ligand interactions. Several groups have previously identified the importance of an arginine residue found in transmembrane region VII of the EP3 receptor and conserved throughout prostanoid receptors (9-11). Substitution of Arg329 in transmembrane VII to either Ala or Glu led to a loss of detectable [3H]PGE2 binding and receptor-mediated inhibition of [cAMP]i (9).
Comparisons of the amino acid sequence between the rabbit EP3 receptor and the other cloned prostanoid receptors have identified several regions of conservation (9). Fourteen conserved amino acid residues were identified outside the putative transmembrane regions, including six amino acid residues clustered in the amino-terminal portion of the second extracellular loop. We hypothesized that conserved extracellular regions of the EP3 receptor affect receptor/ligand interactions either directly or indirectly, analogous to the proposed interactions between the extracellular regions and ligands of peptide-binding GPCRs such as neurokinin-1 (13), thyrotropin (14), or [Arg8]vasopressin receptors (15). To test whether this conserved primary structure plays a role in receptor-ligand interaction, a series of point mutants were generated and assayed for their ability to bind a panel of natural and synthetic prostanoid analogs. Findings presented herein provide evidence that the second extracellular loop of the prostaglandin E2 EP3 receptor plays a role in ligand selectivity.
Misoprostol and misoprostol-free acid were gifts of Dr. Paul Collins (Searle). All other prostanoid analogs were purchased from Cayman Chemical (Ann Arbor, MI). Isoproterenol, ascorbic acid, and indomethacin were purchased from Sigma. [3H]PGE2 was purchased from DuPont NEN. LipofectAMINE, and Opti-MEM were purchased from Life Technologies, Inc.
Site-directed Mutagenesis of the ReceptorMissense
mutations were introduced using the polymerase chain reaction (PCR) as
described previously (9). The sequence of the flanking oligonucleotides
was as follows: upstream oligonucleotide (EP3 nucleotide
501 coding), 5 TG GTG TAC CTA TCC AGG 3
; downstream oligonucleotide
(EP3 nucleotide 963 coding), 5
CCA GGG ATC CAA TAT CTG G
3
.
The internal oligonucleotides used to introduce missense mutations are
listed as follows (with underlining indicating nucleotide substitutions): QA198: 5 A CAG TAC ACG ATC GCG
TGG CCC GG 3
; WA199, 5
TAC ACC ATC CAA GCT
CCC GGG ACG 3
; PS200, 5
ATC CAG TGG TCA GGT
ACC TGG TGC TTC 3
; TA202, 5
TC CAG TGG CCT
GGT GCT TGG TGC TTC 3
; WA203, 5
G TGG
CCT GGT ACC GCA TGC TTC
ATC AGC 3
; CA204, 5
CAG TGG CCT GGT
ACC TGG GCT TTC ATC AGC3
; FA205, 5
GT TGG CCC
GGT ACG TGG TGC GCA ATC AGT AC 3
.
PCR fragments encoding the target amino acid substitutions were then subcloned into the hemagglutinin-tagged 77A isoform of the EP3 receptor in plasmid 77A hemagglutinin wt pRC/CMV, generating the full-length EP3 77A receptor (9). Two independent clones bearing each amino acid substitution were then isolated and characterized in subsequent experiments. The identity of the mutations was confirmed by sequencing both strands of the PCR amplified region using a ThermoSequenase kit (Amersham Life Science, Inc.).
Expression of EP3 cDNAs in Cell CultureHEK293tsA201 cells were transiently transfected with plasmids bearing wt or mutant EP3 cDNA as described (9). Cells were cultured for 72 h, and the medium was replaced every 24 h. At 72 h cells were lysed and membranes prepared as described (16). Protein concentrations were determined by the BCA assay (Pierce).
Ligand Binding AssaysFor saturation binding isotherm experiments, 10-40 µg of membrane protein, representing 20 fmol of receptor, was incubated with various concentrations of [3H]PGE2, and reactions were stopped by filtration onto glass fiber filters as described (9). For competition binding assays, 10-40 µg of membrane proteins were incubated with 1 nM [3H]PGE2 and varying concentrations of unlabeled competitor and assayed as described above.
cAMP MeasurementsHEK293tsA201 cells were transiently
co-transfected with plasmids containing the human 2AR
and either the wt or mutant EP3 receptor. cAMP measurements
were performed by radioimmunoassay as described (9).
Saturation binding isotherms, competition binding isotherms, and dose-dependent responses of [cAMP]i were analyzed using Prism (GraphPad, San Diego, CA). Ki values were calculated using the method of Cheng and Prusoff (17). Statistical analyses were performed using Instat (GraphPad).
Sequence alignments of the cloned rabbit
EP3 receptor was performed against all cloned prostanoid
receptors (9). A cluster of six conserved amino acid residues was
identified in the putative second extracellular loop (Fig.
1). The sequence was identified as
Q198WPGTWCF, where bold
characters represent conserved amino acids. Pro200 is
conserved throughout all cloned prostanoid receptors with the exception
of the FP receptor. We tested whether this conserved portion of the
receptor plays a role in the prostaglandin EP3 receptor
function and/or structure.
Mutations of Trp199 and Thr202 Cause an Increase in the Affinity of Methyl Ester Compounds
TA202
displayed markedly increased affinities for methyl ester compounds of
the E series as compared with the wild type receptor despite displaying
similar dissociation constants for [3H]PGE2
(Kdwt = 1.4 ± 0.3 nM, KdTA202 = 1.3 ± 0.1 nM). As shown in Fig.
2A, TA202 resulted in a 128-fold increase in
affinity for misoprostol as compared with the wild type receptor
(Kiwt = 1600 ± 350 nM, KiTA202 = 13 ± 3 nM). In contrast, the affinity of TA202 for
the carboxylate derivative misoprostol-free acid increased a modest
2-fold (Kiwt) = 6.5 ± 1.9 nM, KiTA202 = 3.3 ± 0.6 nM). The Ki values for
other natural and synthetic prostanoid agonists with a carboxylate at
the C1 position were not statistically different from wild type (Table
I). Sulprostone, which has a sulfonamide moiety at C1,
had a modest 3-fold increase in affinity. To test if the increased
affinity for misoprostol could be extended to other E series methyl
ester compounds, TA202 was assayed with a panel of paired methyl
ester/carboxylate prostanoid analogs that differed in their
substituents at positions which differ between PGE2 and
misoprostol: substitution at the C15 and C16 position and presence or
absence of a double bond between C5 and C6. For each compound tested
TA202 displayed increased affinity for the methyl ester compound from
38- to 128-fold as compared with wild type; however, the affinities for
the carboxylate analog of each pair was not statistically different
from wt (Table II). Fig. 2B summarizes the
selectivity ratios for the various drugs tested.
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The identity of Trp199 is not highly conserved among prostanoid receptors as tyrosine, phenylalanine, or alanine may be found at this position. Nonetheless, substitution of Trp199 with alanine resulted in a similar phenotype to the TA202 receptor. WA199 displayed a 9-fold increase in affinity for misoprostol and a 3-fold increase in affinity for sulprostone while the Ki values for other compounds tested remained unaffected (Table I).
Mutation of Pro200 Affects Ligand SelectivityProline 200 is conserved among the EP1,
EP2, EP3, EP4, DP, IP, and TP
receptors across species. In the FP receptor, this position is occupied
by a serine residue. Pro200 of the EP3 receptor
was mutated to Ser, and its binding characteristics were determined.
The KdPS200 = 4.1 ± 0.5 nM versus
Kdwt = 1.4 ± 0.3 nM suggested a modest loss of
[3H]PGE2 affinity for PS200 (two-tailed
p value < 0.01). Competition binding isotherms
resulted in a loss of specificity for PS200 as determined using
PGE2, PGD2, and PGF2. The PS200
receptor displayed a 4-fold loss in affinity for PGE2;
however, it gained 3- and 5-fold in affinity for PGF2
and PGD2, respectively (Table I, Fig. 3).
Similarly, results obtained with the synthetic PGE analogs sulprostone
and misoprostol-free acid demonstrated a 5-7-fold decrease in
affinity, while misoprostol displayed a 5-fold increase in affinity.
Thus, the overall pattern for the PS200 substitution was a loss in
affinity for compounds that bind with high affinity to wt and an
increase for compounds that bind with lower affinity to wt receptor.
These results suggest Pro200 plays a crucial role
maintaining ligand binding selectivity.
Substitution of Gln198, Trp203, Cys204, and Phe205 Does Not Affect Ligand Selectivity
Substitution with alanine at the positions Gln198, Trp203, Cys204, and Phe205 did not affect the binding profile of these receptors despite absolute conservation of the primary structure at these positions among all cloned prostanoid receptors. Membranes prepared from cells transfected with each receptor were tested in saturation binding (data not shown) and competition binding isotherms with the panel of natural and synthetic prostanoid analogs, and no statistically significant changes in affinity or order of agonist potency were observed (Table I). These results argue against the role of each individual residues in receptor-ligand interactions.
Receptor-mediated Inhibition of [cAMP]i FormationSignal transduction properties of each receptor variant were determined. As compared with the wild type receptor (EC50 = 480 pM), the various receptor EC50 values were: QA198 = 550 pM, WA199 = 360 pM, PS200 = 500 pM, TA202 = 370 pM, WA203 = 400 pM, CA204 = 370 pM, FA205 = 200 pM) and did not display any statistically significant differences in receptor evoked signaling in three independent experiments.
Using site-directed mutagenesis studies of the prostaglandin EP3 receptor, the present results demonstrate that the second extracellular loop plays an integral role in receptor-ligand interaction and particularly in ligand selectivity. The gain of agonist affinity observed for several mutant receptor phenotypes argues strongly against gross perturbation of receptor structure by these amino acid substitutions as the cause of the altered ligand binding phenotypes. The precise role of the second extracellular loop in receptor-ligand interaction is unknown. One possible explanation for the observed phenotype is that the second extracellular loop forms part of the binding pocket and is in direct contact with the bound ligand. Alternatively, the effects of these mutations may be the result of an indirect role of the second extracellular loop. This is analogous to the Ca2+, Na+, or K+ channels where it has been proposed that extracellular loops fold into the ion channel pore and interact with the transmembrane helices (18). A third possibility is that the loop is important for the overall conformation of the receptor without direct interaction with the transmembrane helices. This interpretation may be consistent with the idea that modification of Trp199, Pro200, or Thr202 induces a general relaxation of the receptor conformation, preventing it from discriminating structurally related prostanoid analogs.
Several models supporting a direct role of extracellular domains of peptide/amino acid binding GPCRs have been described in the literature. In the case of the receptor for parathyroid hormone, it was suggested that amino acid residues near the extracellular surface of the transmembrane helices play a "filter" role allowing discrimination between various ligands (19). This concept could be extended to the EP3 receptor, where it is conceivable to consider Thr202 or Trp199 as filters which reduce affinity for compounds with a C1 methyl ester. Elimination of Thr202 or Trp199 side chains results in the elimination of a "gate" preventing methyl ester prostanoids from accessing the binding cleft. This model argues in favor of a two-step process in terms of receptor-ligand interaction. It has been suggested for the metabotropic glutamate receptor, mGLUR1, that the extracellular region of the receptor may act as a primary point of receptor-ligand interaction or "bait" and subsequently facilitate presentation to the transmembrane ligand pocket (20). Similarly, this model may be applied to the second extracellular loop of the EP3 receptor, whereby Trp199, Pro200, and/or Thr202 attract ligands and present them to the binding pocket. The filter and bait models are not mutually exclusive and may be complementary to one another.
It is interesting to note that mutation of Cys204, which is conserved among all cloned prostanoid receptors, caused no detectable change in ligand binding affinity or receptor activation. These results are in sharp contrast to the results presented for the thromboxane A2 receptor, where replacement of Cys183 to Ser (analogous position as Cys204) led a complete loss of binding and signaling (6). These authors suggested that a critical disulfide bond existed between Cys183 and Cys105 (transmembrane domain III) of the thromboxane A2 receptor; however, results presented here argue against the importance of a putative disulfide bridge involving Cys204 in the EP3 receptor.
It is also of interest that within this highly conserved amino-terminal portion of the second extracellular loop, substitutions at the other absolutely conserved positions Gln198, Trp203, Cys204, and Phe205 did not detectably affect ligand binding or signal transduction. It may be that substitutions at several positions are required to disrupt receptor function. Alternatively, an untested function (e.g. internalization/recycling) may have been altered.
We propose a revised version of the model currently described for the EP3 receptor (21). We had previously shown that the EP3 and EP4 receptors displayed increased affinities for carboxylate compounds versus their methyl ester derivatives (9, 12). Furthermore, a large body of literature has suggested that the carboxylate moiety of C1 interacts with the positive side chain of arginine in transmembrane VII. The above results with TA202 suggest that a negative charge on C1 of prostanoids is not required for high affinity interactions with the EP3 receptor as shown for the TA202 receptor. Based on the high degree of homology of the second extracellular loop among cloned prostanoid receptors, it is conceivable these findings may be generalized to some or all of the prostanoid receptors. This may suggest that a revised three-dimensional model incorporating the extracellular loop regions is required to interpret receptor-ligand interactions.
We thank Lee Limbird and Brett Stillman for helpful discussions. We thank Matt Breyer and Ron Emeson for critical reading of the manuscript.