©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Selective Coupling of Prostaglandin E Receptor EP3D to G and G through Interaction of -Carboxylic Acid of Agonist and Arginine Residue of Seventh Transmembrane Domain (*)

Manabu Negishi (§) , Atsushi Irie , Yukihiko Sugimoto , Tsunehisa Namba (1), Atsushi Ichikawa

From the (1)Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences Department of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Prostaglandin (PG) E receptor EP3D is coupled to both G and G. To examine the roles of the interaction of -carboxylic acid of PGE and its putative binding site, the arginine residue in the seventh transmembrane domain of EP3D, in receptor-G protein coupling, we have mutated the arginine residue to the noncharged glutamine. PGE with a negatively charged -carboxylic acid and sulprostone, an EP3 agonist with a noncharged modified -carboxylic acid, inhibited the forskolin-stimulated adenylate cyclase activity via G activation in the EP3D receptor in the same concentration-dependent manner. In contrast, the adenylate cyclase stimulation via G activation by sulprostone was much lower than that by PGE. On the other hand, both PGE and sulprostone showed potent G activity but failed to show G activity in the mutant receptor. EP3D receptor showed a high affinity binding for PGE in the form coupled to either G or G. Although the mutant receptor showed high affinity binding when coupled to G, it lost high affinity binding in the condition of G coupling. Furthermore, sulprostone bound to the G-coupled EP3D receptor with higher affinity than the G-coupled receptor. Among various EP3 agonists, -carboxylic acid-unmodified agonists showed both G and G activities, but the modified agonists showed only G activity. These findings suggest that the interaction between the -carboxylic acid of PGE and the arginine residue of the receptor regulates the selectivity of the G protein coupling.


INTRODUCTION

Hormonal actions are initiated by agonist binding to receptors. The agonist-bound receptor is associated with and activates a G protein to a state in which it can regulate effectors(1) . A number of rhodopsin-type receptors can be coupled to multiple G proteins, and then a single agonist produces a variety of hormonal actions through modulation of multiple signal transduction systems(2) . The ligand binding is believed to be determined by the transmembrane domains, and multiple sites in transmembrane domains have been suggested to contribute to the formation of a ligand binding pocket in various receptors; several amino acid residues in the transmembrane domains may be involved in agonist-induced receptor activation(3) . However, the precise molecular mechanisms through which the agonist, upon receptor binding, induces G protein activation are still poorly understood. Especially, it is not known how an agonist can modulate multiple G proteins through the binding to a receptor.

Prostaglandin (PG)()E produces a broad range of biological actions in diverse tissues through its binding to specific receptors on plasma membranes(4, 5) . The pharmacological actions of PGE are diverse among tissues; PGE causes contraction or relaxation of vascular and nonvascular smooth muscle and stimulates or suppresses the secretion of neurotransmitters or hormones. The diversity of the actions of PGE is due to the coupling of PGE receptors to a variety of signal-transduction pathways. PGE receptors are pharmacologically subdivided into four subtypes, EP1, EP2, EP3 and EP4, on the basis of their responses to various agonists and antagonists(6, 7) . We have recently revealed the primary structures of mouse EP1, EP3, and EP4 receptors(8, 9, 10) . They belong to the G protein-coupled rhodopsin-type receptor superfamily with seven transmembrane domains and are coupled to the activation of the Ca channel, inhibition, and activation of adenylate cyclase, respectively. In addition, we have identified three isoforms of the mouse EP3 receptor (EP3, -, and -) and four isoforms of the bovine EP3 receptor (EP3A, -B, -C, and -D) with different COOH-terminal tails, which are produced through alternative splicing and differ in the specificity of coupling to G proteins. EP3, and A are coupled to G, while EP3B and C are coupled to G and EP3D and are coupled to both G and G(11, 12, 13) . Among these isoforms, EP3D and - belong to a receptor coupled to multiple G proteins. The diversity of the cellular responses to PGE is in part based on the existence of functionally different EP3 isoforms coupled to various signal transduction pathways in addition to subtypes. Thus, functional analysis of agonist-binding sites of PGE receptors is an urgent need for understanding diverse physiological roles of PGE.

There are several regions conserved specifically among the prostanoid receptors, and the most highly conserved regions are found in the seventh transmembrane domain(3) . In rhodopsin, the hydrocarbon chain of retinal was shown to be attached to Lys-296 in the seventh transmembrane domain(14) . The positively charged arginine residue within the seventh transmembrane domain, which is conserved in all of the prostanoid receptors, was proposed to be the binding site of the negatively charged -carboxyl group of prostanoid molecules by analogy to the retinal binding site of rhodopsin(3) . Consistent with this hypothesis, a point mutation at this arginine residue in the seventh transmembrane domain of the human thromboxane A receptor has been shown to result in the loss of ligand binding activity(15) . A variety of PGE analogues with potent agonist activity have been developed, but some EP3 agonists have the structural features of substitution at C-1, where -carboxylic acid is replaced by a variety of esters or methanesulfonamido groups, its negative charge being blocked. This suggests that the charge-charge interaction of the -carboxylic acid of PGE with the arginine residue of EP3 receptors appear not to be critical for some agonist activities through EP3 receptors. To assess the role of the interaction of -carboxylic acid of PGE with the arginine residue of EP3 receptors in coupling to G proteins, we chose the EP3D receptor, coupled to multiple G proteins, and mutated the arginine in the seventh transmembrane domain to noncharged glutamine. In the present study, we report that the arginine residue of the EP3D receptor is not essential for G coupling but is necessary for G coupling.


EXPERIMENTAL PROCEDURES

Materials

EP3 agonists were generous gifts from Dr. K.-H. Thierauch of Schering (sulprostone), Dr. P. W. Collins of Searle (misoprostol), Dr. M. P. L. Caton of Rhone-Poulenc Ltd. (M& 28767), Dr. S. Kurozumi of Teijin Ltd. (TEI-3356), and Dr. B. M. Bain of Glaxo Group Research Ltd. (GR 63799X). [5,6,8,11,12,14,15-H]PGE (179 Ci/mmol), [-P]GTP (6,000 Ci/mmol), and the I-labeled cAMP assay system were obtained from Amersham Corp. PGE was obtained from Cayman Chemical (Ann Arbor, MI); pertussis toxin (PT) was from Seikagaku Kogyo (Tokyo, Japan), cholera toxin (CT) was from Funakoshi Pharmaceuticals (Tokyo), and rabbit antiserum against G (RM/1) and that against G and G (AS/7) were from DuPont NEN. Sources of other materials are shown in the text.

Construction and Stable Expression of the Mutated Receptor

A polymerase chain reaction-mediated mutagenesis (16) was used to convert arginine (Arg-332) of EP3D cDNA to glutamine (Gln-332). The sequence of the mutated region in the constructed cDNA for the mutant receptor (EP3D-R332Q) was confirmed by sequence analysis by the dideoxynucleotide chain-termination method. cDNA transfection was performed by lipofection, essentially as described previously(17) . Briefly, Chinese hamster ovary (CHO) cells deficient in dihydrofolate reductase activity (CHO-dhfr) were transfected with the mutated cDNA inserted into eukaryotic expression vector pdKCR-dhfr containing a mouse dihydrofolate reductase gene as a selection marker (18). Selection was performed using the -modification of Eagle's medium, lacking ribonucleosides and deoxyribonucleosides, with 10% dialyzed fetal bovine serum (Cell Culture Laboratories). Clonal cell lines were obtained by single-cell cloning and were screened by RNA blotting.

Adenylate Cyclase Assay

CHO cells expressing EP3D or EP3D-R332Q were exposed to 1 ng/ml PT or 5 µg/ml CT for 12 h. The harvested cells were permeabilized in Hepes-NaOH (pH 7.5) containing 0.015% digitonin, 2 mM MgCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and 0.1 M NaCl essentially according to the method of Lohse et al.(19) . Under these conditions, the leakage of lactate dehydrogenase, an enzyme that is confined to the cytosol, was complete (data not shown). After the permeabilization, the permeabilized cells (50 µg) were incubated for 5 min at 37 °C with 1 mM ATP, 10 µM GTP, 2 mM creatine phosphate, 1 unit of creatine phosphate kinase, and 100 µM Ro-20-1724, and the reactions were terminated by the addition of 5% trichloroacetic acid. The cAMP formed was determined by radioimmunoassay with an Amersham cAMP assay system.

PGEBinding Assay

CHO cells expressing EP3D or EP3D-R332Q were exposed to 1 ng/ml PT or 5 µg/ml CT for 12 h. The harvested cells were homogenized using a Potter-Elvehjem homogenizer in 20 mM Tris-HCl (pH 7.5), containing 0.25 M sucrose, 10 mM MgCl, 1 mM EDTA, 20 µM indomethacin and 0.1 mM phenylmethylsulfonyl fluoride. After centrifugation at 800 g for 5 min, the supernatant was further centrifuged at 250,000 g for 20 min. The pellet was washed, suspended in 20 mM Tris-HCl (pH 7.5) containing 10 mM MgCl and 1 mM EDTA, and was used for the [H]PGE binding assay. The membrane (20 µg) was incubated with various concentrations of [H]PGE at 30 °C for 1 h, and [H]PGE binding to the membrane was determined as described previously(11) . Nonspecific binding was determined using a 1,000-fold excess of unlabeled PGE in the incubation mixture. The specific binding was calculated by subtracting the nonspecific binding from the total binding.

GTPase Activity

GTPase activity was assayed by incubating the membrane at 37 °C for 5 min in a total volume of 100 µl of 20 mM Hepes-NaOH (pH 7.5) containing 2 mM MgCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 0.1 M NaCl, 1 mM App(NH)p, 0.2 mM ATP, and 0.1 µM [-P]GTP (0.5 µCi). The reactions were initiated by the addition of the membrane (5 µg) and stopped by the addition of 0.9 ml of ice-cold 5% Norit A and 0.1% bovine serum albumin in 20 mM sodium phosphate (pH 7.0). The mixtures were centrifuged (2,000 g, 5 min, 4 °C), and the radioactivity of [P]P released into the supernatant (150 µl) was determined. Nonspecific GTP hydrolysis was determined using a 1,000-fold excess of unlabeled GTP in the incubation mixture. The specific low KGTPase activity was calculated by subtracting the nonspecific hydrolysis from the total hydrolysis. Under these conditions, the time course was linear over a 5-min incubation period.


RESULTS

Effects of PGEand Sulprostone on Gand GActivities of EP3D and EP3D-R332Q Receptors

To assess the role of the arginine residue in the seventh transmembrane domain of the EP3D receptor in agonist-mediated signal transduction, we constructed a mutant EP3D receptor (EP3D-R332Q) with mutation of arginine (Arg-332) to glutamine. CHO cells were transfected with EP3D-R332Q cDNA, and cells stably expressing EP3D-R332Q were established. Sulprostone, a widely used PGE analogue replaced at -carboxylic acid by methanesulfonamide, has almost the same agonist potency as PGE(20) . Thus, we examined the effects of PGE, an agonist with negatively charged -carboxylic acid, and sulprostone, an agonist with noncharged modified -carboxylic acid, on the adenylate cyclase system of EP3D- or EP3D-R332Q-expressing CHO cells. The EP3D receptor shows a biphasic, inhibitory and stimulatory response in the adenylate cyclase system due to functional coupling to both G and G(13) . To ablate receptor coupling to one or the other G protein, the cells were treated with PT or CT. These treatments of the cells with PT and CT completely eliminated further ADP-ribosylation by PT and CT in the in vitro assay, respectively (data not shown). As Fig. 1A shows, PGE and sulprostone concentration dependently inhibited the forskolin-stimulated adenylate cyclase activity in CT-treated cells expressing the EP3D receptor, the half-maximal concentrations for the inhibition (10 nM) and levels of the maximal inhibition (75% inhibition) being the same. PGE and sulprostone also inhibited adenylate cyclase activity in the EP3D-R332Q receptor concentration-dependently, but the half-maximal concentrations for the inhibition (100 nM) were 1 order of magnitude higher than those in the EP3D receptor. On the other hand, the level of the maximal inhibition in the EP3D-R332Q receptor was the same as that in the EP3D receptor. PGE concentration-dependently stimulated basal adenylate cyclase activity, but sulprostone showed less potent stimulation in PT-treated cells expressing the EP3D receptor (Fig. 1B). By contrast, neither PGE nor sulprostone stimulated it in the EP3D-R332Q receptor.


Figure 1: Effects of PGE and sulprostone on adenylate cyclase activity in CT- or PT-treated permeabilized cells expressing EP3D or EP3D-R332Q receptor. A, G activity; CT-treated permeabilized cells expressing the EP3D (, ) or EP3D-R332Q receptor (, ) was assayed for adenylate cyclase activity with the indicated concentrations of PGE (, ) or sulprostone (, ) in the presence of 3 µM forskolin, as described under ``Experimental Procedures.'' Values are expressed as a percentage of the control (41.5 ± 3.2 pmol/min/mg) obtained with the cells in the absence of agonist. B, G activity; PT-treated permeabilized cells expressing each receptor were assayed for adenylate cyclase activity with the indicated concentrations of PGE (, ) or sulprostone (, ). The values are means of triplicate experiments, which varied by less than 5%.



We next examined the agonist-mediated G protein activation in the PT- or CT-treated cell membrane expressing each receptor. Fig. 2A shows the GTPase activity of the CT-treated cell membrane, this treatment blocking the GTPase activity of G but allowing that of G. PGE and sulprostone stimulated the GTPase activity in the CT-treated cell membrane expressing the EP3D receptor, their concentration dependence profile being the same. PGE and sulprostone also stimulated it in the EP3D-R332Q receptor concentration-dependently, but the half-maximal concentrations for the stimulation (100 nM) were 1 order of magnitude higher than those in the EP3D receptor. On the other hand, the level of the maximal stimulation in the EP3D-R332Q receptor was the same as that in the EP3D receptor. Fig. 2B shows the GTPase activity of the PT-treated cell membrane, this treatment blocking the receptor-mediated activation of GTPase activity of G but allowing that of G. PGE concentration-dependently stimulated the GTPase activity in the PT-treated cell membrane expressing the EP3D receptor, but the stimulation by sulprostone was much lower than that by PGE. By contrast, the agonist-induced stimulation of GTPase activity was not observed with the EP3D-R332Q receptor. To assess the selective determination of GTPase activity of each type of G protein, we examined the ability of antisera against G and G to affect agonist-stimulated GTPase activity in the CT- and PT-treated cell membranes, respectively. As shown in , the antiserum against G attenuated the PGE-stimulated GTPase activity in the CT-treated cell membrane expressing the EP3D or EP3D-R332Q receptor. The antiserum against G attenuated the stimulation in the PT-treated cell membrane expressing the EP3D receptor. Thus, the PGE-stimulated GTPase activities in CT- and PT-treated cell membranes are ascribed to G and G, respectively, two receptors are exclusively coupled to G in the CT-treated cell membranes, and the EP3D receptor is coupled to G in the PT-treated cell membrane.


Figure 2: Effects of PGE and sulprostone on GTPase activity in the membrane of CT- or PT-treated cells expressing EP3D or EP3D-R332Q receptor. The membrane of CT- (A) or PT- (B) treated cells expressing the EP3D (, ) or EP3D-R332Q receptor (, ) was assayed for GTPase activity with the indicated concentrations of PGE (, ) or sulprostone (, ), as described under `` Experimental Procedures.'' The values are means of triplicate experiments, which varied by less than 5%.



Agonist Binding to EP3D or EP3D-R332Q Receptor Coupled to Either Gor G

We next examined the PGE binding affinities of EP3D and EP3D-R332Q receptors in the conditions of their selective coupling to either G or G. Fig. 3shows the Scatchard analysis of PGE binding to CT- or PT-treated cell membrane expressing the EP3D or EP3D-R332Q receptor. EP3D receptor coupled to either G or G had an apparently single high affinity binding site, the K value (3.8 nM) of the receptor coupled to G being slightly lower than that (5.0 nM) of the receptor coupled to G. On the other hand, EP3D-R332Q receptor had also an apparently single high affinity binding site (K = 10.2 nM) when coupled to G, but it showed very low affinity (K> 200 nM) in the condition of coupling to G.


Figure 3: Scatchard analysis of PGE binding to the membrane of CT- or PT-treated cells expressing EP3D or EP3D-R332Q receptor. The specific binding of [H]PGE (0.25-100 nM) to the membrane of CT- () or PT- () treated cells expressing EP3D (A) or EP3D-R332Q receptor (B) was determined, as described under ``Experimental Procedures.'' The Scatchard plot was transformed from the values of specific [H]PGE binding.



We further analyzed the binding affinities of EP3D and EP3D-R332Q receptors, coupled to either G or G, for PGE and sulprostone by assessing the displacement of [H]PGE binding to the receptors. Fig. 4A shows the binding to the EP3D receptor. PGE inhibited the [H]PGE binding to either the CT- or PT-treated cell membrane expressing the EP3D receptor, their half-maximal concentrations for the inhibition (10 nM) being the same. Sulprostone also inhibited the binding to the CT-treated cell membrane with the same half-maximal inhibition as that of PGE, but the displacement curve of sulprostone shifted 1 order toward the right in the PT-treated cell membrane. In contrast to PGE, the binding affinity of the G-coupled receptor for sulprostone was lower than that of the G-coupled receptor. Fig. 4B shows the binding to the EP3D-R332Q receptor. Displacement curves of PGE and sulprostone were the same in the CT-treated cell membrane. On the other hand, we could not obtain displacement curves of PGE and sulprostone in the PT-treated cell membrane because the membrane did not exhibit significant specific [H]PGE binding at this [H]PGE concentration (4 nM) used, as suggested by the Scatchard analysis (Fig. 3B).


Figure 4: Displacement of [H]PGE binding by PGE and sulprostone in the membrane of CT- or PT-treated cells expressing EP3D or EP3D-R332Q receptor. The membrane of CT- (, ) or PT- (, ) treated cells expressing EP3D (A) or EP3D-R332Q receptor (B) was incubated with 4 nM [H]PGE and the indicated concentrations of PGE (, ) or sulprostone (, ). Specific [H]PGE binding was determined as described under ``Experimental Procedures.'' Values are expressed as percentages of the control (1.85 ± 0.078 pmol/mg; PT-treated EP3D; 1.70 ± 0.044 pmol/mg, CT-treated EP3D; 1.02 ± 0.037 pmol/mg, CT-treated EP3D-R332Q). The values are means of triplicate experiments, which varied by less than 5%.



Effects of Various EP3 Agonists on Gand GActivities of EP3D and EP3D-R332Q Receptors

We examined the effects of various EP3 agonists with a negatively charged -carboxylic acid or noncharged modified -carboxylic acid on the adenylate cyclase system of EP3D and EP3D-R332Q receptors. PGE, M& 28767(21) , and TEI-3356 (22) are EP3 agonists with negatively charged unmodified -carboxylic acid, but instead M& 28767 and TEI-3356 are modified at the 11-hydroxyl and 9-carbonyl groups, respectively. On the other hand, sulprostone, misoprostol(21) , and GR 63799X (23) are EP3 agonists with a noncharged modified -carboxylic acid; their -carboxylic acids being substituted by methanesulfonamide, methylester, and phenyl groups, respectively. As shown in , agonists with a negatively charged -carboxylic acid not only inhibited the forskolin-stimulated adenylate cyclase activity in the CT-treated cells expressing EP3D receptor but also stimulated the basal adenylate cyclase activity in the PT-treated cells. By contrast, agonists with noncharged modified -carboxylic acid strongly inhibited the forskolin-stimulated adenylate cyclase activity, but they only very weakly stimulated the basal adenylate cyclase activation. In the EP3D-R332Q receptor, all of the EP3 agonists used showed only G activity. These findings suggest that the interaction between -carboxylic acid of PGE and the arginine residue of EP3D receptor plays an important role for the G activation but not for the G activation.


DISCUSSION

A variety of rhodopsin-type receptors have been reported to transduce multiple signals by interacting with different G proteins (2). The three tachykinin receptors and the thyrotropin receptor stimulate both phospholipase C and adenylate cyclase through G and G, respectively(24, 25) . The M1 muscarinic receptor and thrombin receptor mediate both adenylate cyclase inhibition and phospholipase C activation through G and G, respectively(26, 27) . The -adrenergic receptor is simultaneously coupled to G and G, signaling the stimulation and inhibition of adenylate cyclase, respectively(28) . Concerning ligand binding sites, several charged amino acid residues in the transmembrane domains of -adrenergic receptor, coupled to both G and G, have been shown to be essential for agonist binding according to site-directed mutagenesis studies(29) . However, the residues involved in the activation of each G protein are not understood.

The EP3D receptor is also a receptor coupled to multiple G proteins (13). In the present study, we demonstrated that the arginine residue in the seventh transmembrane domain of EP3D receptor, a putative binding site of -carboxylic acid of PGE, is essential for the PGE-induced adenylate cyclase stimulation but not for the inhibition (Fig. 1), and this is due to the different requirement for the arginine residue in the receptor for PGE-induced activation of G and G (Fig. 2). In the receptor-G coupling, the EC value of PGE for the G activation in EP3D-R332Q receptor was about 1 order of magnitude higher than that in the EP3D receptor (Fig. 2). This difference can be probably attributed to the difference between the Kvalues of the G-coupled receptors for PGE (Fig. 3). The point mutation at the arginine may induce a somewhat conformational change of the receptor structure as a whole, leading to a decrease in the efficiency of the agonists for G activation. However, it is clear that the mutant receptor has the ability to fully activate G because the maximal levels of PGE-induced G activation in the two receptors were the same. In the receptor-G coupling, EP3D-R332Q receptor almost completely lost the activation of G (Fig. 2), and this is due to loss of high-affinity binding of the receptor for PGE in the condition of coupling to G (Fig. 3B). Thus, the arginine residue in the seventh transmembrane domain is essential for high-affinity binding complex formation of the receptor with G but not for that of the receptor with G.

We examined, in turn, the contribution of the -carboxylic acid of agonists to the G protein activation. PGE with a negatively charged -carboxylic acid activated both G and G (Fig. 2). On the other hand, sulprostone with a noncharged modified -carboxylic acid activated G equally as well as did PGE, but it did not activate G as well as it did PGE (Fig. 2). This difference was also observed in the PGE binding displacement by sulprostone; the G-coupled receptor showed lower binding affinity for sulprostone than the G-coupled one (Fig. 4A). PGE has several structural features participating in its agonist activity, -carboxylic acid, 15-hydroxyl group, 9-carbonyl group, and cyclopentane ring(6) . Among these features, the modification site in sulprostone is -carboxylic acid, and other sites are not modified. Thus, the low potency of sulprostone for G activation may be ascribed to the modification of -carboxylic acid. Furthermore, this weak effect on G activity was also observed with other EP3 agonists with various modifications at the -carboxylic acid (). On the other hand, EP3 agonists with an unmodified -carboxylic acid had both potent G and G activity, even if they had modification at the sites other than -carboxylic acid. These findings indicate that -carboxylic acid is important to the G-receptor coupling, and this modification significantly affects coupling. Although the agonists with modified -carboxylic acid had a much lower potency for G activity than PGE, they still showed significant activation (). Although -carboxylic acid is important for determination of G coupling, other group(s) of PGE structure may also contribute to this determination in concert with -carboxylic acid, or there might be noncharged interaction of the modified -carboxylic acid of the agonists with the arginine, such as hydrogen bonding. Sulprostone is a well-characterized PGE analogue widely used as a very valuable and potent EP3 agonist(6) . Our finding of preferential activation of G by sulprostone suggests that a single receptor has a functionally different pharmacological profile depending on the ligand structure. PGE at lower concentrations was previously demonstrated to inhibit vasopressin-induced cAMP accumulation but at high concentrations to stimulate cAMP accumulation in the cortical collecting duct, resulting in dual regulation of water reabsorption (30). In contrast to PGE, sulprostone prevented the vasopressin-induced cAMP accumulation but failed to stimulate cAMP accumulation(30) . Considering selective expression of EP3 receptors, including EP3D-type isoform, among various PGE receptor subtypes in the cortical collecting duct(31, 32) , the stimulatory activity of PGE would be mediated by EP3D, and this idea is consistent with the weak effect of sulprostone to activate G.

The present study of the point mutation at the arginine residue and actions of various EP3 agonists suggests that charge-charge interaction of -carboxylic acid of PGE and the arginine residue of the receptor is required for the receptor-G coupling but not for the receptor-G coupling, and this interaction is correlated with G protein selectivity. Functional analysis of the agonist-binding site of the M3 muscarinic receptor suggested that Thr-234 and Tyr-509, which are located at fifth and sixth transmembrane domains, respectively, were the binding sites of acetylcholine and also played a role in agonist-induced receptor activation(33) . These transmembrane domains are connected by the third intracellular loop, a region known to be critical for G protein recognition and activation by the M3 muscarinic receptor(34) . These two binding residues have been proposed to be involved in triggering the agonist-induced conformational change in the fifth and sixth transmembrane domains required for the functional activation of the third intracellular loop. In EP3 receptors, we demonstrated that EP3 receptor isoforms with different COOH-terminal tails, which are produced through alternative splicing, are coupled to different G proteins and showed that the COOH-terminal tail determines the selectivity of G protein coupling (12, 13). The seventh transmembrane domain is directly connected with the COOH-terminal tail. Thus, a conformational change of the COOH-terminal tail may regulate the selectivity of G protein coupling, and the EP3D receptor may be able to form two types of conformation, which can selectively be associated with either G or G, depending on the interaction of -carboxylic acid and the arginine residue. Concerning coupling of receptors with G proteins, Lefkowitz and co-workers have proposed a two-state model in which receptors are in equilibrium between the inactive conformation and a spontaneously active conformation that can couple to G protein, and classic agonists increase the concentration of the latter conformation of the receptors(35) . Although our present study suggests that the EP3D receptor has two active conformations, coupling different G proteins, it is not known whether the selective coupling of the receptor to a particular G protein is a consequence of the interaction of the ligand with the receptor or whether the G protein coupling of the receptor influences its affinity for a particular ligand.

Agonist-specific coupling of rhodopsin-type receptors to multiple second messenger systems has been reported in the Drosophila octopamine/tyramine receptor (36) and type 1 pituitary adenylate cyclase-activating polypeptide receptor(37) , but key amino acid residues of the receptor structures responsible for selectivity of G protein coupling have not yet been studied. In addition to these reports, our finding provides support for the idea that receptors form multiple conformations, which are differentially coupled to multiple G proteins.

In conclusion, in this report we have shown that the arginine residue in the seventh transmembrane domain of the EP3D receptor, a putative binding site of -carboxylic acid of PGE, contributes to the selectivity of G protein coupling, and -carboxylic acid of PGE plays an important role in the G protein coupling selection, perhaps through interaction with the arginine residue. This study will contribute not only to understanding the diverse PGE actions, but it will also help to elucidate the molecular mechanism of receptor-mediated multiple G protein activation.

  
Table: Functional attenuation of G or G coupling by G or G antiserum

The membrane (5 µg) of CT- or PT-treated cells expressing EP3D or EP3D-R332Q receptor was incubated with 2 µl of normal serum (NS), G antiserum (AS/7), or G antiserum (RM/1) for 1 h at 4 °C, after which the membrane was assayed for GTPase activity with or without 10 µM PGE, as described under ``Experimental Procedures.'' Values are means ± S.E. for triplicate experiments.


  
Table: 0p4in Noncharged modified -carboxylic acid.


FOOTNOTES

*
This work was supported in part by Grants-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (05304027, 05454568, 05671816 and 05771875) and by grants from the Mochida Memorial Foundation for Medical and Pharmaceutical Research and the Sankyoh Life Science Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-75-753-4547; Fax: 81-75-753-4557.

The abbreviations used are: PG, prostaglandin; G protein, heterotrimeric GTP-binding protein; CHO, Chinese hamster ovary; PT, pertussis toxin; CT, cholera toxin; App(NH)p, 5`-adenylyl-,-imidodiphosphate.


ACKNOWLEDGEMENTS

We thank A. Harazono for technical assistance and Drs. K.-H. Thierauch of Schering, P. W. Collins of Searle, M. P. L. Caton of Rhone-Poulenc Ltd., S. Kurozumi of Teijin Ltd., and B. M. Bain of Glaxo Group Research Ltd. for providing sulprostone, misoprostol, M& 28767, TEI-3356 and GR 63799X, respectively.


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