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. 1999 Jan;126(1):365–371. doi: 10.1038/sj.bjp.0702291

Identification of a region of the C-terminal domain involved in short-term desensitization of the prostaglandin EP4 receptor

Murat Bastepe 1, Barrie Ashby 1,*
PMCID: PMC1565795  PMID: 10051157

Abstract

  1. The prostaglandin EP4 receptor, which couples to stimulation of adenylyl cyclase, undergoes rapid agonist-induced desensitization when expressed in CHO-K1 cells.

  2. Truncation of the 488-amino acid receptor at residue 350 removes the carboxy-terminal domain and abolishes desensitization.

  3. To further delineate residues involved in desensitization, the receptor was truncated at position 408, 383 or 369. Receptors truncated at position 408 or 383 underwent PGE2-induced desensitization, whereas the receptor truncated at position 369 displayed sustained activity, indicating that the essential residues for desensitization lie between 370 and 383.

  4. The six serines in the 14-amino acid segment between residues 370 and 383 were mutated to alanine, retaining the entire C-terminal domain. Desensitization was absent in cells expressing this mutant.

  5. The results indicate involvement of serines located between 370 and 382 in rapid desensitization of the EP4 receptor.

Keywords: Prostaglandin EP4 receptor, cyclic AMP, agonist-induced desensitization, deletion mutagenesis, site-directed mutagenesis

Introduction

Prostaglandin E2 (PGE2) is an important modulator in many physiological and pathophysiological events (Campbell & Halushka, 1996), binding to four distinct G protein-coupled receptors (Negishi et al., 1995). The EP1 receptor couples to phospholipase C, the EP2 and EP4 receptors couple to stimulation of adenylyl cyclase, and the EP3 receptor couples to inhibition of adenylyl cyclase. All the EP receptor subtypes have been cloned. The EP3 receptor has six isoforms, generated from alternative splicing of a single gene, that differ in their C-terminal region (Regan et al., 1994a; An et al., 1994; Adam et al., 1994; Schmid et al., 1995; Kotani et al., 1995). The EP3 isoforms display differences in their degree of cyclase inhibition (Regan et al., 1994a; An et al., 1994) and susceptibility to desensitization (Negishi et al., 1993).

PGE2-induced increase in the cyclic AMP level is mediated by two receptors: EP2 and EP4. Both receptors display a broad tissue distribution with similar expression patterns (Katsuyama et al., 1995), whereas each has distinctive structural and functional characteristics. EP2 is a 358-amino acid protein with relatively short third intracellular and C-terminal domains (Regan et al., 1994b). In contrast, EP4, which has ∼30% sequence identity to EP2, consists of 488 amino acids, and has a long third intracellular loop and a long cytoplasmic tail that comprises almost one third of the receptor (An et al., 1993; Bastien et al., 1994). In addition, the two receptors display different sensitivities to metabolic inactivation of PGE2, in that the first PGE2 metabolite 15-keto-PGE2 is more potent at the EP2 receptor than EP4 (Nishigaki et al., 1996). Furthermore, the EP2 receptor lacks agonist-induced short-term desensitization, whereas EP4 desensitizes rapidly (Nishigaki et al., 1996).

Agonist-induced desensitization is a common biological phenomenon that involves reduction of responsiveness despite continuous agonist challenge. Recent pharmacological evidence suggests that EP4 may mediate several actions of PGE2. Regulation by PGE2 of matrix metalloproteinase activities in the RNK-16 cell line, a model for natural killer cells, is mediated by EP4 (Zeng et al., 1996) so that short-term desensitization may provide a tight regulatory control over the PGE2-elicited chemokinesis. The EP4 receptor has also been implicated in neonatal remodelling of the cardiovascular system triggered by a drop in PGE2 level (Nguyen et al., 1997), which is among actions of PGE2 utilized in clinical therapy (Campbell & Halushka, 1996). Desensitization may play an important role in sensing the drop of PGE2 level by the EP4 receptor, which is critical for closure of the ductus arteriosus.

Structural determinants of desensitization have been studied extensively in the case of β-adrenergic receptors (see Bohm et al., 1997 for a review). Short-term agonist treatment results in uncoupling of the receptor from its G protein, followed by receptor sequestration. The uncoupling event involves phosphorylation of the receptor by cyclic AMP-dependent protein kinase (PKA) and/or β-adrenergic receptor kinase at certain serine and threonine residues located in the third intracellular and/or C-terminal domains. Sequestration involves internalization of the receptor through endocytosis.

We have shown that the C-terminal domain is essential for agonist-induced desensitization of EP4 (Bastepe & Ashby, 1997). We generated a mutant EP4 severely truncated at its C-terminus, and demonstrated that the mutant, although intact in terms of ligand binding and G protein coupling, is resistant to short-term desensitization. Supporting our findings, the C-terminal domain of EP4 has recently been shown to confer agonist-induced receptor desensitization in a chimeric receptor with the EP3β receptor (Neuschaferrube et al., 1997). Figure 1 shows the membrane topography of the EP4 receptor, indicating the 36 serine and threonine residues in the C-terminus and the position of the truncation at residue 350 that results in an active receptor that does not undergo agonist-induced short-term desensitization. In an effort to map the amino acid residues involved in agonist-induced desensitization of EP4, we generated a series of mutants with successive deletions at their C-terminus, and one that carries serine-to-alanine point mutations at certain positions. Here we report that six serines within a 14-amino acid stretch in the proximal third of the cytoplasmic tail are essential residues involved in agonist-induced desensitization of EP4.

Figure 1.

Figure 1

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Membrane topography of the human EP4 receptor. Amino acid sequences within the proposed transmembrane domains were determined by hydropathy analysis of the sequence. Asterisks indicate serine and threonine residues in the carboxy-terminal domain. The arrow indicates the position of the truncation at residue 350 that results in an active receptor that does not undergo short-term desensitization.

Methods

Materials

Restriction enzymes were from New England Biolabs (Beverly, MA, U.S.A.). [3H]PGE2, [2,8-3H]adenine and [14C]cyclic AMP were from DuPont NEN (Boston, MA, U.S.A.). Other chemicals and reagents were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). CHO-K1 cells were from the American Type Culture Collection (Rockville, MD, U.S.A.). The plasmid pRc/CMV was from Invitrogen (San Diego, CA, U.S.A.). DNA sequencing was carried out by automated sequencing at the University of Pennsylvania DNA Sequencing Facility (Philadelphia, PA, U.S.A.). Custom-order oligonucleotides were from Life Technologies Inc. (Gaithersburg, MD, U.S.A.).

Deletion mutagenesis

Construction of the plasmid pRc/CMV-hEP4-wt was described previously (Bastepe & Ashby, 1997). A fragment of pRc/CMV-hEP4-wt, extending from the unique SacII site at nucleotide 694 to the desired truncation point (nucleotides 1224, 1149 and 1107 for hEP4-t408, hEP4-t383 and hEP4-t369, respectively), was amplified by PCR. Forward primer was 5′-CCA CCG CGG CCG CCT CGG TTG CCT CC-3′ (underlined bases indicate the SacII site). Three different stop codons located in three different reading frames and an ApaI site were introduced in the 3′ end of the PCR product via reverse primer, which was 5′-GGA GGG CCC TAT TTA TTC AGC CAT TTT CAC TGA GGT CTG-3′, 5′-GGA GGG CCC TAT TTA TTC ACC GGG AGA TGA AGG AGC GAG-3′ or 5′-GAG GGG CCC TAT TTA TTC ATG TCC TTT GAC TGT CTG AG-3′ (underlined bases indicate the ApaI site; bases in bold-type indicate stop codons) for hEP4-t408, hEP4-t383 or hEP4-t369, respectively. Following purification and restriction enzyme digestion with SacII and ApaI, each PCR product was ligated with the 6773-bp linearized piece of the SacII/ApaI digestion products of pRc/CMV-hEP4-wt. The plasmids, designated pRc/CMV-hEP4-t408, pRc/CMV-hEP4-t383 and pRc/CMV-hEP4-t369, were sequenced in order to check for undesired mutations and sequence integrity. To maintain consistency in plasmid structures, the 3′-untranslated region of the wild-type EP4 cDNA was deleted by a similar strategy using the reverse primer: P4=5′-GGA GGG CCC TAT TTA TTC ATA TAC ATT TTT CTG ATA AGT TCA G-3′ (underlined bases indicate the ApaI site; bases in bold-type indicate stop codons).

Site-directed mutagenesis

Serine-to-alanine mutations were carried out by oligonucleotide-directed mutagenesis according to Higuchi et al. (1988). Initially, Ser370Ala and Ser371Ala mutations were introduced. Two overlapping fragments of pRc/CMV-hEP4-wt were amplified by PCR using the primers: P1=5′-CCA CCG CGG CCG CCT CGG TTG CCT CC-3′ and P2=5′-GGC AGC AGC TGT CCT TTG ACT G-3′, or P3=5′-CAG TCA AAG GAC AGC TGC TGC C-3′ and P4. One of the PCR products extended from the unique SacII site to nucleotide 1116 and the other from nucleotide 1095 to the ApaI site adjacent to the termination codon. Both PCR products carried the same T to G mutations at nucleotides 1108 and 1111 which were introduced via mismatch in primers P2 and P3. Small aliquots of the overlapping PCR products were combined, denatured, reannealed and subjected to additional 30 cycles of PCR using primers P1 and P4. Following purification and restriction enzyme digestion with SacII and ApaI, the resultant PCR product was substituted with the corresponding region of pRc/CMV-hEP4-wt. The construct was sequenced, and used as template in the PCR for generation of the cDNA encoding the mutant hEP4 with serine-to-alanine mutations at positions 370, 371, 374, 377, 379 and 382. The first overlapping PCR product, generated using primers P1 and P5 (5′-GGC GAT GAA GGC GCG AGC GTG GCC TGC CAT GGC AGA AG-3′), extended from the unique SacII site to nucleotide 1146 and the second, generated using primers P6 (5′-TGG CAG GCC ACG CTC GCG CCT TCA TCG CCC GGG AG-3′) and P4, extended from nucleotide 1118 to the ApaI site adjacent to the termination codon. Both PCR products carried the same T to G mutations at nucleotides 1120, 1129, 1135, and 1144, introduced via mismatches in primers P5 and P6. The final plasmid construct, designated pRc/CMV-hEP4-S370-382A, was generated as described above and the mutations confirmed by sequencing.

Expression in CHO-K1 cells

Stable transfection of CHO-K1 cells with each cDNA plasmid and isolation of clonal cells were performed as previously described (Bastepe & Ashby, 1997). Positive clones were identified by measuring the PGE2-induced cyclic AMP formation and selected clonal cells maintained in medium containing 10% foetal bovine serum, 350 μg ml−1 G-418 sulphate and 1 mM acetyl salicylic acid (used to inhibit endogenous PGE2 formation). For transient expression, CHO-K1 cells were seeded at a density of 5×106 cells per 100-mm culture plate. Twenty-four hours later, transfection was carried out with 8 μg of plasmid DNA and 12.5 μg ml−1 Lipofectamine reagent (Life Technologies Inc.) according to the manufacturer's instructions. Cells were assayed 48 h after the start of transfection.

Determination of adenylyl cyclase activity

Cells grown in 6-well 35-mm culture plates were labelled overnight with 2 μCi ml−1 of [3H]adenine (25 Ci mmol−1). Labelling medium was removed and cells incubated for 10 min with fresh medium containing 2 mM 3-isobutyl-l-methylxanthine (IBMX). Cells were challenged with forskolin (30 μM) or PGE2 (10 μM) for various times. Reactions were stopped by the replacement of the medium with a solution containing HCl (0.2 M), 0.2% sodium dodecyl sulphate and 2000 c.p.m. of [14C]cyclic AMP as recovery standard. [3H]cyclic AMP was determined according to Salomon (1979) as percentage of the total labelled adenine nucleotides. Kinetic data obtained from PGE2 challenge of each clonal cell line were normalized to initial rate of cyclic AMP formation determined over the first 2 min and expressed as percentage of cyclic AMP expected to accumulate within 30 min at the initial rate.

Membrane preparation and binding experiments

Cells grown in 100-mm culture plates were washed and scraped in hypotonic lysis buffer containing (in mM) Tris-HCl 20 (pH 7.5), MgCl2 10, EDTA 1, EGTA 1, Leupeptin (10 μM) and soybean trypsin inhibitor (10 μg ml−1). Following incubation on ice for 30 min, the lysate was sonicated and centrifuged at 80,000×g for 30 min at 4°C. The pellet was washed once with (in mM) Tris-HCl 50 (pH 7.5), MgCl2 10, EDTA 1, and 10 μM Leupeptin (Buffer A), re-centrifuged and resuspended in Buffer A. Protein concentrations were determined by the Coomassie® Plus protein assay reagent (Pierce, Rockford, IL, U.S.A.). Membranes were incubated with 1 nM [3H]PGE2 (200 Ci mmol−1) in the presence of varying concentrations of non-labelled PGE2 in Buffer A for 60 min at room temperature. Binding was determined as described previously (Kunapuli et al., 1994a) except the filters were presoaked in Tris-HCl 50 mM, (pH 7.5) containing 0.1 mg ml−1 bovine serum albumin and 0.2% polyethylenimine and washed with ice-cold Tris-HCl 50 mM, (pH 7.5) buffer. Data were analysed using EBDA and LIGAND softwares (Biosoft, Cambridge, U.K.) (Mcpherson, 1983; Munson & Rodbard, 1980).

Results

Truncation mutants of the EP4 receptor

Figure 2A shows a schematic representation of the deletion mutants in comparison with wild-type EP4. The cytoplasmic C-terminal domain of the wild-type EP4 receptor consists of 156 amino acids. In the mutants, designated hEP4-t408, hEP4-t383 and hEP4-t369, the number of amino acid residues deleted are 80, 105 and 119, respectively. Eighteen, 25 and 31 of the 38 serine and threonine residues fall within the deleted fragment in hEP4-t408, hEP4-t383 and hEP4-t369, respectively.

Figure 2.

Figure 2

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Schematic representation of the putative seventh transmembrane and C-terminal domains of wild-type EP4, hEP4-t408, hEP4-t383 and hEP4-t369. The C-terminal serines (S) and threonines (T) are indicated at proportional distance from each other with less than 10% error. (B) Schematic representation of hEP4-S370-382A. Hatched area represents residues 370–383, which are illustrated underneath. Alanine substitutions are indicated by asterisks.

We expressed the truncated receptors transiently in CHO-K1 cells to characterize their agonist binding properties. Specific binding of [3H]PGE2 to membranes of transfected cells was saturable for each receptor and Kd values were similar to the wild-type EP4 receptor (Table 1).

Table 1.

Specific PGE2 binding to wild-type and mutant EP4 receptors

graphic file with name 126-0702291t1.jpg

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Functional features of the truncated receptors were studied in clonal CHO-K1 cells stably expressing wild-type or each of the mutant receptors. The time-course of cyclic AMP formation was examined in selected clonal cells challenged with PGE2 in the presence of the phosphodiesterase inhibitor IBMX, which inhibits break-down of cyclic AMP, so that a decrease in the rate of cyclic AMP synthesis represents a decline in adenylyl cyclase activity. As a means of assessing desensitization, this method has been validated by detailed studies of prostaglandin receptor desensitization in intact cells (Ashby, 1989; 1990). As shown in Figure 3A, cells stably expressing wild-type EP4, hEP4-t408 and hEP4-t383 displayed similar kinetic patterns of 10 μM PGE2-induced cyclic AMP formation. The time-course, which appeared linear over the first 2–3 min of agonist stimulation, curved off with time and reached a plateau within 15 min, indicating a decline in rate of cyclic AMP synthesis. In contrast, the kinetic pattern obtained in the clonal cells expressing hEP4-t369 was significantly different from the others, in that the cyclic AMP formation increased in a sustained manner over the course of the experiment (Figure 3A). The kinetic pattern of adenylyl cyclase activation, similar to that observed in cells expressing the previously-reported hEP4-t350 (Bastepe & Ashby, 1997), demonstrates a lack of agonist-induced short-term desensitization of hEP4-t369.

Figure 3.

Figure 3

Figure 3

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Time-course of PGE2 (A) or forskolin (B)-induced cyclic AMP formation in CHO-K1 cells stably expressing the wild-type EP4 receptor or each of the deletion mutants. Clonal hEP4-wt/CB9, hEP4-t408/C7, hEP4-t383/E2, hEP4-t369/BD10 or hEP4-S370-382A/AC8 cells were stimulated with PGE2 (10 μM) or forskolin (30 μM) for indicated times in the presence of IBMX (2 mM). Reactions were stopped and cyclic AMP measured as described in Methods. Values are representative of at least three separate experiments with similar results. [3H] cyclic AMP was determined as percentage of total labelled adenine nucleotides. The level obtained with PGE2 stimulation was normalized for each clonal cell to the initial rate of cyclic AMP formation determined over the first 2 min. After subtraction of the base-line, each value was expressed as a percentage of the amount of cyclic AMP expected to accumulate within 30 min at the initial rate of synthesis, giving an indication of the degree of desensitization. Note that all of the constructs are represented in Figure 3B but the data are hidden by other symbols since they are so similar.

To assure that the differences in the patterns of cyclic AMP formation obtained from individual clonal cells did not arise from clonal differences among intrinsic adenylyl cyclase activities, we examined the time-course of cyclic AMP formation in response to forskolin, a direct activator of adenylyl cyclase. Unlike those obtained in response to PGE2 stimulation, the kinetic profiles observed in response to forskolin (30 μM) in the presence of IBMX were similar in the clonal cells regardless of receptor type (Figure 3B), indicating that the specific activities of the cyclase systems of the clonal cell lines are similar to each other.

To confirm our findings, we repeated the experiments with two other clonal cell lines for wild-type EP4, hEP4-t383 and hEP4-t369. Figure 4 shows the average 2- or 20-min level of PGE2-induced cyclic AMP in different clonal cell lines expressing the same type of receptor. Expressed as percentage of the hypothetical 30-min level reached at the initial rate of synthesis, the levels attained after 2 min were similar in all the selected clonal cells. However, those reached after 20 min displayed significant differences. Although the average level of cyclic AMP accumulated in the clonal cell lines expressing wild-type EP4 and the hEP4-t383 mutant were still similar to each other, the amount accumulated in cells expressing hEP4-t369 was significantly higher than the others (Figure 4). The observation that the mutant terminating at Thr369 (hEP4-t369) does not desensitize in response to short-term agonist challenge, unlike the one terminating at Arg383 (hEP4-t383), indicates that the residues of the EP4 receptor involved in agonist-induced desensitization are embedded in a 14 amino-acid stretch located between positions 370 and 383 within the proximal one third of the C-terminal domain.

Figure 4.

Figure 4

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PGE2-induced cyclic AMP formation in clonal CHO-K1 cells expressing hEP4-wt, hEP4-t383, hEP4-t369, or hEP4-S370-382A. Clonal cells were stimulated with PGE2 (10 μM) for indicated time periods in the presence of IBMX (2 mM). Reactions were stopped and cyclic AMP measured as described in Methods. Values represent means±s.e.mean of 4–6 separate experiments with a total of three clonal cell lines for each receptor. [3H] cyclic AMP was determined as a percentage of total adenine nucleotides, and normalized, for each clonal cell, to the initial rate of cyclic AMP formation determined over the first 2 min as described in Methods. *P<0.001 compared with wild-type or hEP4-t383 at the same time point according to Student's t-test.

We also determined specific binding of PGE2 to the wild-type and truncated receptors expressed in the clonal cells to assure that receptor expression levels were comparable. As presented in Table 1, the amount of [3H]PGE2 specifically bound to membranes prepared from individual clonal cells displayed little variation. The average amount of specifically-bound [3H]PGE2 in each group of clonal cells expressing the same type of receptor was comparable to one another, with no indication of correlation with susceptibility to receptor desensitization.

Site-directed mutagenesis of the EP4 receptor

There are six serines within the identified region of the C-terminal domain of the EP4 receptor which are potential targets for phosphorylation. To ascertain the importance of these serines in desensitization of EP4, we generated another mutant, designated hEP4-S370-382A, that retained the entire C-terminal domain with serine-to-alanine substitutions at positions 370, 371, 374, 377, 379 and 382 (Figure 2B).

The agonist affinity of hEP4-S370-382A expressed transiently in CHO-K1 cells was determined by measuring binding of [3H]PGE2 to membranes and the Kd value was similar to that of wild-type receptor (Table 1). We examined the time-course of PGE2-induced cyclic AMP response in clonal cells stably expressing hEP4-S370-382A (Figure 3A). The kinetic pattern indicated a sustained increase in the level of cyclic AMP, which was similar to that observed in clonal cells expressing hEP4-t369 but significantly different from that observed in clonal cells expressing wild-type EP4. This finding clearly shows that desensitization is substantially impaired by serine-to-alanine substitutions at positions 370, 371, 374, 377, 379 and 382.

We examined the time-course of PGE2-induced cyclic AMP formation in two additional clonal cell lines expressing hEP4-S370-382A. A sustained elevation of cyclic AMP formation was observed in both clonal cells. As shown in Figure 4, whereas the average cyclic AMP levels were similar after 2 min of agonist stimulation in multiple clonal cells expressing wild-type EP4, hEP4-t369 or hEP4-S370-382A, the level attained after 20 min was significantly higher in cells expressing hEP4-S370-382A than those expressing wild-type EP4. We also examined the forskolin-stimulated adenylyl cyclase activity in each of the clonal cells, and showed that it was similar to those measured in cells expressing wild-type or truncated EP4 receptors (data not shown). [3H]PGE2 binding in the clonal cell lines was comparable with each other and with wild-type or the truncated receptors (Table 1).

Discussion

The EP4 subtype of the PGE2 receptor is a member of the G protein-coupled receptor superfamily. Agonist stimulation of EP4 leads to activation of adenylyl cyclase and the receptor is susceptible to agonist-induced short-term desensitization. In this study, we measured the time-course of cyclic AMP formation in CHO-K1 cells expressing wild-type or mutant EP4 receptors in order to determine the structural features involved in receptor desensitization.

The desensitization process examined here is essentially complete by 10 min which predominantly reflects a short-term desensitization process rather than down-regulation as is evident from the work of Nishigaki et al. (1996). These authors compared desensitization of EP2 and EP4. They showed rapid desensitization of EP4 (complete after 10 min) but no rapid desensitization of EP2, even after 30 min. By contrast, both EP2 and EP4 showed loss of receptor binding with half times on the order of 1 h or longer. While it is clear that some down-regulation takes place during the time period that we observe, the data of Nishigaki et al. (1996) show that it is possible to differentiate between short- and long-term desensitization by conducting experiments of 30 min or shorter duration.

Mechanisms of short-term receptor desensitization have been extensively studied in the β2-adrenergic receptor. The putative third intracellular and C-terminal domains have been highlighted as the most important structural features involved in agonist-induced short-term desensitization (Bohm et al., 1997). Earlier, we reported that the C-terminal domain of EP4 is required for desensitization by showing that deletion of 138 amino acids at the C-terminus results in a mutant EP4 receptor that is unable to undergo desensitization (Bastepe & Ashby, 1997).

In the present study, our findings from mutant EP4 receptors carrying successive deletions in their cytoplasmic tail indicate that, despite the lengthiness of the C-terminal domain, residues of importance with respect to rapid desensitization reside within a short stretch of amino acids. Even though deletion of the last 105 amino acids in the C-terminus (hEP4-t383) does not alter the susceptibility of EP4 to agonist-induced desensitization, removal of an additional 14 residues (hEP4-t369) results in the loss of receptor desensitization upon short-term agonist stimulation.

Desensitization of G protein-coupled receptors in response to short-term agonist exposure entails receptor/G protein uncoupling mediated by phosphorylation of the receptor at certain serine or threonine residues located in the third intracellular loop and/or C-terminal domain (Bohm et al., 1997). The kinases involved include cyclic AMP-dependent protein kinase, which leads to heterologous desensitization, and specific GRKs, which lead to homologous desensitization. Potential targets for phosphorylation in the identified 14-amino acid region of the EP4 receptor are six serine residues at positions 370, 371, 374, 377, 379 and 382. We eliminated these residues in another mutant by substituting each with an alanine. Our results, from experiments involving functional analysis of the latter mutant, strongly suggest that at least one of the six mutated serines is indeed involved in agonist-induced short-term desensitization of the EP4 receptor. The mutant receptor, which differs from wild-type by only 6 amino acids, displays a significantly impaired agonist-induced desensitization, characterized by a sustained increase in the PGE2-induced cyclic AMP formation.

The kinases involved in desensitization of G protein coupled receptors include cyclic AMP-dependent protein kinase, which leads to heterologous desensitization, and specific G protein-coupled receptor kinases (GRKs), which lead to homologous desensitization. Among the six serines identified, Ser370 and/or Ser371 seem likely candidates for phosphorylation by PKA. Although the PKA consensus sequence, Arg-Arg-Xaa-Ser, does not exactly appear in this region, a similar sequence, Gln-Arg-Thr-Ser370-Ser371, is present in the human receptor (An et al., 1993; Bastien et al., 1994). Moreover, the corresponding regions of the mouse and rat EP4 receptors bear the exact consensus sequence, carrying an arginine (Arg395 and Arg370, respectively) instead of the glutamine in human (Honda et al., 1993; Sando et al., 1994). Nevertheless, a recent study has shown that heparin, a compound with known non-specific GRK inhibitory activity, but not the PKA inhibitor peptide (PKI), prevents agonist-induced short-term desensitization of EP4 (Nishigaki et al., 1996), suggesting involvement of GRKs, but not PKA.

A consensus sequence for GRK-mediated phosphorylation has yet to be unequivocally identified. It appears from studies using synthetic peptides that, whereas some GRKs, including rhodopsin kinase and β-adrenergic receptor kinase, may actively phosphorylate serines flanked N- or C-terminally by acidic residues (Onorato et al., 1991) others may display higher activity for serines C-terminal to basic residues (Kunapuli et al., 1994b; Loudon & Benovic, 1994). In the C-terminal domain of the EP4 receptor, there are multiple serine residues, most of which are closely flanked by acidic or basic residues. Those within the identified region, however, appear to be predominantly flanked by basic residues, which may be helpful in future studies to identify the GRKs involved in desensitization of EP4. Demonstration of agonist-induced phosphorylation by use of receptor-specific antibodies and over-expression of individual GRKs should provide more detail of EP4 desensitization.

The primary difference between the two PGE2 receptors EP2 and EP4 is their susceptibility to agonist-induced short-term desensitization (Nishigaki et al., 1996). This distinction suggests that, whereas EP2 may be involved in mediating more sustained actions of PGE2, EP4 may mediate rapidly waning events. Although boundaries between the physiological roles of the EP2 and EP4 receptors remain to be clarified, agonist-induced short-term desensitization is clearly a considerable regulatory element in EP4-mediated signalling, but not in EP2-mediated signalling. Identification of structural features of EP4 involved in this response should prove useful for future investigations involving the physiology and pharmacology of these receptors.

Acknowledgments

We thank Drs Lee-Yuan Liu-Chen and Satya P. Kunapuli for helpful discussion and critical review of this manuscript. The work was supported by NIH Grant RO1-HL 48114 (B.A.), and a Pre-doctoral fellowship from the Southeastern Pennsylvania Affiliate of the American Heart Association and a NATO Pre-doctoral Fellowship from the Scientific and Technical Research Council of Turkey (M.B.).

Abbreviations

CHO-K1

Chinese hamster ovary K1

GRK

G protein coupled receptor kinase

IBMX

3-isobutyl-l-methylxanthine

PGE2

prostaglandin E2

PKA

protein kinase A

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