Introduction

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Prostaglandin E₂ receptors (EP-Rs), like most prostanoid receptors, belong to the class of G protein-coupled ectoreceptors (GPCR) with seven transmembrane domains (Negishi, 1994). There are four subtypes of E-prostaglandin receptors (EP-Rs) that differ in their affinity to synthetic ligands and their G protein coupling specificity. EP1-Rs are linked to G_q and increase inositol trisphosphate (InsP₃) and, hence, cytosolic Ca²⁺ concentration. EP2-Rs and EP4-Rs are coupled to G_s and increase intracellular cAMP. EP3-Rs are coupled to G_i and decrease hormone-stimulated cyclic AMP (cAMP) formation (Fig. 1) (Coleman, 1994). These receptors display an overall sequence homology of about 50%; the putative transmembrane domains are the most conserved (Coleman, 1994). Analysis of naturally occurring variants (Negishi, 1993a,b; Hasegawa, 1996), of receptor hybrids between different prostanoid receptors (Dorn, 1997; Kobayashi, 1997; Neuschäfer-Rube, 1997a,b), and of receptors in which single amino acids or groups of amino acids have been replaced or removed (Funk, 1993; Irie, 1994; Audoly and Breyer, 1997; Bastepe and Ashby, 1997; Chang, 1997) provided insight into the structure-function relationship of prostanoid receptors. The transmembrane domains (Funk, 1993; Chang, 1997) and some parts of the extracellular loops (Audoly and Breyer, 1997; Dorn, 1997) of the prostanoid receptors seem to be responsible for ligand binding and binding specificity, the carboxyl-terminal domains have been implicated in control of signal transduction (Irie, 1994), G protein-coupling specificity (Negishi, 1993a), and agonist-induced receptor desensitization (Negishi, 1993b; Nishigaki, 1996). Splice variants of the mouse or the bovine EP3-Rs that differ only in their carboxyl-terminal portions couple to different G proteins (Negishi, 1993a). Truncation of the murine EP3-R carboxyl-terminal domain leads to constitutive activation (Irie, 1994). Only one of the mouse EP3-R carboxyl-terminal splice variants, the EP3-R, showed agonist-dependent desensitization (Negishi, 1993b). Similarly, of the two G_s-linked EP-Rs (EP2-R and EP4-R), only the EP4-R, which has a long serine- and threonine-rich carboxyl-terminal domain, shows rapid agonist-induced desensitization (Nishigaki, 1996). Agonist-induced desensitization but not G protein coupling was lost by truncation of the carboxyl-terminal domain of the human EP4-R (Bastepe and Ashby, 1997).

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Hypothetical structure of the Gi-coupled rat PGE2-receptor (rEP3-R), the Gs-coupled human PGE2 receptor (hEP4-R), a chimeric rEP3hEP4-Ct receptor (rEP3hEP4R-Ct-R), and their functions. Glycosylation sites in the amino-terminal domains and the second extracellular loops have been omitted for clarity. Ct, carboxyl-terminal domain. Numbers represent the numbers of amino acids in the respective extra- or intracellular domains.

In a previous study, to elucidate the role of the EP-R carboxyl-terminal domain in G protein coupling and agonist-induced receptor desensitization, a receptor hybrid was generated consisting of the amino-terminal main portion of the G_i-coupled, nondesensitizable rat EP3-R (rEP3-R) up to the end of the seventh transmembrane domain and the carboxyl-terminal portion of the G_s-coupled, desensitizable human EP4-R (hEP4-R) (Neuschäfer-Rube, 1997a,b). This receptor hybrid retained the binding specificity of the EP3-R (i.e., it bound the EP3-R-specific agonist M&B28767 with higher affinity than prostaglandin (PG) E₂ (PGE₂). When stably expressed in HepG2 cells, the hybrid receptor exclusively coupled to G_i (Neuschäfer-Rube, 1997a). The carboxyl-terminal domain of the EP4-R in the receptor hybrid was necessary and sufficient to confer agonist-induced receptor desensitization (Fig. 1) (Neuschäfer-Rube, 1997b). The molecular events responsible for this effect are currently unknown. Rapid termination of signaling by GPCRs is typically initiated by receptor phosphorylation events catalyzed either by second messenger-activated kinases [such as cAMP-dependent protein kinase (PKA) or Ca²⁺/phospholipid-dependent protein kinase (PKC)] or by GPCR kinases (GRK) (Lohse, 1993; Premont, 1995). Currently, GRKs form a family of six such kinases (GRK1-6) that can phosphorylate serine and/or threonine residues in the carboxyl-terminal domains of ligand-occupied GPCRs (Premont, 1995).

To test the hypothesis that GRK-mediated phosphorylation of the hEP4-R carboxyl-terminal domain is responsible for receptor desensitization, hEP4-R (which may activate PKA), rEP3-R, and the chimeric rEP3hEP4-Ct-R (Ct indicates the carboxyl-terminal domain), which do not activate second messenger-dependent kinases, were tagged with a FLAG-epitope and expressed in COS-7 cells. Receptor phosphorylation and sequestration promoted by specific agonists or second messenger kinase activators, such as phorbol-12-myristate-13-acetate (PMA) or forskolin, were then investigated.

The FLAG-hEP4-R and the FLAG-rEP3hEP4-Ct-R but not the FLAG-rEP3-R were phosphorylated by agonist stimulation. The FLAG-rEP3hEP4-Ct-R phosphorylation was augmented by GRK overexpression. In addition, GRK overexpression enhanced agonist-induced FLAG-rEP3hEP4-Ct-R sequestration. These results indicate that GRK-mediated phosphorylation of the carboxyl-terminal domain of the hEP4-R in the FLAG-rEP3hEP4-Ct-R is possibly involved in the sequestration of this receptor.

	Experimental Procedures

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Materials. All materials were of analytical grade and from commercial sources. M&B28767 was a generous gift from Rhone-Poulenc Rorer (Dagenham, United Kingdom). [³H]PGE₂ was obtained from Amersham (Braunschweig, Germany); unlabeled PGE₂, forskolin, 3-isobutyl-1-methylxanthine (IBMX), and PMA were purchased from Calbiochem-Novabiochem (Bad Soden, Germany). Cell culture media were obtained from Gibco-BRL (Eggenstein, Germany) and ³²P_i from ICN (Meckenheim, Germany). Primers (Table 1) were synthesized by NAPS (Göttingen, Germany). Sepharose Cl-4B, protein-G Sepharose FF, glutathione agarose, and BrCN-activated Sepharose were purchased from Pharmacia (Freiburg, Germany). Trypsin (10,800 U/mg) and monoclonal antibody (mAb) factor FLAG-M2 were from Sigma Chemical Co. (Heidelberg, Germany). The sources of other materials are given in the text.

View this table: [in this window] [in a new window]   TABLE 1 Sequence and location of the PCR primers used to incorporate the FLAG-epitope into the rEP3, hEP4-R- and the hybrid rEP3hEP4-Ct receptors and to amplify the cDNA for the hEP4-R carboxyl-terminal domain. The locations given are the sequence positions in the data files retrieved from GenBank under the accession numbers indicated. The reverse primers are the sequences complementary to the indicated positions. Primers are shown in 5' to 3' direction.

FLAG-Epitope Tagging of the rEP3-R, hEP4-R, and Chimeric rEP3hEP4-Ct-R. Cloning of the rEP3-R (Neuschäfer-Rube, 1994) and hEP4-R (Blaschke, 1996) cDNAs was carried out as described previously. The cDNA for the chimeric rEP3hEP4-Ct-R was constructed by recombinant polymerase chain reaction (PCR) technology. The protocol for the construction of the rEP3hEP4-Ct-R cDNA has been described in detail elsewhere (Neuschäfer-Rube, 1997a). A FLAG-octapeptide sequence (N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C) recognized by the mAb FLAG-M2 was inserted after the initiator methionine of the rEP3-R, hEP4-R, and the rEP3hEP4-Ct-R by PCR using Silver Star Taq-polymerase (Eurogentec, Seraing, Belgium). The forward primers were FLAG-EP4 for the hEP4-R cDNA and FLAG-EP3 for the two other cDNAs (Table 1) The reverse primers were P2 for the rEP3-R, P3 for the rEP3hEP4-Ct-R, and P4 for the hEP4-R (Table 1). All PCRs were performed with 10-ng template (pcDNAI/rEP3-R, pcDNAI/rEP3hEP4-Ct-R, and pcDNA/AMP hEP4-R) and 35 cycles of the following temperature profile: 1 min at 95°C, 1 min at 60°C, and 2 min at 72°C. The resultant cDNA fragments for the FLAG-rEP3-R and the FLAG-rEP3hEP4-Ct-R were cloned with the T/A cloning method into PUC57/T (MBI-Fermentas, Vilnius, Lithuania) and verified by DNA sequencing. The 1100-base-pair (bp) NotI/XbaI FLAG-rEP3-R fragment and the 1600-bp NotI FLAG-rEP3hEP-Ct-R fragment were further subcloned in the eukaryotic expression vector pcDNAI (Invitrogen, de Schelp, The Netherlands). The cDNA fragment for the FLAG-hEP4-R was cleaved with HindIII and XbaI and cloned into pcDNAI/AMP.

Cell Culture and Transfection. COS-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal calf serum (FCS) and penicillin (100 units/ml) and streptomycin (100 µg/ml) as antibiotics. COS-7 cells were seeded at a density of 1 × 10⁶ cells/10-cm-diameter plate and transiently transfected after 24 h using the DEAE-dextran method (Levesque, 1991) with pcDNAI/FLAG-rRP3-R, pcDNA/AMP/FLAG-hEP4-R, or pcDNAI/FLAG-rEP3hEP4-Ct-R (5 µg/plate) alone or together with pcDNAI or pcDNAI expression vectors for GRK2, GRK3, GRK5, or GRK6 (5 µg/plate) (Oppermann, 1996a). Twelve hours after transfection, cells were split into 6-well plates at a density of 2 × 10⁵ cells/well and assays were performed 72 h after transfection.

Membrane Isolation and PGE₂ Binding Assay. For membrane preparations, transfected cells were scraped into a homogenization buffer containing 25 mM Tris·HCl (pH 7.5) 250 mM sucrose, 10 mM MgCl₂, 1 mM EDTA and 0.2 mM Pefabloc SC (Biomol, Hamburg, Germany), 10 µg/ml leupeptin, and 10 µg/ml soybean trypsin-inhibitor as protease inhibitors. After homogenization of the cells in a Dounce homogenizer, a crude membrane fraction was prepared by centrifugation of the homogenate at 100,000g. The resulting pellet was suspended in binding buffer containing 25 mM 4-morpholine-ethanesulfonic acid/NaOH (MES/OH) (pH 6.2), 10 mM MgCl₂, and 1 mM EDTA and stored at 70°C. For PGE₂ binding, membranes (20-50 µg of protein) were incubated with 5 nM [³H]PGE₂ in 100 µl of binding buffer for 1 h at 20°C. Nonspecific binding was determined in the presence of 25 µM PGE₂. Bound and unbound ligands were separated by rapid vacuum filtration through GF 52 filters (Schleicher & Schüll, Dassel, Germany). Filters were washed five times with 4 ml of ice-cold binding buffer. Radioactivity retained on the filter was counted in 5 ml of Hydroluma (Baker, Deventer, Netherlands). Binding constants were calculated by nonlinear regression analysis (LIGAND; Rovati, 1988).

Generation of a glutathione S-transferase (GST)/hEP4-R-Ct Fusion Protein. A cDNA fragment encoding the carboxyl-terminal domain of the hEP4-R from the end of the seventh transmembrane domain was amplified by PCR using Silver Star Taq-polymerase. The forward primer was EP4-Ct F (Table 1) and the reverse primer was P3. PCR was performed with 10-ng template (pcDNA/AMP/FLAG-hEP4-R) and 35 cycles of the following temperature profile: 1 min at 95°C, 1 min at 60°C, and 2 min at 72°C. The resultant 521-bp cDNA fragment was cloned blunt-end into PUC18 (Pharmacia) and sequenced. The cDNA fragment was further subcloned into the BamHI/EcoRI site of the prokaryotic expression vector pGEX-5X-3 (Pharmacia) to fuse the hEP4-R carboxyl-terminal domain to the carboxyl terminus of the isopropyl--D-thiogalactopyranoside (IPTG)-inducible GST that was encoded by the vector. Escherichia coli strain BL-21 was transformed with this fusion-plasmid. Synthesis of the fusion protein was induced by 1 mM isopropyl--D-thiogalactopyranoside for 5 h. The fusion protein was enriched from crude cell extracts by glutathione-agarose affinity chromatography according to the manufacturer's instructions and purified to apparent homogeneity by preparative SDS-polyacrylamide gel electrophoresis (PAGE) using the PrepCell 491 (Bio-Rad, Munich, Germany). The purified GST-hEP4-R-Ct fusion protein was concentrated using centricon 10 (Amicon, Beverly, MA), dialyzed against PBS, and used as antigen to raise polyclonal antibodies in rabbits.

Purification of Polyclonal Anti-hEP4-R-Ct IgG. Four weeks after the last immunization, the rabbit was bled. IgG was purified on protein G Sepharose FF-beads. IgGs were eluted with 50 mM glycine/HCl (pH 2.7) neutralized and dialyzed against PBS. Antibodies against GST were absorbed on GST-Sepharose, which was prepared by coupling recombinant GST to BrCN-activated Sepharose (10 mg of GST/ml of Sepharose).

Western Blotting. Membrane proteins (20-50 µg) of transfected or control cells were solubilized in Laemmli sample buffer under reducing or nonreducing conditions for 60 min at 37°C and 10 min at 60°C with vigorous shaking, separated on 10% SDS-polyacrylamide gels, and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA) by semidry blotting. The PVDF membrane was first blocked in 5% (w/v) skim milk in PBS, 0.1% (v/v) Tween 20 (PBS-T), and FLAG-tagged receptor proteins were detected by treating immunoblots overnight with 0.3 µg/ml mAb FLAG-M2 in 1% (w/v) skim milk in PBS-T at 4°C followed by horseradish peroxidase-conjugated antimouse IgG (1:20,000 dilution; Bio-Rad). FLAG-hEP4-R was also detected by overnight incubation with 0.5 µg/ml rabbit anti-hEP4-R-Ct IgG in 1% (w/v) skim milk in PBS-T at 4°C followed by horseradish peroxidase-conjugated antirabbit IgG (1:10,000 dilution; Amersham) for 60 min at room temperature. Antigen-antibody complexes were visualized with the enhanced chemiluminescence system (Amersham) according to the instructions of the company.

Intact Cell Phosphorylation. Transfected COS-7 cells in 6-well plates were washed two times with phosphate-free DMEM and prelabeled for 60 min with 150 µCi/ml [³²P]orthophosphate in 500 µl of the same medium containing 10% (v/v) FCS that had been dialyzed extensively against 10 mM HEPES buffer (pH 7.5) containing 150 mM sodium chloride. After cell-labeling, various agents, in a volume of 500 µl in phosphate-free, 10% (v/v) dialyzed FCS containing DMEM, were added for 10 min at 37°C unless otherwise indicated. Where indicated, cells were treated with 400 nM staurosporine 20 min before stimulation. After stimulation, the medium was removed and the cells were washed twice with ice-cold PBS before immunoprecipitation.

Tryptic Receptor Cleavage in Intact Cells. Intact naive or PGE₂-stimulated cells expressing FLAG-hEP4-R were treated for 5 min at 37°C with 400 µl of 0.25% (w/v) trypsin in PBS to cleave trypsin cleavage sites in the extracellular domains. Digestion was stopped by the addition of 400 µl of FCS. Cells were transferred to microfuge tubes and collected by centrifugation (5 min at 160g) to remove tryptic activity before receptor solubilization.

Immunoprecipitation. For immunoprecipitation, cells expressing FLAG-rEP3-R or the FLAG-rEP3hEP4-Ct-R were scraped into lysis buffer (50 mM HEPES, pH 7.5, 5 mM EDTA, 10 mM sodium fluoride and 10 mM sodium pyrophosphate, with 0.2 mM Pefabloc SC), 10 µg/ml leupeptin, and 10 µg/ml soybean trypsin-inhibitor as protease inhibitors, transferred to microfuge tubes, and lysed by vigorous vortexing. Membranes were collected by centrifugation (10 min at 14,000g) and solubilized in 40 µl of lysis buffer containing 1% (w/v) SDS for 15 min at 65°C. After chilling to 4°C, 760 µl of concentrated detergent/salt solution was added to yield a final concentration of 1% (v/v) Triton X-100, 0.05% (w/v) SDS and 150 mM sodium chloride in lysis buffer (IP buffer). Cells expressing FLAG-hEP4-R were directly scraped in 800 µl of IP buffer and lysed by vigorous vortexing. Insoluble material was removed by centrifugation (30 min at 20,000g), and the supernatant was precleared with 100 µl of 10% (v/v) Sepharose 4B in IP buffer containing 0.1% (w/v) bovine serum albumin for 60 min at 4°C. The precleared supernatant was incubated for 2 h at 4°C with an immune complex of 15 µg of mAb FLAG-M2 or anti-hEP4-R-Ct IgG and 100 µl of 10% (v/v) protein-G Sepharose FF in IP buffer containing 0.1% (w/v) bovine serum albumin, which was preformed by incubation for 60 min at 4°C. Immune complexes were collected by centrifugation and washed five times with ice-cold IP buffer. Samples were suspended in Laemmli sample buffer and prepared for SDS-PAGE as outlined above.

Sequestration Assay. For receptor sequestration studies, transfected cells were washed three times with 5 ml of HEPES buffer (pH 7.4) containing 140 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl₂, 1.2 mM KH₂PO₄, 11 mM glucose, and 15 mM HEPES (incubation buffer) and then preincubated for 10 min in the same buffer with or without 100 nM M&B28767. The agonist was removed by two washes with incubation buffer, an acid wash with 3 ml of 50 mM glycine and 150 mM NaCl (pH 3) for 1 min, and an additional two washes with incubation buffer. Cells were then detached from the tissue culture plates with 250 µl of ice-cold Ca²⁺-free incubation buffer containing 1 mM EDTA. Of this cell suspension, 50 µl were incubated in a total volume of 100 µl with 5 nM [³H]PGE₂ for 30 min at 37°C. The reaction was stopped and cell-bound radioactivity was measured as outlined above in Membrane Isolation and PGE₂ Binding Assay.

	Results

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Expression of FLAG-rEP3-R, FLAG-hEP4-R, and FLAG-rEP3hEP4-Ct-R in COS-7 cells. COS-7 cells were transiently transfected with the cDNAs for the FLAG-rEP3-R, FLAG-hEP4-R, and FLAG-rEP3hEP4-Ct-R cloned in the eukaryotic expression vectors pcDNAI or pcDNA/AMP. Competition binding studies with membrane preparations of transfected cells showed that all receptors had a similar affinity for PGE₂ (FLAG-rEP3-R, K_d = 11 ± 1 nM; FLAG-hEP4-R, K_d = 5.9 ± 3 nM; FLAG-rEP3hEP4-Ct-R, K_d = 12 ± 4 nM; not shown). The K_d values were in the same range as those of the untagged wild-type receptors (Neuschäfer-Rube, 1994, 1997a), which indicates that the amino-terminal FLAG-tag did not alter the receptor binding properties. FLAG-rEP3-R and FLAG-rEP3hEP4-Ct-R were expressed to a comparable very high level (FLAG-rEP3-R, B_max = 7.2 ± 0.3 pmol/mg of protein; FLAG-rEP3hEP4-Ct-R, B_max = 5 ± 1 pmol/mg of protein) whereas FLAG-hEP4-R expression was somewhat lower (B_max 0.9 ± 0.2 pmol/mg of protein).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Detection of FLAG-rEP3-R, FLAG-rEP3hEP4-Ct-R, and FLAG-hEP4-R by immunoblotting. Membrane proteins (20-30 µg/lane) of control and transfected cells were resolved by SDS-PAGE under reducing or nonreducing conditions and transferred to PVDF membrane. Receptor proteins were detected with a sandwich of the mAb FLAG-M2 (A) or polyclonal anti-hEP4-R-Ct IgG (B) and horseradish-peroxidase-conjugated antimouse or antirabbit antiserum visualized with enhanced chemiluminescence as described in Experimental Procedures.

Agonist-Induced Phosphorylation of the FLAG-hEP4-R and the FLAG-rEP3hEP4-Ct-R. To study agonist-induced phosphorylation of the FLAG-rEP3-R, FLAG-hEP4-R, and FLAG-rEP3hEP4-Ct-R, COS-7 cells expressing FLAG-tagged receptors were labeled with ³²P_i and stimulated either with the EP3-R agonist M&B28767 (100 nM; FLAG-rEP3-R and FLAG-rEP3hEP4-Ct-R) or PGE₂ (1 µM; FLAG-hEP4-R) for 10 min. A phosphoprotein was immunoprecipitated with the FLAG-M2 antibody from solubilized proteins of cells transfected with the FLAG-rEP3hEP4-Ct-R (Fig. 3C) and FLAG-hEP4-R (Fig. 3E). These proteins comigrated with the FLAG-rEP3hEP4-Ct-R and the FLAG-hEP4-R detected by Western blotting. Phosphorylation of the FLAG-rEP3hEP4-Ct-R and the FLAG-hEP4-R were significantly enhanced upon agonist-stimulation (i.e., after pretreatment with M&B28767 or PGE₂). In contrast, no phosphoprotein was immunoprecipitated from cells transfected with FLAG-rEP3-R either with or without agonist-stimulation (Fig. 3A).

View larger version (62K): [in this window] [in a new window]   Fig. 3.   Phosphorylation of the FLAG-rEP3-R, FLAG-rEP3hEP4-Ct-R, and FLAG-hEP4-R by PGE2 or M&B28767 and activators and inhibitors of second-messenger-dependent kinases. 32Pi-labeled COS-7 cells expressing the FLAG-rEP3-R, FLAG-rEP3hEP4-Ct-R, or the FLAG-hEP4-R were preincubated without or with 400 nM staurosporine for 20 min at 37°C before cellular stimulation. Cells were then stimulated for 10 min at 37°C with 1 µM PGE2, 100 nM M&B28767, 2 mM PMA, or 50 µM forskolin + 1 mM IBMX. Receptors were immunoprecipitated with FLAG-M2 antibody, resolved under nonreducing conditions by SDS-PAGE, and phosphorylation was determined by PhosphorImager analysis after 2 days. Top, a representative PhosphorImager analysis; bottom, the quantitative analysis from pooled data from three independent experiments. Receptor phosphorylation is expressed as a percentage of phosphorylation of untreated cells in absence of staurosporine. Data are means ± S.E.; *p < .05 compared with basal levels.

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Concentration dependence of M&B28767-stimulated FLAG-rEP3hEP4-Ct-R phosphorylation. 32Pi-labeled COS-7 cells expressing the FLAG-rEP3hEP4-Ct-R were stimulated for 10 min at 37°C with the concentrations of M&B28767 indicated. Receptor phosphorylation was analyzed as described in the legend to Fig. 3, untransfected COS-7 cells. Top, a representative PhosphorImager analysis. Bottom, the quantitative analysis from pooled data from three independent experiments. Receptor phosphorylation is expressed as a percentage of phosphorylation in unstimulated FLAG-rEP3hEP4-Ct-R cells. Data are means ± S.E.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Time-dependence of M&B28767-stimulated FLAG-rEP3hEP4-Ct-R phosphorylation. 32Pi-labeled COS-7 cells expressing the FLAG-rEP3hEP4-Ct-R were stimulated for the times indicated at 37°C with 100 nM M&B28767. Receptors phosphorylation was analyzed as described in the legend to Fig. 3, untransfected COS-7 cells. Top, a representative PhosphorImager analysis. Bottom, the quantitative analysis from pooled data from three independent experiments. Receptor phosphorylation is expressed as a percentage of phosphorylation in unstimulated FLAG-rEP3hEP4-Ct-R cells. Data are means ± S.E.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   PGE2-induced phosphorylation of FLAG-hEP4-R carboxyl-terminal domain. 32Pi-labeled FLAG-hEP4-R-expressing COS-7 cells were stimulated with 1 µM PGE2 for 10 min and then treated with 0.25% trypsin for 5 min at 37°C. Trypsin was removed from intact cells. Receptor and receptor fragments were solubilized, immunoprecipitated with polyclonal anti-hEP4-R-Ct IgG, and resolved by SDS-PAGE, and phosphorylation was determined by PhosphorImager analysis after 2 days. The figure shows one of two identical experiments performed.

Inhibition by Staurosporine of Second Messenger Kinase but Not Agonist-Induced Phosphorylation of the FLAG-rEP3hEP4-Ct-R and FLAG-hEP4-R. To investigate which kinases effect agonist-induced FLAG-rEP3hEP4-Ct-R and FLAG-hEP4-R phosphorylation or are capable of phosphorylating the FLAG-rEP3-R, cells were treated with agonist or with both specific activators and inhibitors of the second messenger-dependent kinases PKC or PKA. Forskolin, an activator that increases intracellular cAMP concentration and activates PKA, had no significant effect on either FLAG-rEP3-R, FLAG-rEP3hEP4-Ct-R, or FLAG-hEP4-R phosphorylation (Fig. 3, A, C, and E). In contrast, the phorbol ester PMA, a potent PKC activator, led to a slight increase in FLAG-rEP3-R phosphorylation (Fig. 3A) and induced a strong phosphorylation of the FLAG-rEP3hEP4-Ct-R and the FLAG-hEP4-R (Fig. 3, C and E). PMA induced FLAG-rEP3hEP4-Ct-R and FLAG-hEP4-R phosphorylation to a comparable extent as the agonists M&B28767 and PGE₂.

Enhanced Agonist-Promoted Phosphorylation of the FLAG-rEP3hEP4-Ct-R by GRK Overexpression. As second messenger-activated kinases seemed not to be involved in agonist-induced FLAG-hEP4-R and FLAG-rEP3hEP4-Ct-R phosphorylation, it was assumed that the activated receptor serves as a substrate for GRK. To test this hypothesis, GRKs 2, 3, 5, and 6 were co-overexpressed with the FLAG-rEP3hEP4-Ct-R and agonist-induced receptor phosphorylation was determined. Western-blotting with antibodies specific for GRKs 2 or 3, or GRKs 5 or 6 (Oppermann, 1996a) revealed that COS-7 cells contained little intrinsic GRKs and that all GRKs were over-expressed to a comparably high level (not shown). GRK over-expression did not influence maximal [³H]PGE₂ binding by the FLAG-rEP3hEP4-Ct-R (not shown). Overexpression of GRKs 2, 3, and 5 augmented basal and agonist-stimulated FLAG-rEP3hEP4-Ct-R phosphorylation by about 2-fold, whereas the ratio between basal and agonist-stimulated receptor phosphorylation remained constant (Fig. 7). GRK 6 was less effective. There was no significant difference in the capacity of GRKs 2, 3, and 5 to phosphorylate the receptor. These results show that under the conditions used in this cellular assay, the FLAG-rEP3hEP4-Ct-R seemed susceptible to agonist-promoted phosphorylation mediated by GRKs.

View larger version (61K): [in this window] [in a new window]   Fig. 7.   Augmentation of M&B28767-stimulated FLAG-rEP3hEP4-Ct-R phosphorylation by GRK co-overexpression. COS-7 cells were cotransfected with pcDNAI/FLAG-rEP3hEP4-Ct-R and either pcDNA I or pcDNAI expression vectors for GRK2, GRK3, GRK5, or GRK6. Cells were metabolically labeled with 32Pi and stimulated with 100 nM M&B28767 for 10 min. Receptor phosphorylation was analyzed as described in the legend to Fig. 3. Top, a representative PhosphorImager analysis. Bottom, the quantitative analysis from pooled data from three independent experiments. Receptor phosphorylation is expressed as a percentage of phosphorylation in unstimulated FLAG-rEP3hEP4-Ct-R cells. Data are means ± S.E. *, p < .05 compared with stimulated cells without GRK overexpression; , p < .05 compared with naive control cells without GRK overexpression.

Enhanced Sequestration of the FLAG-rEP3hEP4-Ct-R by GRK Overexpression. To correlate GRK-mediated FLAG-rEP3hEP4-Ct-R phosphorylation with agonist-induced desensitization of the receptor previously observed in HepG2/rEP3hEP4-Ct-R cells (Neuschäfer-Rube, 1997b), GRK2 was coexpressed with the FLAG-rEP3hEP4-Ct-R or the nondesensitizable FLAG-EP3-R as an internal control and sequestration of receptors as a response to agonist-exposure was examined. Receptor sequestration was measured as a loss of [³H]PGE₂ binding sites on the plasma membrane of transfected COS-7 cells after a 10-min preincubation period with 100 nM M&B28767. In cells transfected with the FLAG-rEP3-R, agonist exposure did not cause significant receptor sequestration either in the absence or in the presence of overexpressed GRK2 (Fig. 8). In contrast, preincubation of FLAG-rEP3hEP4-Ct-R-expressing cells with the agonist M&B28767 significantly decreased the number of [³H]PGE₂ binding sites on the cell surface by about 15%. This agonist-induced receptor sequestration was augmented to about 30% when GRK2 was co-overexpressed with the FLAG-rEP3hEP4-Ct-R (Fig. 8). These results indicate that there may be a direct link between GRK-mediated phosphorylation and sequestration of the FLAG-rEP3hEP4-Ct-R induced by the EP3-R agonist M&B28767.

View larger version (44K): [in this window] [in a new window]   Fig. 8.   Augmentation by GRK2 of M&B28767-stimulated FLAG-rEP3hEP4-Ct-R sequestration. COS-7 cells were transfected with pcDNAI/FLAG-rEP3-R or pcDNAI/FLAG-rEP3hEP4-Ct-R and either pcDNA I (Control) or pcDNAI/GRK2 (GRK2). In a preincubation period, cells were exposed to 100 nM M&B28767 for 10 min. The agonist was then removed by an acid wash followed by extensive washing with incubation medium (see Experimental Procedures). For binding assays, cells were detached from the tissue culture plates with an EDTA buffer. Binding of 5 nM [3H]PGE2 was measured in the cell suspension after 30 min at 37° with 25 µM unlabelled PGE2 to determine nonspecific binding. Values are mean percentages ± S.E. of control cells that were not exposed to the agonist in the preincubation period. *, p < .05; , p < .05 compared with cells without GRK2 overexpression.

	Discussion

Top Summary Introduction Experimental Procedures Results Discussion References

The biological effects elicited by prostanoid receptors, like those of many other GPCRs, are regulated by an attenuation of their intracellular signal transduction in response to short- or long-term ligand exposure (Nordstedt, 1988; Sakai, 1996). Recent studies showed that the carboxyl-terminal domain of prostanoid receptors plays an important role in this desensitization process. Variation of the EP3-R carboxyl-terminal domain created by alternative splicing resulted in different rapid agonist-induced receptor desensitization (Negishi, 1993b). The role of the carboxyl-terminal domain in desensitization is also underscored by the recent finding of a loss of desensitization in a carboxyl-terminally truncated EP4-R (Bastepe and Ashby, 1997). As previously shown, the carboxyl-terminal domain of the EP4-R is not only necessary but also sufficient to confer rapid agonist-induced desensitization in a hybrid receptor with the nondesensitizable rEP3-R (Fig. 1) (Neuschäfer-Rube, 1997a). By contrast, the third intracellular loop of the EP4-R was neither necessary nor sufficient to mediate agonist-induced desensitization (Neuschäfer-Rube, 1997a).

Desensitization by Phosphorylation. Agonist-induced receptor desensitization of GPCRs may be mediated by receptor phosphorylation. Several lines of evidence support this postulated link between agonist-stimulated desensitization and phosphorylation also for the hEP4-R and the rEP3hEP4-Ct-R. First, PGE₂ induced both the desensitization of the hEP4-R (Bastepe, 1997) and phosphorylation of the FLAG-hEP4-R expressed in COS-7 cells (Fig. 3E). The EP3-R agonist M&B28767 did not induce desensitization of the rEP3-R, stably expressed in HepG2 cells (Neuschäfer-Rube, 1997b), and was also inefficient in the induction of FLAG-rEP3-R phosphorylation (Fig. 3A). By contrast, the agonist stimulated both the desensitization of the rEP3hEP4-Ct-R in HepG2 cells (Neuschäfer-Rube, 1997b) and phosphorylation of the FLAG-rEP3hEP4-Ct-R transiently expressed in COS-7 cells (Fig. 3C). Second, the time course of the M&B28767-induced FLAG-rEP3hEP4-Ct-R phosphorylation (Fig. 5) fit well with the kinetics of agonist-induced rEP3hEP4-Ct-R desensitization, measured as a reduced inhibition of forskolin-stimulated cAMP formation (Neuschäfer-Rube, 1997b). Both effects were rapid, with an onset after 1 min and a maximum at 10 to 30 min.

Phosphorylation Sites. Because the only structural difference between the rEP3-R and the rEP3hEP4-Ct-R is the carboxyl-terminal domain, agonist-induced phosphorylation in the latter receptor most likely occurred in the hEP4-R carboxyl-terminal domain. This assumption was supported by the finding that a tryptic fragment of the hEP4-R, which had an apparent molecular weight that corresponded to the predicted molecular mass of a tryptic fragment ranging from the third extracellular loop to the end of the carboxyl-terminal domain, was phosphorylated by PGE₂-stimulation (Fig. 6). However, additional phosphorylation, which depends on the presence of the hEP4-R carboxyl-terminal domain, of other parts of the receptor (i.e., Ser or Thr residues in the first, second, or third intracellular loops), cannot be ruled out. rEP3-R and hEP4-R carboxyl-terminal domains contain different numbers of target sequences for protein kinases. The rEP3-R carboxyl-terminal domain contains only five serines, two of which are potential PKC-phosphorylation sites [consensus sequence S/TXR (Pearson and Kemp, 1991)] and no threonine. By contrast, the carboxyl-terminal domain of the hEP4-R contains 27 serine and 10 threonine residues, five of which are potential PKC-phosphorylation sites and nine of which might be defined as potential phosphorylation sites for GRKs; however, GRKs have no strict recognition sequence, but they seem to prefer Ser or Thr that are preceded by an Asp or Glu at a distance of three amino acids (Onorato, 1991; Fig. 1).

Phosphorylation by GRKs. Agonist-dependent receptor phosphorylation could be mediated either through second messenger-dependent kinases or GRKs. There are examples for both mechanisms. The human prostacyclin receptor, which increases cAMP-formation at low iloprost concentrations and InsP₃-formation at high iloprost concentrations, was phosphorylated and desensitized by high iloprost concentrations only (Smyth, 1996). Phosphorylation and desensitization were inhibited by staurosporine, which implies a PKC-dependent mechanism. Conversely, GRK-dependent phosphorylation of Ser and Thr residues in the carboxyl-terminal domain has been shown to mediate receptor desensitization of the 1-adrenergic receptor (Freedman, 1995), the endothelin receptor (Freedman, 1997), the -opioid receptor (Pei, 1995), the thrombin receptor (Ishii, 1994), the type-1A angiotensin II receptor (Oppermann, 1996b), and the adenosine A3 receptor (Palmer, 1995), to name but a few. Several lines of evidence support the involvement of GRKs in agonist-stimulated hEP4-R and rEP3hEP4-Ct-R phosphorylation and desensitization: