©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning and Characterization of the Human Prostanoid DP Receptor (*)

(Received for publication, March 8, 1995)

Yves Boie Nicole Sawyer Deborah M. Slipetz Kathleen M. Metters Mark Abramovitz (§)

From theDepartment of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec H9R 4P8, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A cDNA encoding a functional human prostanoid DP (hDP) receptor has been constructed from a genomic clone and a fragment cloned by 3`-rapid amplification of cDNA ends-polymerase chain reaction. The hDP receptor consists of 359 amino acid residues with a predicted molecular mass of 40,276 and has the putative heptahelical transmembrane domains characteristic of G-protein-coupled receptors. The deduced amino acid sequence of the hDP receptor, when compared with all other members of the prostanoid receptor family, shows the highest degree of identity with the hIP and hEP(2) receptors, followed by the hEP(4) receptor. Radioreceptor binding studies using membranes prepared from mammalian COS-M6 cells transiently transfected with an expression vector containing the DP receptor cDNA showed that the rank order of affinities for prostaglandins and prostaglandin analogs, in competition for [^3H]prostaglandin D(2) (PGD(2)) specific binding sites, was as predicted for the DP receptor, with PGD(2) PGE(2) > PGF = iloprost > U46619. The signal transduction pathway of the cloned hDP receptor was studied by transfecting the hDP expression vector into HEK 293(EBNA) cells. Activation of the hDP receptor with PGD(2) resulted in an elevation of intracellular cAMP and in mobilization of Ca, but did not lead to generation of inositol 1,4,5-trisphosphate. Northern blot analysis of human tissues showed that the hDP receptor has a very discrete tissue distribution and was detectable only in retina and small intestine. In summary, we have cloned and expressed a functional cDNA for the hDP receptor.


INTRODUCTION

Prostaglandin D(2) (PGD(2)) (^1)is formed in a variety of tissues including brain, spleen, lung, bone marrow, stomach, skin (1, 2) , and also in mast cells. PGD(2) has been implicated in many physiological events both in the central nervous system and peripheral tissues. In the central nervous system, PGD(2) has been shown to affect the induction of sleep(3) , body temperature(4) , olfactory function (2 and references within), hormone release (2 and references within), and nociception (2 and references within). Since PGD(2) is the major prostanoid released from human mast cells upon immunological challenge(5) , it is also considered to be an important mediator in allergic disorders such as allergic rhinitis(6) . In addition, it has been tested as a potential intraocular pressure lowering agent for treatment of glaucoma(7, 8) . Other roles for PGD(2), such as effects on platelet aggregation (1 and references within), systemic vasodilation, pulmonary constriction, and bronchoconstriction (1 and references within) appear, however, to be restricted to specific species. In addition, several of the physiological effects assigned to PGD(2), including bronchoconstriction, may be attributable to the cross-reactivity of PGD(2) with the prostaglandin F and/or thromboxane A(2) receptors(9) .

The physiological and pathophysiological actions of PGD(2) are mediated through interaction with the prostanoid DP receptor. (^2)Specific binding sites for the DP receptor have been studied using membranes from human platelets (10) , rat brain(11) , human basophils(12) , and mouse mastocytoma P-815 cells(13) . Activation of the DP receptor has been shown to result in an increase in both intracellular cAMP ([cAMP]) (14, 15) and Ca ([Ca])(16) , and the receptor is potentially regulated by phosphorylation events(13) . DP receptors are thought to be localized in platelets (17 and references within), neutrophils(18, 19, 20) , non-chromaffin cells from adrenal medulla(16) , and smooth muscle cells from several tissues and nervous tissue, including the central nervous system(17) . However, the DP receptor is the least abundant of the prostanoid receptors and is, therefore, the least well characterized.

The study of the human prostanoid receptors is rapidly advancing with the recent cloning of the human (h) thromboxane A(2) (TP)(21) , prostaglandin F (FP)(22, 23) , and prostacyclin (IP) (24, 25, 26) receptors and four distinct subtypes of the prostaglandin E(2) (EP(1)(27) , EP(2)(28) , EP(3)(29, 30, 31) , and EP(4)(32, 33) (^3)) receptor. Here we report the cloning of the human prostaglandin D(2) (DP) receptor, and the nucleotide and deduced amino acid sequences are described. The radioligand binding and functional characteristics of the cloned and expressed hDP receptor have been addressed, in addition to its tissue distribution.


MATERIALS AND METHODS

Human Genomic Library Screening

In order to clone the hDP prostanoid receptor, a mouse DP receptor DNA probe was first generated using the polymerase chain reaction as described below. Two oligonucleotides based on the mouse DP receptor amino acid sequence (34) were designed and synthesized (Research Genetics, Cambridge, MA). The 5`-sense 128-fold degenerate oligonucleotide (5`-ATG AA(T,C) GA(G,A) (T,A)(G,C)I TA(T,C) (A,C)GI TG(T,C) CA-3`) was based on the first eight amino acids (MNESYRCQ), and the 3`-antisense 16-fold degenerate oligonucleotide (5`-AA (G,A)CA CCA IGT ICC IGG (G,A)CA (G,A)TA (T,C)TG-3`) was based on nine amino acids (QYCPGTWCF) which occur in the second extracellular loop of the mouse DP receptor. The polymerase chain reaction protocol for the amplification, performed on a DNA thermal cycler 480 (Perkin Elmer, Montreal, Canada) using Taq polymerase (Boehringer Mannheim, Laval, Quebec) and 1.5 µg of mouse genomic DNA (Clontech) as template, was as follows: denaturation at 95 °C for 60 s, annealing at 52 °C for 30 s, and extension at 72 °C for 40 s, for 35 cycles. The 549-bp amplified product was subsequently isolated from a low-melt agarose gel, random prime labeled (T7 Quick Prime Kit, Pharmacia, Baie D'urfe, Quebec) with [P]dCTP (DuPont NEN, Mississauga, Ontario), and added to a 50% formamide hybridization buffer to screen 1.2 10^6 plaques from a EMBL3 T7/SP6 Sau3AI human genomic library (Clontech). Two positive phage clones, hDPgc.1 and hDPgc.2, were plaque-purified, and DNA was prepared by the plate lysate method(35) .

Restriction mapping of hDPgc.1 and hDPgc.2 DNA was performed using XhoI, SacI, SfiI, EcoRI, XbaI, BamHI, HindIII, and Asp718I. Restriction digest analysis showed that both clones were identical. Southern blot analysis of the digested DNA was performed with either the P-labeled mouse DP genomic DNA fragment or a 29-mer 4-fold degenerate oligonucleotide, hDP-VII(+) (5`-ATIGTIGA(T,C)CCITGGATITT(T,C)ATIATITT-3`), based upon 10 amino acids from transmembrane domain (TMD) VII (IVDPWIFIIF) from the mouse DP receptor(34) . Results from the Southern analysis identified a 3-kb EcoRI fragment which hybridized with the 549-bp mouse DP DNA probe but not the oligonucleotide probe and, in addition, a 4.5-kb XhoI/EcoRI fragment originating from the 3`-end of the genomic insert which hybridized with the hDP-VII(+) oligonucleotide probe but not the mouse DP DNA probe. These fragments were isolated and subcloned into Bluescript pKS vectors (Stratagene) for sequence analysis using an ABI 373 Stretch automated sequencer. The first 1.5 kb of the 3-kb EcoRI fragment was sequenced on both strands using KS and SK primers or primers generated from the determined sequences. This sequence was found to contain the 5` end of the coding region up to and including an exon/intron splice site located near the end of TMD VI as well as part of the intron. The 3` end of the 4.5-kb XhoI/EcoRI fragment was sequenced as above and found to contain the continuation of the coding region from the end of TMD VI through to the end of TMD VII, which was also the end of the genomic clone.

3`-RACE-PCR Cloning

To obtain the missing 3`-coding region of the hDP receptor, 3`-RACE-PCR was performed on human small intestine, lung, thymus, kidney, and uterus cDNA libraries (Clontech). Anchored primers, complementary to gt10 or gt11 DNA sequences at their multiple cloning site junctions, and sense primers, AP-1 (5`-CCTGGCCATGGCACTGGAGTGCTGG-3`) (end of TMD III), AP-2 (5`-GCTGTGCAACCTCGGCGCCATGCGC-3`] (end of TMD V), and AP-3 (5`-CTCCGAGCCTTGCGATTTCTATCTG-3`) (beginning of TMD VII), based on the sequence obtained from the hDP receptor genomic clone were either purchased from Clontech or synthesized as above. The polymerase chain reaction protocol for all rounds of amplification was as follows: denaturation at 95 °C for 60 s, annealing at 55 °C for 60 s, and extension at 72 °C for 60 s, for 35 cycles. For the first reaction, 2 µl (10^7 plaque-forming units/µl) of the cDNA library was amplified using a gt primer and AP-1. For the second amplification, 1/50 (2 µl) of the first polymerase chain reaction was reamplified with the same gt primer and either AP-2 or AP-3. Amplified products were then analyzed by Southern blotting according to standard protocols (35) using the P-labeled oligonucleotide hDP-VII(+). Hybridizing polymerase chain reaction fragments were subsequently cloned into Bluescript vector pKS and sequenced as above. An amplified fragment (510 bp) from the small intestine cDNA library was found to extend an additional 157 bp (including an in-frame putative stop codon) as compared with the sequence at the 3` end of the hDP genomic clone. Shorter amplified fragments of similar size were also obtained from the lung and uterus cDNA libraries. The lung fragment contained only 53 bp of additional sequence information and did not extend to the putative stop codon.

Construction of a cDNA Encoding the Human DP Receptor

The following strategy was used in order to construct a cDNA containing the full-length coding region for hDP: the 5` end from the 3-kb EcoRI genomic fragment in pKS was digested with SmaI and PstI releasing a 0.75-kb fragment which encoded amino acid residues 1 to 232 of the hDP receptor. The 0.75-kb fragment was then ligated into the pKS construct, previously digested with SmaI and PstI, which contained the small intestine 3`-RACE-PCR fragment encoding amino acid residues 233 to 359. This construct was designated pKS-hDP.

Construction of pcDNA3-hDP and pCEP4-hDP Mammalian Expression Vectors

Digestion of pKS-hDP with NotI and XhoI released the hDP 1.2-kb cDNA which was subcloned into the same restriction sites of both the pcDNA3 and pCEP4 mammalian expression vectors (Invitrogen). The correct orientation was verified by BglII digestion. These vectors, termed pcDNA3-hDP and pCEP4-hDP, were subsequently used in transfection experiments for receptor binding and signal transduction assays, respectively.

Northern Blot Analysis

Poly(A) RNA samples (3 µg) from 19 different human tissues (Clontech) were subjected to electrophoresis in a 1.2% agarose gel containing 0.22 M formaldehyde and were then transferred to a nylon membrane (Nytran, Schleicher and Schuell). The nylon membrane was hybridized at 42 °C overnight in 50% formamide, 4 SSPE, 5 Denhardt's solution, 0.5% SDS, 100 mg/ml sonicated salmon sperm DNA containing 1 10^6 cpm/ml of a P-random-prime-labeled 0.9-kb SmaI hDP fragment originating from the 3-kb EcoRI genomic fragment. The nylon membrane was washed with 0.1 SSC containing 0.1% SDS at 55 °C and then exposed at -80 °C for 14 days with two intensifying screens.

pcDNA3-hDP Expression in COS-M6 Cells and [^3H]PGDBinding Assays

The pcDNA3-hDP vector was transfected into COS-M6 cells by cationic liposome-mediated transfer using LipofectAMINE reagent (36) (Life Technologies, Inc./BRL, Burlington, Ontario). The cells were maintained in culture for 48 h, harvested, and subjected to lysis by nitrogen cavitation(37) , and membranes were prepared by differential centrifugation (1000 g for 10 min, then 100,000 g for 30 min). [^3H]PGD(2) binding assays were performed in 10 mM BES/KOH (pH 7.0) containing 1 mM EDTA, 10 mM MnCl(2), 0.8 nM [^3H]PGD(2) (115 Ci/mmol; DuPont NEN), and 60 µg of protein from the 100,000 g membrane fraction. Incubations were conducted for 60 min at 30 °C prior to separation of the bound and free radioligand by rapid filtration through GF/B filters presoaked in 10 mM BES/KOH (pH 7.0) containing 0.01% (w/v) bovine serum albumin. Filters were washed with 16 ml of soaking buffer, and the residual [^3H]PGD(2) bound to the filter was determined by liquid scintillation counting. Specific binding was defined as the difference between total binding and nonspecific binding which was determined in the presence of 1 µM PGD(2).

pCEP4-DP Expression in HEK 293(EBNA) Cells

Stable expression of the hDP receptor was achieved by transfection of the pCEP4-hDP plasmid into HEK 293(EBNA) cells (maintained under selection with GENETICIN® (G418) (Life Technologies, Inc.)), using LipofectAMINE® reagent as described above. Cells were maintained in culture for 48 h post-transfection and then grown in the presence of 200 µg/ml hygromycin B (Calbiochem) for 3-4 weeks to select for resistant cells expressing the hDP receptor. These cells were subsequently used for signal transduction assays.

cAMP Assays in HEK 293(EBNA) Cells Expressing the Human DP Receptor

hDP-HEK 293(EBNA) cells were maintained in culture in Dulbecco's modified Eagle's medium growth medium (Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 20 units/ml penicillin G, 20 µg/ml streptomycin sulfate, 250 µg/ml G418, and 200 µg/ml hygromycin B). The medium was replaced 16-24 h prior to harvesting the cells (at approximately 80% confluence). The cells were harvested by incubation in enzyme-free cell dissociation buffer (Life Technologies, Inc./BRL, Burlington, Ontario), washed with HEPES-buffered Krebs-Ringer solution (1.25 mM MgSO(4), 1.5 mM CaCl(2), 5 mM KCl, 124 mM NaCl, 8 mM glucose, 1.25 mM KH(2)PO(4), and 25 mM HEPES/KOH (pH 7.4)) and then resuspended in the same buffer. The cAMP assay was performed in a final volume of 0.2 ml of HEPES-buffer Krebs-Ringer solution containing a 100 µM concentration of the phosphodiesterase type IV inhibitor RO-20-1724 (BIOMOL Research Laboratories, Plymouth Meeting, PA) to prevent hydrolysis of cAMP. RO-20-1724 and the compounds under evaluation were added to the incubation mixture in dimethyl sulfoxide (Me(2)SO) or ethanol to a final vehicle concentration, 0.3% (v/v), which was kept constant in all samples. The reaction was initiated by the addition of 1.5 10^5 cells per incubation. Samples were incubated at 37 °C for 15 min, and the reaction was then terminated by immersing the samples in boiling water for 3 min. Cell viability was always geq96% as determined by trypan blue exclusion. Measurement of cAMP was performed by radioimmunoassay using I-cAMP (Amersham, Oakville, Ontario).

Intracellular Calcium Assays in HEK 293(EBNA) Cells Expressing the Human DP Receptor

Intracellular Ca ([Ca]) measurements were performed using the hDP-HEK 293(EBNA) stable cell line. The cells were harvested with enzyme-free cell dissociation buffer, recovered by centrifugation at 300 g for 5 min at 22 °C, and resuspended at 2 10^6 cells/ml in Dulbecco's modified Eagle's medium growth medium without G418 or hygromycin B. The cells were incubated at 37 °C, 6% CO(2) for 45 min with 2 µM fura-2/AM (Molecular Probes). Unincorporated fura-2/AM was removed by three washes in Hanks' buffered saline solution (1.3 mM CaCl(2), 5 mM KCl, 0.3 mM KH(2)PO(4), 0.9 mM MgCl(2), 138 mM NaCl, 4 mM NaHCO(3), 0.3 mM Na(2)HPO(4), 5.6 mM glucose) followed by centrifugation at 300 g for 5 min at 22 °C. The cells were then resuspended at a final concentration of 10^6 cells/ml in Hanks' buffered saline solution. [Ca] was measured following an agonist challenge of 2 10^6 fura-2/AM loaded cells using a Perkin-Elmer LS-5 spectrophotometer essentially as described previously(38) . Cells were viable at the end of each experiment, as monitored by the large increase in [Ca] observed upon the addition of a 1 µM concentration of the calcium ionophore ionomycin (Sigma) followed by quenching of the [Ca] response with 1 mM EDTA.

Inositol 1,4,5-Trisphosphate Assays in HEK 293(EBNA) Cells Expressing the Human DP Receptor

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) measurements were performed using the hDP-HEK 293(EBNA) and hEP(1)-HEK 293(EBNA) stable cell lines and control HEK 293(EBNA) cells. The cells were harvested by incubation in enzyme-free cell dissociation buffer, washed with Hanks' buffered saline solution, and then resuspended in the same buffer. The Ins(1,4,5)P(3) assay was performed in a final volume of 0.4 ml of Hanks' buffered saline solution containing 10 mM LiCl. The compounds under evaluation were added to the incubation mixture in Me(2)SO(4) to a final concentration of 0.5% (v/v), which was kept constant in all samples. The reaction was started with the addition of 5 10^5 cells per incubation. Samples were incubated at 37 °C for 10 min, and the reaction was terminated by the addition of 20% ice-cold trichloroacetic acid. Ins(1,4,5)P(3) was extracted and measured by [^3H]Ins(1,4,5)P(3) radioreceptor assay (DuPont NEN) according to the manufacturer's instructions except that the bound and free radioligand was separated by rapid filtration through GF/C filters presoaked in 10 mM HEPES/KOH (pH 8.0). Filters were washed with 4 ml of 10 mM HEPES/KOH (pH 8.0), and the residual [^3H]Ins(1,4,5)P(3) bound to the filter was determined by liquid scintillation counting.


RESULTS AND DISCUSSION

Cloning and Construction of a cDNA Coding for the Human DP Receptor

In order to clone the hDP receptor, a mouse DP receptor fragment was first amplified from mouse genomic DNA using the polymerase chain reaction as described under ``Materials and Methods.'' This mouse DP DNA probe was subsequently used to screen a human genomic library, and a hDP genomic clone was identified. The clone was, however, missing coding information from the carboxyl-terminal end of the receptor. The additional coding sequence was derived by 3`-RACE-PCR of a human small intestine cDNA library which made it possible to construct the entire coding region for the hDP receptor from the hDP gene and the 3`-RACE-PCR fragment (Fig.1A). The hDP genomic clone was found to contain an intron with consensus donor and acceptor splice site sequences (39) near the end of TMD VI (Fig.1B). This position is identical with that found in the mouse DP gene (Hirada) and homologous in position to that of the hTP receptor gene (40) as well as other members of the human prostanoid receptor gene family. (^4)This suggests that the prostanoid receptor family arose from a single ancestral gene.


Figure 1: Nucleotide and deduced amino acid sequence of the human prostanoid DP receptor. A, the relationship between the genomic clone, hDPgc.1, and the 3`-RACE-PCR product derived from the small intestine cDNA library is shown. The coding regions in hDPgc.1 are indicated by open boxes and expanded directly below with the intron removed and the seven putative transmembrane domains numbered in Roman numerals. The full-length coding region of the hDP receptor was obtained by joining the SmaI/PstI genomic fragment to the 3`-RACE-PCR fragment at the common PstI site (see ``Materials and Methods'' for details). B, the nucleotide and deduced amino acid sequences of the human DP receptor gene. The nucleotides and amino acids, shown below, are numbered according to the initiator methionine. The end of the genomic clone is indicated by an arrow above the last nucleotide. The upstream in-frame stop codon, TAG, at position -171, is in bold, and an asterisk denotes the position of the translation stop codon. The nucleotides in the intron are in small letters. The positions of the putative transmembrane segments I-VII (based on the hydropathicity profile) are underlined. N-Glycosylation sites (up triangle, filled) and potential protein kinase C phosphorylation sites (black square) are indicated below the amino acids. The two SmaI and one PstI restriction sites are double-underlined.



The hDP cDNA contains a 1,080-bp open reading frame coding for a 359-amino-acid protein (Fig.1B) with a calculated molecular mass of 40,276. The ATG assigned as the initiator codon matches reasonably well to the Kozak consensus sequence (4/5 nucleotides) for translation initiation(41) . The genomic clone also contains an in-frame TAG stop codon 171 bp upstream of the predicted start codon (Fig.1B). The hDP protein contains three potential N-glycosylation sites, Asn-10, Asn-90, and Asn-297, in the NH(2) terminus and the first and third extracellular loops, respectively. There are also two potential protein kinase C phosphorylation sites, Ser-50 and Thr-145, located in the putative first and second cytoplasmic loops, respectively. In addition, there are six more potential phosphorylation sites in the carboxyl-terminal tail, comprised of five serine residues and one threonine residue which may be important in receptor desensitization(42) . Hydropathy analysis (43) of the deduced amino acid sequence of the hDP receptor confirmed the presence of the seven putative TMDs which are characteristic of G-protein-coupled receptors (GPCR)(44) . The hDP receptor also shares limited, but significant, amino acid identity with members of the rhodopsin-like family within the GPCR superfamily(44) .

Expression of the Human DP Receptor in COS-M6 Cells

[^3H]PGD(2) binding assays were performed using membranes prepared from transient transfection of COS-M6 cells with pcDNA3-hDP. Preliminary receptor binding assays, performed to establish assay conditions, demonstrated that [^3H]PGD(2) specific binding was optimal at pH 7.0 and was maximally stimulated in the presence of 10 mM Mn. In addition, Scatchard analysis showed that [^3H]PGD(2) specific binding to the hDP receptor was of high affinity and conformed to a one-site model (Accufit Two-Site saturation software, Beckman Instruments) with an equilibrium dissociation constant (K) of 1.5 nM (Fig.2A). [^3H]PGD(2) specific binding was saturable, with a maximum number of specific binding sites (B(max)) of approximately 1.2 pmol/mg of membrane protein. Scatchard analysis was also performed in the presence of the slowly hydrolyzable GTP analog GTPS (100 µM) (Fig.2A). In this case, the affinity of [^3H]PGD(2) for hDP was reduced approximately 20-fold (K = 28 nM) demonstrating that dissociation of the associated G-proteins from the hDP receptor converts the receptor to a low affinity state with respect to ligand binding, although the B(max) (1.3 pmol/mg of protein) was unchanged.


Figure 2: Competition for [^3H]PGD(2) specific binding to the human DP receptor expressed in COS-M6 cells. Radioligand membrane binding assays were conducted as described under ``Materials and Methods.'' A, Scatchard analysis of [^3H]PGD(2) specific binding to membranes from COS-M6 cells expressing hDP. Receptor binding assays were performed over a concentration range of 0.4-10 nM [^3H]PGD(2) in the presence (black square) and absence (bullet) of 100 µM GTPS. At each radioligand concentration, total and nonspecific binding was determined in the absence and presence of 1 µM PGD(2). The deduced specific binding saturation isotherm was obtained by subtracting nonspecific from total binding, and linear transformation was performed according to the method of Scatchard using Accufit Two-Site saturation software (Beckman Instruments). B, equilibrium competition binding assays were conducted in the presence of 0.03-1000 nM BW 245C (bullet), PGD(2) (black square), and BW A868C (diamond, filled) and 0.3 nM-100 µM PGE(2) (up triangle, filled), PGF (▾), iloprost (), and U46619 (). BW 245C and BW A868C were generous gifts from The Wellcome Foundation Ltd. Data points are the mean of duplicate experimental values and are representative data from two experiments giving similar results.



Equilibrium competition binding assays were conducted in order to determine the relative affinities of prostaglandins and related synthetic analogs for the hDP receptor (Fig.2B). The most effective competing ligand was PGD(2) which displayed a K value of 1.1 nM. The selective DP agonist BW 245C (45) and the selective DP antagonist BW 868C (46) were equipotent with K values of 0.9 nM and 1.7 nM, respectively. PGE(2) was approximately 100-fold less effective as a competing ligand with a K of 101 nM, while PGF, the IP agonist iloprost, and the thromboxane A(2) mimetic U46619 were less active in competition for [^3H]PGD(2) specific binding to the hDP receptor. The rank order of affinities for prostaglandins and related synthetic analogs at the hDP receptor was therefore: BW 245C = PGD(2) = BW 868C PGE(2) > PGF = iloprost > U46619, as predicted for the DP receptor from pharmacological studies(47, 48) . There was no detectable [^3H]PGD(2) specific binding observed using COS-M6 cell membranes from mock-transfected cells under these experimental conditions.

Increases in cAMP Production in HEK 293(EBNA) Cells Expressing the Human DP Receptor

[cAMP] production was increased in HEK 293(EBNA) cells stably expressing the hDP receptor following challenge with prostaglandins and related synthetic analogs (Fig.3). The DP-selective agonist BW 245C was the most potent compound to increase cAMP production with an EC value of 0.7 nM. In comparison, PGD(2) was 8-fold less potent with an EC value of 6 nM. The putative DP-selective antagonist BW A868C marginally increased cAMP, producing 20 pmol of cAMP/1.5 10^5 cells versus 140 pmol of cAMP/1.5 10^5 cells for PGD(2) at concentrations of 1 µM. Finally, PGE(2), PGE(1), PGF, iloprost, and U46619 all had EC values of >500 nM. The rank order of potency of prostaglandins and related synthetic analogs was: BW 245C > PGD(2) PGE(2) > PGE(1) > PGF > iloprost approx U46619. This rank order of potency has been predicted for the DP receptor from previous pharmacological studies(17) . The increase in cAMP production upon agonist stimulation of hDP-HEK 293(EBNA) cells is mediated through the hDP receptor since these responses did not occur in comparable experiments performed in control HEK 293(EBNA) cells. BW 245C, PGD(2), BW A868C, PGF, iloprost, and U46619 did not increase cAMP production in control cells when tested at concentrations up to 1 µM (data not shown). In contrast, PGE(2) and PGE(1) increased cAMP production in HEK 293(EBNA) cells in a concentration-dependent manner. The magnitude of these responses, however, was small (6 pmol of cAMP/1.5 10^5 cells), approximately 20-fold lower than the responses for these ligands obtained in HEK 293(EBNA) cells expressing the DP receptor. These data suggest that HEK 293(EBNA) cells contain a small population of endogenous EP receptor(s) capable of responding to PGE(2) and PGE(1). In summary, the hDP receptor signals through coupling to elevation in [cAMP] production.


Figure 3: Increases in cAMP in HEK 293(EBNA) cells expressing the human DP receptor. DP-HEK 293(EBNA) cells were incubated in HEPES-buffered Krebs-Ringer buffer and 100 µM RO-20-1724 as described under ``Materials and Methods.'' The incubation medium also contained, as shown in this figure: BW 245C (bullet), PGD(2) (black square), BW 868C (diamond, filled), PGE(2) (up triangle, filled), PGE(1) (Delta), PGF (▾), Iloprost (), and U46619 () over a concentration range up to 1 µM. The reaction was initiated by addition of 1.5 10^5 cells, and the samples were incubated at 37 °C for 15 min prior to termination of the reaction by immersing the samples in boiling water for 3 min. Measurement of cAMP in the samples was performed by radioimmunoassay. Results have been expressed as picomoles of cAMP produced per 1.5 10^5 cells at each ligand concentration. Data points are the mean of duplicate experimental values and are representative data from two experiments giving similar results.



Increases in [Ca]in Fura-2/AM Loaded HEK 293(EBNA) Cells Expressing the hDP Receptor

Challenge of fura-2/AM loaded HEK 293(EBNA) cells expressing the hDP receptor with 1-1000 nM PGD(2) or BW 245C increased [Ca]mobilization in a concentration-dependent manner (Fig.4A). In addition, there was no change in [Ca] when the cells were challenged with the vehicle (Me(2)SO(4) at 0.2% v/v). The addition of 1000 nM PGD(2) or BW 245C to control HEK 293(EBNA) cells did not provoke any change in [Ca] (data not shown) which demonstrates that increases in [Ca] after agonist challenge in hDP-HEK 293(EBNA) cells is mediated through the hDP receptor. However, challenge of the fura-2/AM loaded hDP-HEK 293(EBNA) cells with forskolin to activate directly adenylyl cyclase also evoked a [Ca] response (data not shown) which was similar to that achieved with PGD(2). This also occurred in control HEK 293(EBNA) cells which did not express the DP receptor. These data suggest that the changes in [Ca] in the DP-HEK 293 (EBNA) cells may be provoked through increases in [cAMP] and not through DP receptor activation of the phospholipase C/inositol phosphate generation pathway. This was investigated further by measuring Ins(1,4,5)P(3) generation following activation of the DP receptor. Challenge of DP-HEK 293(EBNA) cells with 1 µM PGD(2) did not increase the level of Ins(1,4,5)P(3) above the basal level observed in the absence of agonist (Fig.4B). Similar results were obtained using control HEK 293(EBNA) cells. In contrast, challenge of hEP(1)-HEK 293(EBNA) cells with 300 nM PGE(2) resulted in a 4-fold increase in Ins(1,4,5)P(3) when compared with the basal level (p < 0.001). The signal transduction pathway of the hEP(1) receptor is known to result in activation of phospholipase C and the subsequent generation of Ins(1,4,5)P(3). The DP receptor, however, does not appear to signal through this pathway. This is supported by studies showing that PGD(2) can cause a cAMP-dependent Ca influx in non-chromaffin cells from bovine adrenal medulla(16) . The precise mechanism through which the hDP receptor mediates the effect on calcium mobilization in HEK 293(EBNA) cells is, therefore, being characterized in more detail.


Figure 4: A, increases in [Ca] in fura-2/AM loaded HEK 293(EBNA) cells expressing the DP receptor. hDP-HEK 293(EBNA) cells were harvested and prepared as detailed under ``Materials and Methods.'' hDP-HEK 293(EBNA) cells (2 10^6) were then challenged with vehicle (Me(2)SO(4) at 0.2% (v/v) final) (A) or 1.0 nM (B), 10 nM (C), 100 nM (D), and 1000 nM (E) BW 245C. Similar results were also obtained with PGD(2) over the same concentration range (data not shown). At the end of each experiment, cells were challenged with 1 µM ionomycin to release [Ca] followed by quenching of the [Ca] released with 1 mM EDTA. These are representative data from two experimental observations giving similar results. The relative excitation ratio of 340/380 has been adjusted to 0 after the establishment of the baseline at time = 80 s. B, Ins(1,4,5)P(3) measurements in hDP-HEK 293(EBNA) and hEP(1)-HEK 293(EBNA) cells. The cells were harvested and prepared as detailed under ``Materials and Methods.'' hEP(1)-HEK 293(EBNA) and hDP-HEK 293(EBNA) cells (5 10^5) were then challenged with either 300 nM PGE(2) or 1 µM PGD(2), respectively (hatched bars), or with vehicle (Me(2)SO(4) at 0.5% v/v final) (solid bars). The reaction was terminated by the addition of 20% (v/v) ice-cold trichloroacetic acid, and Ins(1,4,5)P(3) was measured as detailed under ``Materials and Methods.'' Results have been expressed as picomoles of Ins(1,4,5)P(3) per 1.25 10^5 cells for each cell type and ligand concentration. Data points are the mean ± S.E. of triplicate experimental values and are representative data from three experiments giving similar results.



Northern Blot Analysis

Northern blot analysis was performed on poly(A) RNA from 19 different human tissues. Hybridizing bands were detected only in the small intestine and retina (Fig.5). Three different size transcripts were detected in the retina which may result from the use of different polyadenylation signals in the 3`-untranslated region. The inability of the probe to detect DP message in the other tissues supports previous data in the literature (47) and suggests that expression of the receptor may be limited and, in addition, may be restricted to specific cell types within tissues. In the brain, for example, PGD(2) is the most potent sleep-promoting prostanoid known(4) , and the sleep-promoting zone has been localized in the rat brain to the region in the ventral surface of the rostral basal forebrain(3) . Techniques such as in situ hybridization will therefore be necessary in the elucidation of the precise localization of the hDP receptor.


Figure 5: Northern blot analysis of human tissue RNA. Poly(A) RNA (3 µg) from 19 different human tissues, listed above each lane, was applied. Hybridization analysis was carried out using a P-labeled hDP cDNA fragment as described under ``Materials and Methods.'' The positions of the RNA markers on the gel are indicated.



Comparison of the Human DP Receptor with Other Prostanoid Receptors

The hDP and mouse DP receptors are 73% identical at the amino acid level which increases to 85% in the TMDs. Amino acid sequence comparison of all members of the human prostanoid receptor family is shown in Fig.6. In general, the prostanoid family shows 28% to 45% amino acid sequence identity between any two members. There are a total of 28 amino acid residues conserved in all prostanoid receptors, 8 of which are also conserved in all rhodopsin-like GPCRs. Of the 28 residues, only 6 of them occur outside of putative TMDs, and this includes the invariant and unique tripeptide, WCF, found in the second extracellular loop. The cysteine residue within the tripeptide forms a putative disulfide bridge with the conserved cysteine residue in the first extracellular loop. TMD VII is the most highly conserved TMD within the prostanoid receptor family, and it contains a conserved arginine residue, which has been postulated to act as the counterligand for the alpha-carboxyl moiety occurring in the prostanoid ligands(49) . TMD VII also contains another invariant tripeptide, DPW, which includes the proline residue conserved in all GPCRs(44) .


Figure 6: Amino acid sequence alignment of the human prostanoid receptor family. The deduced amino acid sequences of the human DP, IP, EP(2), EP(4), EP, TP, FP, and EP(1) prostanoid receptors are shown aligned using GCG Wisconsin DNA software. Identical amino acids in at least three sequences are boxed. Dashes indicate gaps introduced in the sequences for alignment purposes. The TMDs are indicated by overlines. Conserved amino acids in all prostanoid receptors are indicated by a up triangle, filled, and those conserved in all rhodopsin-like GPCRs are indicated by a bullet.



Phylogenetic comparison of the prostanoid receptor family, shown in Fig.7, reveals two main subfamilies, one comprising EP(1), TP, FP, and EP(3) and the other grouping EP(4), DP, IP, and EP(2). EP(1), TP, and FP are the most closely related within the first subfamily and have been shown to couple to an increase in [Ca]. EP(3) receptors, which are less related within this subfamily, can couple to both elevation of [Ca] and a decrease in [cAMP](50) . EP(2), DP, and IP are the most highly related receptors within the second subfamily of the prostanoid receptors and, together with EP(4), couple to elevation of [cAMP]. Interestingly, the mouse IP receptor has also been shown to couple to phosphatidylinositol metabolism when expressed in CHO cells(51) , although the agonist concentration needed to generate IP(3) was approximately 3 orders of magnitude higher than that required to stimulate cAMP production. It remains to be established whether the subfamily of prostanoid receptors related to IP can all generate IP(3) upon activation and can, therefore, signal through a dual pathway. Moreover, given the relatively high concentrations of agonist required to trigger IP(3) in this recombinant system, the physiological relevance of this signaling pathway remains to be determined.


Figure 7: Phylogram of the human prostanoid receptor family. Alignment of the deduced amino acid sequences of human prostanoid receptors(21, 22, 24, 27, 28, 29, 32) using the Kimura protein distance analysis (53) from the Wisconsin Sequence Analysis Package (Version 8.0). Branch lengths are proportional to calculated distances.



In summary, we have cloned a cDNA for the hDP prostanoid receptor and have shown that this receptor is coupled to stimulation of intracellular cAMP production as a major signaling pathway. In addition, hDP has a discrete distribution as assessed by Northern blot analysis. The availability of the hDP cDNA will facilitate the elucidation of the ligand binding and signal transduction characteristics of this prostanoid receptor and will be essential in delineating the cellular distribution of hDP within tissues.


FOOTNOTES

*
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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank&#174;/EMBL Data Bank with accession number(s) U31332[GenBank], U31098[GenBank], and U31099[GenBank].

§
To whom correspondence and reprint requests should be addressed. Tel: 514-428-8525; Fax: 514-428-8615; abramovit{at}merck.com.

^1
The abbreviations used are: PG, prostaglandin; bp, base pair(s); kb, kilobase(s); TMD, transmembrane domain; RACE, rapid amplification cDNA ends; PCR, polymerase chain reaction; GPCR, G-protein-coupled receptors; Ins(1,4,5)P(3), inositol 1,4,5-trisphosphate; BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; GTPS, guanosine 5`-O-(thiotriphosphate).

^2
Prostanoid receptors are designated following the recommendation of the IUPHAR Commission on Receptor Nomenclature and Classification (52).

^3
The human prostaglandin E(2) (PGE(2)) receptor described by An et al. (33) and Bastien et al. (32) as the EP(2) subtype has now been reclassified as the EP(4) subtype since it has negligible affinity (K = 23 µM) for the EP(2)-selective agonist butaprost. In contrast, the PGE(2) receptor described by Regan et al. (28) displays moderate affinity (K = 1.4 µM) for butaprost and has therefore been designated as the EP(2) subtype.

^4
Y. Boie and M. Abramovitz, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Jilly Evans for critical review of the manuscript.


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