(Received for publication, March 8, 1995)
From the
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 receptors, followed by the hEP
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 [
H]prostaglandin D
(PGD
) specific binding sites, was as predicted for
the DP receptor, with PGD
PGE
>
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
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.
Prostaglandin D (PGD
) (
)is
formed in a variety of tissues including brain, spleen, lung, bone
marrow, stomach, skin (1, 2) , and also in mast cells.
PGD
has been implicated in many physiological events both
in the central nervous system and peripheral tissues. In the central
nervous system, PGD
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
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
, 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
, including
bronchoconstriction, may be attributable to the cross-reactivity of
PGD
with the prostaglandin F
and/or
thromboxane A
receptors(9) .
The physiological
and pathophysiological actions of PGD are mediated through
interaction with the prostanoid DP receptor. (
)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 (TP)(21) , prostaglandin F
(FP)(22, 23) , and prostacyclin (IP) (24, 25, 26) receptors and four distinct
subtypes of the prostaglandin E
(EP
(27) , EP
(28) ,
EP
(29, 30, 31) , and
EP
(32, 33) (
)) receptor. Here
we report the cloning of the human prostaglandin D
(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.
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.
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 (
) and potential protein kinase
C phosphorylation sites (
) 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 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) .
Figure 2:
Competition for
[H]PGD
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
[
H]PGD
specific binding to membranes
from COS-M6 cells expressing hDP. Receptor binding assays were
performed over a concentration range of 0.4-10 nM
[
H]PGD
in the presence (
) and
absence (
) of 100 µM GTP
S. At each radioligand
concentration, total and nonspecific binding was determined in the
absence and presence of 1 µM PGD
. 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 (
), PGD
(
), and BW A868C (
)
and 0.3 nM-100 µM PGE
(
), 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 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
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
mimetic U46619 were less active in
competition for [
H]PGD
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
= BW 868C PGE
> PGF
= iloprost > U46619, as
predicted for the DP receptor from pharmacological
studies(47, 48) . There was no detectable
[
H]PGD
specific binding observed
using COS-M6 cell membranes from mock-transfected cells under these
experimental conditions.
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 (), PGD
(
), BW 868C (
),
PGE
(
), PGE
(
), PGF
(▾), Iloprost (
), and U46619 (
) over a
concentration range up to 1 µM. The reaction was initiated
by addition of 1.5
10
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
cells at each ligand concentration. Data points are the mean of
duplicate experimental values and are representative data from two
experiments giving similar results.
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
) were then challenged with vehicle
(Me
SO
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
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
measurements in hDP-HEK 293(EBNA) and
hEP
-HEK 293(EBNA) cells. The cells were harvested and
prepared as detailed under ``Materials and Methods.''
hEP
-HEK 293(EBNA) and hDP-HEK 293(EBNA) cells (5
10
) were then challenged with either 300 nM PGE
or 1 µM PGD
, respectively (hatched bars), or with vehicle (Me
SO
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
was measured as detailed under
``Materials and Methods.'' Results have been expressed as
picomoles of Ins(1,4,5)P
per 1.25
10
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.
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.
Figure 6:
Amino acid sequence alignment of the human
prostanoid receptor family. The deduced amino acid sequences of the
human DP, IP, EP, EP
, EP
, TP, FP,
and EP
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
, and those conserved in all rhodopsin-like
GPCRs are indicated by a
.
Phylogenetic comparison of the prostanoid
receptor family, shown in Fig.7, reveals two main subfamilies,
one comprising EP, TP, FP, and EP
and the other
grouping EP
, DP, IP, and EP
. EP
,
TP, and FP are the most closely related within the first subfamily and
have been shown to couple to an increase in
[Ca
]
. EP
receptors, which are less related within this subfamily, can
couple to both elevation of [Ca
]
and a decrease in
[cAMP]
(50) . EP
, DP,
and IP are the most highly related receptors within the second
subfamily of the prostanoid receptors and, together with
EP
, 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
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
upon activation and can, therefore, signal through a
dual pathway. Moreover, given the relatively high concentrations of
agonist required to trigger IP
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) U31332[GenBank], U31098[GenBank], and U31099[GenBank].