(Received for publication, August 20, 1996, and in revised form, October 29, 1996)
From the Departments of Pharmacology and Toxicology
and ¶ Physiology, The University of Arizona, Tucson, Arizona 85721 and
Allergan, Inc., Department of Biological Sciences,
Irvine, California 92713-9534
An FP prostanoid receptor isoform, which appears
to arise from alternative mRNA splicing, has been cloned from a
mid-cycle ovine large cell corpus luteum library. The isoform, named
the FPB receptor, is identical to the original isoform, the
FPA, throughout the seven transmembrane domains, but
diverges nine amino acids into the carboxyl terminus. In contrast to
FPA, whose carboxyl terminus continues for another 46 amino
acids beyond the nine shared residues, the FPB terminates
after only one amino acid. The FPA isoform appears to arise
by the failure to utilize a potential splice site, while a 3.2-kilobase
pair intron is spliced out from the FP gene to generate the
FPB isoform mRNA. The two isoforms have
indistinguishable radioligand binding properties, but seem to differ in
functional coupling to phosphatidylinositol hydrolysis. Thus, in COS-7
cells transiently transfected with either the FPA or the
FPB receptor cDNAs, prostaglandin F2
stimulates inositol phosphate accumulation to the same absolute
maximum, but the basal level of inositol phosphate accumulation is
approximately 1.3-fold higher in cells transfected with the
FPB as compared with cells transfected with the
FPA isoform. Using the polymerase chain reaction, mRNA
encoding the FPB isoform was identified in the ovine corpus luteum.
Prostanoids, autacoids formed from arachidonic acid by the actions
of cyclooxygenases, exert diverse physiological effects throughout the
body. For example, in sheep and in many other species, prostaglandin
F2 (PGF2
)1 is
the trigger that initiates luteolysis or regression of the corpus
luteum in the absence of pregnancy (1). There are five primary
prostanoids, prostaglandin D2 (PGD2),
prostaglandin E2 (PGE2), PGF2
,
prostaglandin I2 (PGI2), and thromboxane
A2 (TXA2). Unique G-protein-coupled receptors
have been cloned for each prostanoid, including four receptors specific
for PGE2 (EP1-EP4) (2). The other
cloned receptors are named DP, FP, IP, and TP and bind
PGD2, PGF2
, PGI2, and
thromboxane A2, respectively. In addition, alternative mRNA
splice variants, have been cloned for the EP3 receptor (3,
4) and for the TP receptor (5, 6). In each case, the splice variants
are identical throughout the seven-transmembrane domains, but diverge
approximately 9-12 amino acids into the carboxyl terminus. The
significance of this carboxyl-terminal alternative splicing is not well
understood, but for the EP3 receptor, differences among the
isoforms have been found in localization (7), receptor/G-protein
coupling (3, 8, 9, 10), and desensitization (11).
Phylogenetic analysis of the prostanoid receptors shows that the receptors segregate into two branches (12, 13). One branch contains the DP, IP, EP2, and EP4 receptors. The second branch contains the EP1, EP3, FP, and TP receptors. Since isoforms have been found for two of the four receptors in the second branch, the EP3 and TP receptors, we hypothesized that isoforms might also exist for the FP receptor.
To examine this possibility, we screened an ovine mid-cycle large cell corpus luteum library. Previously, we used this library to clone the ovine homologue (14) of the human FP receptor (15) and found that the message for the FP receptor was highly abundant (~0.1% of the total message). Using a combination of homology-based screening with the ovine FP receptor as a probe and PCR, we cloned a novel isoform of the FP receptor. This isoform, the FPB, is identical to the original (termed here the FPA isoform) throughout the seven-transmembrane domains, but diverges nine amino acids into the carboxyl terminus. Functionally, both isoforms are able to stimulate inositol phosphate (IP) accumulation to the same maximum, but the basal level of hydrolysis is 130% higher for the novel FPB isoform than for the original FPA isoform.
A cDNA containing the complete coding sequence of the ovine FPA receptor (14) was labeled with 32P by nick translation (Life Technologies, Inc.) and an ovine large cell corpus luteum library screened as described previously (14). The library was plated at a density of ~3400 plaques/plate (15 cm), and five plates were screened. From a total of 24 positives, 18 were isolated and each was placed in 250 µl of H2O.
To differentiate possible carboxyl-terminal splice variants from the original FPA isoform, two rounds of PCR were used. In the first round, sense (nt 258-275) and antisense (nt 638-654) primers upstream of the putative sixth transmembrane domain were used. These primers were predicted to be specific for the common region of all FP isoforms. In the second round of PCR, a sense primer (nt 793-810) upstream of the sixth transmembrane domain and an antisense primer (nt 1168-1185) specific for the carboxyl terminus were used that could amplify the original FPA isoform but would be unlikely to yield products (at least of the predicted size) with carboxyl-terminal splice variants. The isolated positive plaques were vortexed for 15 s and lysed by three freeze/thaw cycles consisting of freezing in a dry ice/ethanol bath and thawing at 55 °C for 5 min. For each 50 µl PCR, 26 µl of the phage lysate was added to a 50 µl reaction containing final concentrations of 1 × PCR buffer I (Perkin-Elmer), 10% dimethyl sulfoxide (Sigma), 200 µM dNTPs (Perkin-Elmer), and 1 µM each primer. The samples were heated to 95 °C for 5 min, chilled on ice, 2.5 units of Taq polymerase (Perkin-Elmer) were added, and 30 µl of mineral oil overlaid. The PCR conditions were as follows: an initial denaturation step at 95 °C for 2 min followed by 30 cycles at 95 °C for 30 s, 42 °C for 30 s, and 70 °C for 30 s. The PCRs were held at 4 °C until analyzed by agarose gel electrophoresis.
Clones that gave positive results in the first round of PCR, but negative results in the second round of PCR, were isolated using standard plaque purification techniques. The inserts were subcloned into pBluescript (Stratagene) and sequenced. One clone (KS+/FPB) that was otherwise identical to the FPA but diverged in the carboxyl terminus was identified.
Construction of a Full-length FPB Isoform and Expression VectorKS+/FPB lacked the first 69 bp of
coding sequence, and a full-length clone was obtained by replacing a
HindIII fragment from the original FPA isoform,
KS+/FP (14), with a HindIII fragment from
KS+/FPB. KS+/FP and KS+/FPB were restricted
with HindIII which cuts at nt 638 (Fig. 1, single
underlining) and in the multiple cloning site of KS+ downstream of
the FPB coding sequence. The large fragment containing KS+
plus the 5 end of the FPA receptor and the small fragment
containing the 3
end of FPB from the FPB digestion were isolated using Geneclean II (BIO 101, Inc.), ligated, subcloned, and KS+/FPB/fulcod isolated. To make an
expression vector, KS+/FPB/fulcod was restricted with
NaeI, which cuts at nt 80, and NcoI, which cuts
at nt 1213, filled in using Klenow and subcloned into the blunted
BamHI/HindIII sites of pBC12BI (16) to form
pBC/FPB/fulcod.
PCR on Genomic DNA
PCR of sheep genomic DNA was performed using the Advantage Genomic PCR kit (Clontech). For both the FPA and the FPB receptor, the sense primer corresponded to nt 1018-1044. For the FPA receptor, the antisense primer corresponded to nt 1291-1317, and for the FPB receptor, the antisense primer corresponded to nt 1078-1104. Each 50-µl reaction contained 2 µl of sheep genomic DNA (~200 ng total), 1 × 10 Tth PCR buffer, 1.1 mM Mg(OAC)2, 1 µM each primer, 200 µM dNTPs, and 1 µl of Advantage Tth Polymerase Mix (50 ×). 40 µl of mineral oil was overlaid, and the PCR program used was as follows: 95 °C for 1 min, 35 cycles at 95 °C for 15 s, 68 °C for 12 min, followed by a final extension at 68 °C for 12 min. The reactions were held at 15 °C until analyzed by agarose gel electrophoresis.
Reverse Transcription (RT)-PCRTotal RNA (~5 µg) from a day 10 ovine corpus luteum was reverse-transcribed in a 20-µl reaction using reverse transcriptase (Superscript II, Life Technologies, Inc.) following the manufacturer's instructions using 200 ng of random hexamer primers. After the cDNA synthesis, RNA remaining in RNA-DNA duplexes was degraded by adding ~1 unit of RNase H and incubating for 30 min at 37 °C. For the PCR, 2 µl of the cDNA reaction was added to a 50-µl reaction containing 10% dimethyl sulfoxide, 0.25 mM dNTPs (Perkin-Elmer), 1 µM each primer, 1 × PCR buffer, and 2.5 units of Taq polymerase (Perkin-Elmer). The PCR program was as follows: a 7-min denaturation step at 95 °C, 36 cycles at 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, followed by 7 min at 72 °C, and the reaction was held at 4 °C.
IP Accumulation AssayReceptor-mediated PI hydrolysis was determined by measuring the accumulation of IPs in cells that were preincubated with myo-[2-3H]inositol (17-18 Ci/mmol, Amersham Corp.). COS-7 cells were plated in 10-cm dishes (106 cells/dish) in DMEM with 10% fetal bovine serum) and transfected the following day with the expression plasmids pBC/FPA or pBC/FPB/fulcod (4 µg/dish), using Lipofectin (40 µg/dish, Life Technologies, Inc.) following the manufacturer's instructions. On day 3, the cells were incubated with 3 µC/ml myo-[2-3H]inositol in DMEM, 10% fetal bovine serum. Cells were harvested on the following day, preincubated for 10 min in DMEM containing 25 mM HEPES buffer, 10 mM LiCl in order to block the dephosphorylation of IPs and aliquoted (200 µl) with various concentrations of drug (50 µl) in duplicate. After a 30-min, 37 °C incubation, IPs were extracted with 750 µl of chloroform/methanol/4 N HCl (100:200:2). An additional 250 µl each of the chloroform and 0.5 N HCl were added, and the samples were vortexed. Unincorporated myo-[2-3H]inositol was removed by adding 750 µl of the resulting aqueous layer to a Dowex 1-X8 (formate, 100-200 mesh, Bio-Rad) anion exchange column. Following three washes with 3 ml of 5 mM inositol, the IPs were eluted with 1.5 ml 1.3 M ammonium formate, 0.1 M formic acid and counts/min were determined by liquid scintillation counting.
We screened an ovine mid-cycle large cell luteal library with the
previously cloned ovine FP receptor (14) and used PCR to identify
possible carboxyl-terminal alternative mRNA splice variants. From a
total of 17,000 plaques, one putative alternative splice variant was
identified. We have designated this clone FPB, in contrast
to the original isoform which we now define as FPA. Fig.
1 shows the cDNA and deduced amino acid sequence of
the ovine FPA and FPB isoforms. The
FPA and FPB isoforms share the same sequence
until they diverge nine amino acids into the carboxyl terminus. In the
original isoform, the FPA, the carboxyl terminus continues
for 46 amino acids beyond the splice site, while in the FPB
isoform there is only one additional amino acid. Thus, the
FPB is truncated relative to the FPA isoform.
The immediate 3-untranslated region of the mRNAs encoding these
isoforms differ as well.
To determine how the two FP receptor isoforms arose, PCR was performed
on sheep genomic DNA using primers that spanned the proposed splice
site. The same sense primer (nt 1018-1044) was used in both reactions
in combination with an antisense primer specific to each isoform
(FPA, nt 1291-1317; FPB, nt 1078-1104). A
picture of the agarose gel on which the reactions were electrophoresed is shown in Fig. 2. Lane 1 shows the size of
the product obtained with the FPA-specific primer, and
lane 2 shows the size of the product obtained with the
FPB-specific primer. Thus, amplification with the
FPA-specific primer yielded a product of ~300 bp, which is identical to the size predicted from the cDNA. Amplification with the FPB-specific primer, however, yielded a 3200-bp
product. This product is much larger than the 86-bp product that would be expected from amplification of the FPB cDNA.
To verify that the message for the FPB was expressed in
corpus luteum, RT-PCR was performed on mRNA obtained from a
mid-cycle corpus luteum using primers flanking the coding region of the FPB isoform. The sense primer corresponded to nt 88-111,
and included the codon for the initiator methionine, and the antisense
primer corresponded to nt 1081-1098 of the FPB receptor,
downstream of the stop codon. Fig. 3 shows a photograph
of the ethidium-stained agarose gel with the products of the PCR.
Lane 1 is the product obtained from the ovine corpus luteum
cDNA, lane 2 shows a positive control in which
KS+/FPB/fulcod plasmid DNA was amplified with the same pair
of primers, and lane 3 is the negative control. For both the
ovine corpus luteum cDNA and the plasmid positive control, a
product of the expected size, 1010 bp, was obtained. To verify that
this 1010-bp product encoded the full-length FPB isoform,
the bands from lanes 1 and 2 were isolated and
digested with HindIII, which yielded products of the
expected size, 459 and 551 bp. based on the presence of a
HindIII site at nt 638 (data not shown).
To examine the pharmacology of the FPB clone, radioligand
binding was performed using membranes from COS-7 cells that were transiently transfected with pBC/FPB/fulcod. Fig.
4 shows radioligand binding competition curves using
17-[3H]phenyl-trinor-PGF2 as the
radioligand. The ability of a series of natural and synthetic
prostanoids to displace
17-[3H]phenyl-trinor-PGF2
binding was
measured, and the rank order of potencies of the competitors was as
follows: 17-phenyl-trinor-PGF2
> PGF2
> fluprostenol > PGD2 = PGE2
8-epi-PGF2
. Thus, the synthetic prostanoid,
17-phenyl-trinor-PGF2
, is the most potent with an
EC50 of ~10 nM, while PGF2
is
the most potent natural prostanoid, with an EC50 of ~40
nM, although both PGD2 and PGE2
were also able to displace
17-[3H]phenyl-trinor-PGF2
binding. These
results are nearly indistinguishable from those obtained for the
FPA under similar conditions (14).
Since the FP receptor cloned from other species activates PI hydrolysis
(17, 18), we wanted to determine whether the ovine FPA and
FPB isoforms could also stimulate IP accumulation and whether there were any differences between the isoforms. Fig. 5 shows the results of experiments performed on COS-7
cells that were transiently transfected with the FPA (14)
or the FPB and treated with different concentrations of
PGF2. IP accumulation was normalized to the basal level
of IP accumulation for the FPA. PGF2
did not
stimulate IP formation in untransfected cells. For cells expressing
either the FPA or FPB, PGF2
was
able to elicit the same maximal level of IP accumulation, 220% over basal, and the EC50 was similar for the two isoforms, 8 nM for the FPA and 11 nM for the
FPB isoform. However, the basal level of IP accumulation
was ~130% higher in cells expressing the FPB isoform
than in cells expressing the FPA, which in turn was
~140% higher than the basal level of hydrolysis in untransfected
cells (data not shown). Based on radioligand binding competition
studies, the level of receptor expression was higher for cells
expressing the FPA receptor than for cells expressing the
FPB receptor. In addition, similar experiments were
repeated in COS-P cells, with essentially the same findings.
Previously, we and others reported the cloning of the FP prostanoid receptor from bovine (17), human (15), mouse (18), and ovine (14). Here we report the cloning of the FPB, an isoform of the ovine FP receptor. Isoforms have been reported for the EP3 and TP prostanoid receptors, but this is the first report of isoforms of the FP receptor. Like the EP3 and TP receptor isoforms, the FP receptor isoforms diverge in the carboxyl termini. Thus far, two isoforms of the human TP receptor (6) have been cloned, while a total of 13 different isoforms of the EP3 receptor have been cloned among bovine, human, rabbit, and mouse (2).
The existence of FP receptor isoforms was hypothesized based on previously cloned EP3 and TP receptor isoforms and relationships established by the phylogenetic analysis of the prostanoid receptors. This analysis places the DP, EP2, EP4, and IP receptors in one branch and the remaining EP1, EP3, FP, and TP receptors in the second. With the cloning of the FPB, isoforms have now been found for three of the four receptors in the second branch. Isoforms have not yet been reported for the EP1 receptor, but the phylogenetic relationships suggest that they, too, may exist. Interestingly, the existence of isoforms also seems to correlate with second messenger coupling. Thus, the receptors in the second branch where isoforms have been reported are all primarily coupled to either PI hydrolysis or inhibition of adenylyl cyclase, while isoforms have not been obtained for the receptors in the first branch, which are all coupled to stimulation of adenylyl cyclase.
The TP and EP3 receptor isoforms arise by alternative mRNA splicing at a location that is homologous to the site at which the FP isoforms diverge, suggesting that the FP receptor isoforms are also generated by alternative mRNA splicing. In the case of the TP receptor isoforms, the isoforms arise by the failure to utilize a potential splice site (6). Thus, the sequence of the carboxyl terminus of the endothelial variant is identical to the genomic sequence, while this sequence is spliced out to form the placental variant. It appears that the same mechanism generates the two FP receptor isoforms. Using the primers specific for the FPA receptor, the PCR product obtained from the genomic DNA is identical to the size of the product predicted from the cDNA sequence, indicating that no sequence is spliced out to generate this isoform. However, when primers specific for the FPB are utilized in a PCR on genomic DNA, the product obtained is much larger than predicted from the cDNA. Thus it appears that a ~3.2-kilobase pair intron is spliced out to generate the FPB receptor message. Consistent with the mechanism for the generation of these two isoforms, the highly conserved dinucleotide (GT) from the splice donor site is retained in the sequence of the FPA isoform (nt 1048-1049) (19).
The functional significance of the carboxyl-terminal isoforms has been
most well characterized for the ,
, and
isoforms of the mouse
EP3 receptor. Agonist binding among the isoforms is
virtually indistinguishable (8), but differences among the isoforms
have been found for receptor/G-protein coupling. Traditionally, the
mouse EP3 isoforms are coupled to inhibition of adenylyl
cyclase, but some of the isoforms have been found to stimulate cAMP
formation and/or activate PI hydrolysis (3, 9, 20). In addition, two
isoforms, the EP3
and EP3
, and a
recombinant mutant that is truncated at the splice site have been shown
to have agonist-independent constitutive activity (21). Thus, in the
absence of agonist, the level of forskolin-stimulated cAMP accumulation
is greater in cells expressing the EP3
isoform than in
cells expressing either the EP3
or EP3
isoforms or the truncation mutant. However, maximal inhibition of
forskolin-stimulated cAMP formation in the presence of agonist is the
same for all three isoforms and for the truncation mutant. For the
EP3
isoform, there is agonist-independent constitutive
activity and agonist-dependent inhibition of cAMP
formation, while for the truncation mutant, there is only
agonist-independent inhibition of cAMP formation. Data obtained with
the EP3
isoform suggest that there is agonist-independent stimulation of the G-protein, Gi, but
agonist-dependent stimulation of the G-protein,
Gs (22).
Our data with the FP receptor isoforms indicate that the
receptor/G-protein coupling for the FP receptor isoforms are analogous to the data found for the mouse EP3 and
EP3
isoforms. There is agonist-independent constitutive
IP accumulation in cells expressing either the FPA or
FPB receptor isoforms, but this level is approximately
130% higher for cells expressing the truncated FPB isoform
than for cells expressing the FPA receptor isoform. In
addition, for both the FPA and FPB isoforms,
there is agonist-stimulated IP accumulation which reaches the same
maximal level. This suggests that the FPB isoform is a
naturally occurring truncated receptor that shows agonist-independent
constitutive activity, but is still responsive to agonist. This is in
contrast to the truncation mutant of the EP3 receptor,
which shows only agonist-independent constitutive activity and is
unresponsive to agonist. The data with both the EP3 and FP
receptor isoforms are consistent with mutations made in other G-protein
coupled receptors, including the avian
-adrenergic receptor, which
suggests that sequences in the carboxyl-terminal tail may suppress
G-protein interactions in the absence of ligand (23, 24).
More work will be needed to establish if additional biochemical
differences exist between the FP receptor isoforms. Of particular interest are possible differences in desensitization, localization, and
ability to activate other downstream effectors. The physiological significance of the FPB isoform, and of other prostanoid
receptor alternative splice variants, is also intriguing. In the
mid-cycle large cell corpus luteum library from which the
FPB was cloned, the FPA is far more abundant
than the FPB. This, however, may not be true throughout the
luteal cycle and in other tissues that are also responsive to
PGF2.
We thank Dr. Jon Lomasney and Patrick Graelish (Feinberg Cardiovascular Research Institute, Northwestern University Medical Center, Chicago, IL) for helpful discussions and performing IP assays in COS-P cells and Dr. Karen Kedzie and Heather Krauss (Allergan, Inc., Biological Sciences, Irvine, CA) for their assistance in preparing cells and plasmids.