(Received for publication, October 7, 1996, and in revised form, December 5, 1996)
From the Center for Experimental Therapeutics,
University of Pennsylvania, Philadelphia, Pennsylvania 19104, § Commissariat à l'Energie Atomique (Saclay), Service
de Pharmacologie et d'Immunologie, Saclay 91191 Gif-sur-Yvette,
France, and ¶ INSERM U 348, Hôpital Lariboisière,
75475 Paris Cedex 10, France
Thromboxane A2 (TxA2) is
a potent vasoconstrictor and platelet agonist. Its biological function
is tightly regulated. G protein-coupled membrane receptors transduce
the effects of TxA2. However, although a single thromboxane
receptor (TP) gene has been identified, two splice variants have been
cloned from human placenta and megakaryocytic lines (TP) and from
human endothelial cells (TP
). These differ in the length of their
carboxyl-terminal extensions (15 versus 79 residues), which
contain multiple potential sites for receptor phosphorylation. Given
that TP agonists activate protein kinase C (PKC), it would seem
possible that PKC-dependent phosphorylation of TPs might play a
central role in homologous desensitization of these receptors.
To determine if the TP isoforms were differentially phosphorylated in
response to agonist in vivo, human embryonic kidney (HEK)
293 cells were stably transfected with TP and TP
.
Isoform-specific anti-peptide antibodies were developed and used to
immunoprecipitate the phosphorylated receptors. U46619, a
PGH2/TxA2 mimetic, induced specific
phosphorylation of both isoforms. Phosphorylation of the two isoforms
was similar in dose and time dependence, reaching a plateau at around
100 nM U46619. Inhibition of PKC with either GF 109203X (5 µM) or RO 31-8220 (5 µM) or of protein
kinase A with H-89 (50 µM) marginally influenced
agonist-dependent phosphorylation of either isoform and
failed to modulate homologous desensitization of agonist-induced
stimulation of inositol phosphate formation. Similar results were
obtained when PKC was down-regulated by long term incubation with the
phorbol ester, phorbol myristate acetate. Although short term
stimulation with phorbol myristate acetate caused
PKC-dependent phosphorylation of TPs in vivo,
thrombin stimulation of the TP-transfected HEK cells in
vivo failed to phosphorylate either of the TP isoforms. Thus,
despite the capacity of PKC to phosphorylate TPs in HEK 293 cells and
the likely activation of PKC by TP stimulation, this enzyme, like
protein kinase A, contributes marginally to rapid, agonist-induced
phosphorylation of either TP isoform.
Thromboxane A2
(TxA2)1 is a product of the
sequential metabolism of arachidonic acid by the cyclooxygenases and
TxA2 synthase (1). It is formed upon activation of a
variety of cells, including platelets, macrophages, and vascular smooth
muscle cells and exhibits potent biological activity, causing platelet
aggregation and secretion, mitogenesis, and vasoconstriction (2, 3).
These effects are transduced via membrane receptors, identified
initially with a variety of diverse structural ligands (4). Although
such pharmacological studies suggested diversity among thromboxane receptors (TPs), a single gene, encoding a member of the heptahelical G
protein-coupled receptor (GPCR) family, has been cloned (5). However,
two variants, based on alternative splicing of the carboxyl-terminal tail of the receptor, have been identified. The first, TP, was cloned from a cDNA library derived from placental (6) and also from
megakaryocytic cell lines (7, 8). The second, TP
, was cloned from a
human umbilical endothelial cell cDNA library (9). The precise
biological functions subserved by these isoforms is presently unknown.
For example, mRNAs for both isoforms exist in human platelets (10);
however, it is unknown whether they represent the two forms of
functional TPs identified by ligand binding in human platelets (11).
Similarly, the functional response to TP stimulation in endothelial
cells remains ill defined. The capacity of the isoforms to subserve
distinct biological functions is illustrated by differential coupling
of the expressed isoforms to adenylate cyclase in COS cells (10).
One aspect of TP function that, a priori, may differ between
the isoforms is agonist-dependent desensitization. TP
differs from the a isoform in having a longer (79 versus 15 amino acids) carboxyl-terminal extension, which contains an additional
11 serine and 4 threonine residues. It also contains a tyrosine
residue, absent from the TP
isoform (9). These amino acids are
potential targets for phosphorylation, which is likely to be intrinsic
to the process of desensitization. Given the rapid formation of
TxA2 by activated cells and its potency, regulation of the
response to this eicosanoid by homologous receptor desensitization is
of likely biological importance. Biosynthesis of additional ligands, such as thrombin and growth factors (12-16) may amplify the response to TxA2, perhaps via cross-talk with TPs. Similarly,
TxA2 may evoke formation of counterregulatory ligands, such
as prostacyclin, which may also modulate TP function (17).
Presently, our understanding of the molecular events that underlie TP
desensitization in intact cells is limited. These have largely been
confined to pharmacological studies (18), which suggest differences
between cells but have not discriminated between the cloned isoforms.
We have previously demonstrated (19) that a fusion protein, including
the carboxyl-terminal tail of the TP, may be phosphorylated by
purified PKC and, to a lesser extent, PKA, in vitro. Okwu
et al. (20) have provided evidence that TPs in human
platelets may be subject to phosphorylation.
We now report the characterization of specific, peptide-based
antibodies to TP and TP
and demonstrate that both receptor isoforms are predominantly localized at the plasma membrane of human
embryonic kidney (HEK) 293 cells stably overexpressing TP receptors.
Both isoforms are phosphorylated in response to stimulation with the
prostaglandin H2/TxA2 mimetic, U46619 (21).
Interestingly, the dose and time dependence of agonist-induced receptor
phosphorylation appeared similar for both isoforms in vivo,
and neither PKC nor PKA played a major role in this phenomenon.
The cDNAs for the TP isoforms were kindly
provided by Dr. Colin Funk and Dr. J. Anthony Ware, respectively. The
cDNA and the antibody for human thrombin receptor were kindly
provided by Dr. Lawrence Brass. [32P]Orthophosphate
(~6,000-7,000 Ci/mmol), myo-[2-3H]inositol
(18.3 Ci/mmol) and 14C-protein molecular weight markers
were purchased from Amersham Corp. [3H]SQ29,548 (46 Ci/mmol) was obtained from DuPont NEN. pcDNA3 was obtained from
Invitrogen (San Diego, CA). HEK 293 cells were obtained from ATCC
(Rockville, MD). CNBr-activated Sepharose and E-Z-SEP®
polyclonal kit were obtained from Pharmacia Biotech Inc. Geniticin (G418, specific activity at 750 µg/mg) and all tissue culture media
were purchased from Life Technologies, Inc. Phorbol 12-myristate 13-acetate (PMA), bovine -thrombin (285 units/mg of protein), forskolin (FK), dibutyryl cAMP (Bt2cAMP),
3-isobutyl-1-methylxanthine (IBMX), sodium orthovanadate, sodium
fluoride, sodium pyrophosphate, ATP, sodium deoxycholate, and protease
inhibitors were purchased from Sigma, and
N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89 dihydrochloride) and 4
-PMA were from Biomol Research
Laboratories (Plymouth Meeting, PA). RO-31-8220 and
bisindolylmaleimide I (GF 109203X) were from Calbiochem.
9,11-Dideoxy-9
,11
-methanoepoxyprostaglandin F2
(U46619) and SQ29,548 were obtained from Cayman
Chemical Co., Inc. (Ann Arbor, MI). Anion exchange resin AG 1X-8
(formate form, 200-400 mesh) was from Bio-Rad. All electrophoresis
reagents were from J. T. Baker (Phillipsburg, NJ).
The cDNA encoding the TP isoform was
subcloned into the EcoRI-XbaI sites of the
mammalian expression vector pcDNA3. The cDNA for the TP
isoform subcloned into the EcoRI-EcoRI sites of
the same vector (22) was further digested by BamHI and
Bsu36I and then ligated after creating a blunt end. This
treatment resulted in the truncation of 195 base pairs in the
5
-untranslated region, leaving one ATG start codon. This enhanced the
expression of the receptors in stably transfected cells 4-5-fold
relative to the undigested construct (data not shown). Following
truncation, the stably transfected HEK 293 cells expressed similar
amounts of the TP
and TP
isoforms as assessed by binding studies
(see "Results").
HEK 293 cells were cultured in Dulbecco's modified Eagle's medium
(DMEM) containing 10% fetal bovine serum, 2 mM glutamine, 50 units/ml penicillin, 100 µg/ml streptomycin. HEK 293 cells stably
overexpressing both isoforms were prepared as described previously
(22). Briefly, cells were plated at 0.7 × 106
cells/100-mm culture dish and transfected with 10 µg of
TP-pcDNA3, TP
-pcDNA3, or pcDNA3 using a cationic
liposome-mediated transfer according to the manufacturer's
instructions (DOTAP; Boehringer Mannheim). After 8 h of
transfection, the culture medium was changed, and selection of the
clones was performed in the presence of 1 mg/ml Geneticin (G418).
Receptor expression was assessed by binding of the specific TP
antagonist [3H]SQ29,548 as well as by the agonist
(U46619)-stimulated increase in total inositol phosphate formation.
Binding of [3H]SQ29,548 was performed on intact cells as follows. Briefly, subconfluent adherent cells in 24-well plates (2.5 × 105 cells) were incubated for 30 min at 37 °C in 250 µl of serum-free DMEM, 0.2% bovine serum albumin (BSA) in the presence of increasing concentrations of [3H]SQ29,548. Cells were washed twice in ice-cold phosphate-buffered saline (PBS), containing 0.02% BSA and lysed in 0.5 N NaOH. The protein content was determined by micro-BCA® assay (Pierce) with the microbicinchoninic acid reagent and BSA as a standard. Cell number was assessed in parallel wells. Nonspecific binding was determined by the addition of 10 µM unlabeled SQ29,548 and did not exceed 5-10% of the total binding.
Alternatively, crude membrane fractions were prepared as described (19) with minor modifications. Briefly, cells at confluence in 100-mm dishes were scraped in Hepes buffer (10 mM Hepes, pH 7.6, containing 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, and 10 µM indomethacin), sonicated, and centrifuged for 15 min at 1,000 × g at 4 °C. Supernatants were further centrifuged at 125,000 × g at 4 °C for 1 h, and pellets were resuspended in the same buffer, containing 10% glycerol and 100 mM NaCl. Radioligand binding was performed using 50 µg of membrane protein and increasing concentrations of [3H]SQ29,548. Reactions were carried out in a total volume of 100 µl at 4 °C for 2 h. The reaction was stopped with 4 ml of 10 mM Tris, pH 7.4, followed by filtration through Whatman GF/C glass filters. Filters were subsequently washed twice with the same buffer and counted for radioactivity. Nonspecific binding was determined in the presence of excess unlabeled SQ29,548 (25 µM). It did not exceed 15% of the total binding. For competition binding experiments, 50 µg of membrane protein was incubated with 40 nM [3H]SQ29,548 and increasing concentrations of U46619 (0.1-30 µM). Reactions were carried out under the same conditions as above. The data were subject to Scatchard analysis, and an apparent Kd and Bmax were determined using a computer program (Radlig Biosoft®, Cambridge, UK). The same program was used for analysis of the competition experiments and for calculation of Ki values for U46619.
Total Inositol Phosphate FormationInositol phosphate formation was measured as described previously (23). Briefly, confluent cells (4-5 × 105 cells in 12-well plates) were labeled with 2 µCi of myo-[2-3H]inositol (18.3 Ci/mmol) for 20-24 h in serum-free, inositol-free DMEM, containing 0.5% BSA, 20 mM Hepes. The culture medium was changed, and cells were incubated in the same medium containing 20 mM LiCl. After a 10-min incubation, U46619 was added for 10 min unless otherwise indicated. When SQ29,548 was used, it was added for 10 min prior to the addition of U46619. The reaction was stopped by aspiration of the supernatant and the addition of 0.75 ml of 10 mM formic acid. After incubation for 30 min at room temperature, the solution was collected in 3 ml of 5 mM ammonia hydroxide, giving a pH of 8.5-9. Samples were subjected to an anion exchange AG 1X-8 column. Free inositol and glycerophosphoinositol were washed with a 40 mM concentration of a formate/formic acid buffer, pH 5. Total inositol phosphates were eluted with 4 ml of the 2 M formate/formic acid buffer from which 1 ml was counted. Results were expressed as a percentage of the increase in agonist-stimulated total inositol phosphate formation compared with unstimulated cells.
Ca2+ ReleaseIntracellular Ca2+
levels ([Ca2+]i) were measured in TP- or
TP
-transfected HEK 293 cells in suspension as described previously
(24). Briefly, cells were washed twice with PBS, loaded for 1 h at
37 °C with 5 µM Fura-2/AM (Molecular Probes, Eugene,
OR) in phenol red free-RPMI 1640 culture media. Cells were further
washed and incubated for 5 min in PBS, containing 1 mM EDTA
and 5 mM EGTA, harvested, washed again, and resuspended at
106 cells/ml in RPMI 1640. Cells were allowed to sit for
1 h. Fluorescence was detected in suspended cells diluted at
106 cells/ml, using an SLM/Aminco AB2 spectrophotometer
(Urbana, CA), and approximate values of [Ca2+]i
were calculated using a Kd of 224 nM for Fura-2.
Cells (1 × 106/ml, 1.5 ml) were incubated with 1 µM U46619 for the indicated times in the desensitization experiments. They were then washed and further stimulated with 300 nM U46619. Control cells were treated with vehicle (i.e. ethanol, 0.01%) alone and further assessed in parallel for mobilization of [Ca2+]i.
Generation of Specific Anti-peptide AntibodiesRabbit
polyclonal antibodies were raised to immunoprecipitate the TP receptor
isoforms. These were directed against the last 15 amino acids of the
carboxyl-terminal ends of the isoforms: NH2-SLSLQPQLTQRSGLQ-COOH (amino acid sequence 327-341,
referred to as Ab) for TP
and
NH2-(C)PFEPPTGKALSRKD-COOH (amino acid sequence 355-369,
referred to as Ab
) for TP
. The extra cysteine residue was added
in the TP
peptide to allow coupling to BSA and acetylcholinesterase
(see below). Immunization procedures and the generation of peptides
were performed as described previously (25). Boosters were administered
every 2 months. Sera from the same rabbit and within the same booster
were pooled and kept sterile at 4 °C in the presence of 0.01%
sodium azide. The titer of the different sera was tested in a
competitive enzyme immunoassay, using the corresponding TP receptor
peptides covalently linked to acetylcholinesterase. The antisera Ab
and Ab
had titers of 1/5000-1/10000 and 1/3000-1/5000,
respectively. Immunoaffinity columns of each antibody were prepared for
immunoprecipitation analysis. Antisera were first partially purified
using the E-Z-SEP® kit (Pharmacia) and further incubated
with CNBr-activated Sepharose according to the manufacturer's
instructions. Normal rabbit immunoglobulins, coupled to CNBr-activated
Sepharose, were used for preclearing of cell lysates.
Immunoblot analysis of total cell lysates or of the immunoprecipitates
was performed to characterize the antibodies. Cell membranes were
prepared as described previously, and 50 µg were mixed with 1 volume
of 2 × Laemmli buffer (1 × Laemmli buffer: 4% SDS, 5%
glycerol, 60 mM Tris, pH 6.8, and 0.005% bromphenol blue)
under reducing conditions (75 mM dithiothreitol) and
vortexed. Samples were treated under nonreducing conditions for the
immunoprecipitation experiments. SDS-polyacrylamide gel electrophoresis
was performed using 10% acrylamide for the separating gel. Proteins
were electrotransferred onto nitrocellulose membranes (Schleicher and
Schuell, Keene, NH). Blots were saturated for 2 h in Tris-buffered
saline (50 mM Tris, pH 7.5, 250 mM NaCl, 0.1%
Tween 20) containing 2% BSA. Membranes were further incubated
overnight at 4 °C with Ab (1/2000) or Ab
(1/1000) in
Tris-buffered saline containing 0.5% BSA. Membranes were washed five
times for 10 min in the same buffer without BSA and incubated for
1 h at room temperature with a 1/5000 dilution of a donkey anti
rabbit antibody coupled to horseradish peroxidase (Jackson
ImmunoResearch, West Grove, Pa). Excess antibody was washed, and
positive bands were revealed by ECL (Amersham) according to the
manufacturer's instructions.
HEK 293 cells overexpressing TP
receptor isoforms were subcultured in a two-well Lab-Tek slide culture
chamber (Nunc, Naperville, IL) coated with 10 µg/ml of human
fibronectin (Life Technologies, Inc.). Subconfluent cells were washed
once with PBS buffer and fixed in 100% methanol. Fixed specimens were
blocked for 30 min at room temperature in PBS containing 2% BSA.
Specimens were incubated with the receptor antibodies (diluted at
1/1000 for Ab and 1/500 for Ab
) in PBS, containing 0.5% BSA for
1 h at room temperature followed by fluorescein
isothiocyanate-conjugated donkey anti-rabbit IgG (Jackson
ImmunoResearch) at a dilution of 1/1000 in the same PBS solution.
Slides were mounted in a mounting solution (Vector Laboratories Inc.,
Burlingame, CA). Immunofluorescence staining was examined by an
Olympus® inverted fluorescence microscopy.
Whole cell phosphorylation was performed as described (26) with minor modifications. Briefly, subconfluent cells in 60-mm dishes (2-3 × 106 cells) were washed once in phosphate-free DMEM media, containing 20 mM Hepes, 0.5% BSA as above. Cells were further incubated for 45 min in the same medium, containing 100 µCi/ml of [32P]orthophosphate (6,000-7,000 Ci/mmol). Kinase inhibitors, SQ29,548, or vehicle were added during the labeling period. Stimuli or vehicle were added for 10 min, unless otherwise indicated. Me2SO and ethanol concentrations did not exceed 0.1% and did not modify the pattern of phosphorylation of the TP isoforms. The reaction was quenched by transferring the dishes on ice and aspirating the supernatants. Cells were washed with 2 ml of ice-cold PBS/dish and lysed with 0.8 ml of radioimmune precipitation buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40 (v/v), 0.5% sodium deoxycholate (w/v), 0.1% SDS (w/v) containing 10 mM sodium fluoride, 25 mM sodium pyrophosphate, 10 mM ATP, 1 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 1 mM benzamidine hydrochloride, and 0.5 mM phenylmethylsulfonyl fluoride). Cells were scraped with a rubber policeman after 15 min on ice, passed through a 231/2 gauge needle, and centrifuged for 15 min at 10,000 × g at 4 °C. Supernatants were collected, and total cell protein was measured by micro BCA®, using BSA as a standard. Lysates (0.8 mg of total cell protein in 0.8 ml) were precleared for 1 h at 4 °C with 50 µl of normal rabbit IgG covalently coupled to Sepharose CL-4B. The preclearing step was critical in the phosphorylation assays, since it removed a phosphorylated doublet, which migrates at the same molecular weight as the TP isoforms.
Samples were centrifuged for 3 min at 10,000 × g, and
supernatants were further immunoprecipitated overnight at 4 °C using 50 µl of immunoaffinity Sepharose for either Ab or Ab
, as
described above. The beads were washed four times with 1 ml of
radioimmune precipitation buffer and resuspended in 100 µl of 1 × Laemmli buffer under nonreducing conditions. Samples were vigorously
vortexed for 15 min, centrifuged for 5 min at 10,000 × g, and loaded onto SDS-polyacrylamide gels, as described
above. Quantitative analysis of radioactivity in the samples was
performed using a PhosphorImagerTM 445-SI (Molecular
Dynamics Inc., Sunnyvale, CA) after the gels were dried. Identical
areas were integrated, and results were represented as percentages of
the control values.
To determine whether TP or TP
couples
differentially to downstream signals, we developed HEK 293 cells stably
overexpressing both isoforms. Normal HEK 293 cells as well as cells
transfected with the pcDNA3 vector alone (HEK 293-VEC) showed no
detectable amounts of endogenous TPs in binding assays. Among several
HEK 293 clones stably overexpressing the receptors, we selected clones that exhibited similar levels of receptors as expressed per number of
binding sites per cell or per pmol of receptor per mg of total protein
(Table I). The stably overexpressing clones, TP
-5 and TP
-17, bind [3H]SQ29,548 in a saturable manner.
Scatchard analysis revealed similar dissociation constants
(Kd) 21.9 ± 2.2 nM and 24.4 ± 1.8 nM for the TP
and TP
receptor isoforms,
respectively (Table I). The binding of [3H]SQ29,548 was
displaced with high concentrations of U46619. The Ki
values for U46619 were 1.95 ± 0.32 µM (mean ± S.E., n = 4) for TP
and 1.17 ± 0.17 µM (mean ± S.E., n = 3) for TP
(p value not significant).
|
We
assessed the functionality of the expressed isoforms by measurement of
agonist-induced total inositol phosphate (IP) formation and calcium
mobilization. Both the TP-5 and TP
-17 cell lines exhibited an
increase in IP formation in the presence of U46619, a prostaglandin
H2/TxA2 mimetic. IP formation was similar in
the presence (883 ± 33.2 dpm) and the absence of U46619 (901 ± 180 dpm) in HEK 293 cells transfected with the pcDNA3 vector
alone and corresponded to values observed in unstimulated TP
-5 and TP
-17 clones.
The addition of increasing amounts of U46619 (3-3000 nM)
for 10 min resulted in a 5-8-fold increase in IP formation, which reached a plateau at around 100 nM for TP and 300 nM for TP
(Fig. 1). The EC50
values for U46619 were higher for TP
than TP
(57 ± 6.7 nM versus 11.1 ± 2 nM,
respectively, p < 0.002). U46619 rapidly increased IP
formation via both isoforms (4 min). The increase in IP formation was
linear up to 60 min (data not shown). Preincubation of either TP
-5
or TP
-17 with the antagonist SQ29,548 abolished the ability of
either to transduce IP formation in response to U46619 stimulation
(data not shown).
When TP-5 and TP
-17 were loaded with Fura-2, U46619 induced a
rapid increase in intracellular [Ca2+]i (Fig.
2). In TP
-5 (Fig. 2A, left
part), the increase in [Ca2+]i mobilization
was sustained up to 10 min and was dependent on extracellular sources,
since chelation of extracellular Ca2+ by pretreating the
cells with 2 mM EGTA for 45 s led to a rapid return to
the base line. In contrast, U46619 induced a more transient Ca2+ mobilization in TP
-17 (Fig. 2B,
left part), although the response did not return completely
to base line. Basal [Ca2+]i was 46 ± 13 nM (mean ± S.D., n = 21) for TP
and 51 ± 18 nM (mean ± S.D., n = 13). For both isoforms, the presence of EGTA depressed
Ca2+ mobilization by roughly 60-70%. Thus,
Ca2+ mobilization is dependent on extracellular, as well as
intracellular, pools of Ca2+. Similarly, preincubation with
1 µM U46619 abolished the increase of Ca2+ in
response to a second addition of U46619 in both cell lines (Fig. 2,
A and B, right parts). Homologous
desensitization of the Ca2+ response to U46619 was
demonstrable for concentrations of U46619 as low as 10 nM
and for periods of preincubation as short as 2 min (data not shown). No
significant increase was observed when cells were incubated with
vehicle alone or when they were pretreated with SQ29,548. HEK 293-VEC
showed no Ca2+ mobilization (data not shown).
Immunoblotting and Immunoprecipitation of the Thromboxane Receptors
Anti-peptide antibodies were raised against amino acid
sequences unique to either the human TP or the human TP
isoform. Both antibodies demonstrated specific immunoreactivity toward the
receptors, as assessed by immunoblot analysis and immunoprecipitation of TP
-5 or TP
-17. Fig. 3A represents an
immunoblot analysis of thromboxane receptors in crude membrane
fractions of the TP
-5 and TP
-17 cells. The antiserum Ab
reacted with a broad protein band ranging from 55 to 65 kDa in TP
-5
(Fig. 3A, lane 2). Similar results were obtained
for the Ab
with the TP
-17 membrane fractions (Fig. 3A,
lane 4). No immunoreactivity was revealed in membranes from
cells transfected with vector alone, by either Ab
(Fig. 3A, lane 1) or Ab
(data not shown).
Preincubation of the Ab
or Ab
with the corresponding peptides
suppressed the immunoreactivity in TP
-5 (Fig. 3A,
lane 3) or TP
-17 (Fig. 3A, lane 5),
respectively. The Ab
and Ab
antibodies did not cross-react (data
not shown), indicating their specificity. The capability of these
antibodies to immunoprecipitate the TP isoforms was demonstrated using
immunoaffinity columns prepared with either Ab
or Ab
as described
under "Experimental Procedures." Immunoaffinity columns prepared
with these antibodies immunoprecipitated a broad protein band from
TP
-5 and TP
-17 (Fig. 3B, lanes 3 and
4, respectively), with an apparent molecular weight similar
to that obtained by direct immunoblotting of the membrane fractions. It
is important to note that since Ab
and Ab
are different sera and
could have different characteristics, it is not possible to compare the
quantities of the two isoforms that are immunoprecipitated. Similarly,
immunoprecipitation of a human platelet cell lysate with the Ab
revealed a broad protein band with an apparent molecular weight of
45-50 (Fig. 3B, lane 5). No immunoreactivity
could be detected when immunoprecipitation of the platelet cell lysate
was performed with the Ab
.
Membrane Localization of the TP Receptor Isoforms
The
antibodies Ab and Ab
recognized the receptors in situ
as assessed by immunofluorescence staining of TP
-5 and TP
-17 clones (Fig. 4, A and C,
respectively), Almost all of the cells stained positively for the TPs.
Immunofluorescence staining was uniformly distributed over the cell
surface. No staining was detected in the absence of Ab
or Ab
(Fig. 4, B and D, respectively). Furthermore, no
immunofluorescence was observed when anti-peptide antibodies were
substituted with nonimmune rabbit serum or when the antibodies were
saturated with the corresponding peptides (data not shown).
Specific Phosphorylation of Human TPs by U46619
Incubation of
[32P]orthophosphate-prelabeled TP-5 or TP
-17 with 1 µM U46619 resulted in phosphorylation of a broad
radioactive protein band that migrated at the same molecular weight as
that of the TP isoforms described above (Fig. 5,
A and B, for TP
-5 and TP
-17, respectively).
A very weak signal was detected in the unstimulated cells
(Control) or when HEK 293-VEC were incubated with U46619
(data not shown). The increase in U46619-dependent phosphorylation over unstimulated cells was 4-5-fold
(n = 12) and 3-4-fold (n = 8) for
TP
and TP
, respectively. Preincubation of the cells with 50 µM SQ29,548 prevented phosphorylation of the TPs, whereas
SQ29,548 alone did not induce any phosphorylation. The nature of the
weakly phosphorylated band observed in the unstimulated cells is
difficult to determine, since preclearing of the cell lysates with the
nonimmune rabbit serum eliminated a nonspecific but strongly
radioactive doublet that migrates exactly at the same molecular weight
as that of TP
and TP
. Immunoblot analysis of the
immunoprecipitated receptors in these samples showed no difference in
the quantity of the TP isoforms immunoprecipitated (data not shown).
This indicates that the antisera may recognize both the phosphorylated
and the unphosphorylated isoforms of the receptors. This is important,
since the antibodies are directed against a polypeptide sequence
present in the carboxyl-terminal end, where phosphorylation of the
receptors may occur (9).
Dose- and Time-dependent Phosphorylation of the TP
32P-Labeled TP-5
and TP
-17 were stimulated with increasing concentrations of U46619
(1-1000 nM). Phosphorylation of the TP isoforms was
detectable in TP
-5 cells at concentrations as low as 3 nM U46619 and reached a plateau at ~ 100 nM (Fig. 6A). Similar results
were obtained for TP
-17 cells (Fig. 6B). The
EC50 values for U46619 for phosphorylation of the TP
and TP
isoforms were similar (12 ± 2 nM for TP
,
n = 3; 11.5 ± 1.3 nM for TP
,
n = 4) (Fig. 6B). Phosphorylation of the
TP
and TP
(Fig. 7, A and B,
respectively) receptors was rapidly (1 min) detected at a saturating
concentration of U46619 (300 nM) and reached a plateau
at ~ 30 min. Incubation times of up to 90 min resulted in no
further change in the phosphorylation pattern. Basal phosphorylation
was detected at longer incubation times (>30 min).
Effect of Protein Kinase C Inhibition on U46619-dependent Phosphorylation of the TP Isoforms
To study the role of PKC activation in the
U46619-dependent phosphorylation of the TP receptors, we
first tested the effect of a specific inhibitor of PKC, the
bisindolylmaleimide I (GF 109203X) (27). PMA alone (100 nM)
induced 2-3-fold phosphorylation of the TPs over unstimulated cells
and roughly 2-fold for TP-17. No phosphorylation was observed with
4
-PMA, an isomer of PMA that does not activate PKC. Increasing
concentrations of GF 109203X (2-10 µM) inhibited the
PMA-dependent phosphorylation of the TP isoforms by
70-90% (Fig. 8, A and B).
Although high concentrations of GF 109203X reduced basal
phosphorylation of the both TP isoforms, they inhibited
U46619-dependent phosphorylation by only about 30%.
Similar percentages of inhibition were obtained at lower concentrations
of U46619. Prolonged preincubation with GF 109203X for up to 2 h
or the use of a distinct PKC inhibitor, RO-31-8220 (28), in TP
-5
cells gave similar results (data not shown). Furthermore, thrombin (2 units/ml) failed to induce phosphorylation of the TP isoforms, although
it did cause a significant increase (80-100 nM) in
[Ca2+]i mobilization (data not shown). Similar
results were obtained when the cells were transiently transfected with
the cDNA for the human thrombin receptor despite a 2-fold increase in thrombin receptor expression as analyzed by flow cytometry using
specific anti-peptide antibody against human thrombin receptor (data
not shown).
PKC down-regulation was accomplished by incubating the cells for
24 h in the presence of PMA (200 nM). Under these
conditions, both TP or TP
were still phosphorylated by 1 µM U46619, but not by 100 nM PMA (Fig.
9).
Effect of Protein Kinase A Inhibition on U46619-dependent Phosphorylation of the TP Receptors
Incubation of prelabeled cells with 10 µM
FK in the presence of 0.5 mM IBMX resulted in a weak
phosphorylation of the TP isoforms. The capacity for
PKA-dependent phosphorylation of both TP isoforms was
illustrated when Bt2cAMP was incubated for 30 min in the
presence of IBMX. The phosphorylation of TP
receptors by FK was
strongly inhibited by H-89 (29), a competitive inhibitor of PKA (Fig.
10A). Under these conditions,
U46619-dependent TP receptor phosphorylation was not
inhibited by H-89.
Homologous Desensitization of TP Isoform-mediated Inositol Phosphate Formation
Pretreatment of TP-5 or TP
-17 with 300 nM U46619 for 10 min resulted in 70-90% inhibition of IP
formation in response to a subsequent addition of U46619 (Fig.
11, A and B). Desensitization was
rapid for both receptors, reaching a maximum after 1-2 min of
pretreatment (Fig. 11C). Maximal desensitization was
observed when pretreating the cells with ~100 nM U46619.
Moreover, incubation of cells with 5 µM GF 109203X prior
to pretreatment of the cells with U46619 did not modify the pattern of
desensitization for either the TP
or TP
isoform, as assessed by
IP formation (Fig. 12, A and B,
respectively). Along with these results, the pretreatment of the cells
with either 200 nM PMA for 10 min or 0.5 mM
Bt2cAMP for 30 min did not modify U46619-mediated IP
formation (Table II). Neither PMA or Bt2cAMP
pretreatment modified the extent of homologous desensitization of TPs
under these conditions.
|
TxA2 is an evanescent biological mediator; it exerts potent effects on platelet function and vascular tone in the immediate microenvironment of its formation (18). Given its critical role in determining vascular patency, it would seem likely that its formation and effects would be tightly regulated. TxA2 is not stored, preformed in cells. Rather, it is formed and released rapidly in response to cellular (e.g. platelet) activation by diverse stimuli. Its effects are limited by its hydrolysis to the inactive thromboxane B2, which has a half-life estimated to be 30 s at physiological pH (2) and by homologous desensitization of its membrane receptor-mediated responses (30, 31). This desensitization appears to result initially from uncoupling of the TP from its attendant G proteins; we have previously estimated that the half-life of such a response (coupling to phospholipase C in human platelets) is approximately 120 s. This phenomenon is followed by more gradual loss of binding sites from platelet membranes (30).
A single gene encoding a TP has been cloned (5). This predicts membership of the TPs in the family of GPCRs, consistent with their biochemical characteristics (32). It appears that the TP is subject to alternative splicing in the carboxyl-terminal region, as has been previously described for E prostaglandin receptor type 3 (33). Little is known about the functional significance of this observation. However, pharmacological studies have indicated the possibility of tissue-specific differences in the characteristics of TPs and, indeed, differences in TP binding of ligands within a single cell, platelets (11, 34, 35). Observations involving E prostaglandin receptor carboxyl-terminal isoforms indicate that they are capable of coupling to distinct downstream signaling systems and that they may differ in the rate and extent to which they are subject to homologous desensitization (36). Narumiya and colleagues have recently reported that mRNAs for the two TP isoforms are expressed in human platelets. When the isoforms are overexpressed in COS cells, they may regulate adenylate cyclase activation differentially (10).
Given these observations, we wished to explore the molecular mechanisms that might underlie homologous desensitization of the two TP isoforms. Importantly, they differ in the length and number of potential target residues for phosphorylation (9), raising the possibility of differential tissue responses to TxA2 (e.g. in platelets and the vasculature) based on differential rates of desensitization. To address this possibility and to clarify the role of specific kinases in the desensitization process, we characterized specific, peptide-based antibodies for the two receptors. These confirmed the membrane localization of the two receptor isoforms, as inferred by their sequences.
Interestingly, both isoforms were rapidly phosphorylated following
exposure to agonist. The time and dose dependence of this phenomenon
seemed similar for the two isoforms. Both isoforms coupled to
downstream signaling systems. We focused on phospholipase C-dependent events, since these are thought most relevant
to TxA2-mediated platelet aggregation and vasoconstriction
(11, 34). Again the TP agonist, U46619, could stimulate an increase in
[Ca2+]i and total inositol phosphates via both
isoforms. However, the pattern of the calcium response evoked via the
two isoforms appeared to differ, at least in the stable cell lines that
we established. Thus, whereas U46619 induced a rapid transient,
followed by a delayed plateau in cells expressing TP, the plateau
phase was almost absent in the cells expressing TP
. Activation of
both isoforms appeared to involve release of calcium from intracellular stores. However, the plateau phase of the TP
response appeared to
derive from an extracellular source, raising the possibility of linkage
to a receptor-activated calcium channel (37). Further experiments will
address this possibility and whether a similar distinction is evident
in other cells transfected with the two isoforms.
Given the role of phospholipase C-mediated responses in the biological
consequences of TP activation, it would seem likely that downstream
activation of PKC might play a central role in homologous
desensitization. Indeed, we have previously shown that PKC may
phosphorylate a fusion protein based on the carboxyl-terminal end of
the TP receptor (residues 321-343) in vitro (19).
Peptide competition experiments suggested that this involved sites in the third extracellular loop and the carboxyl-terminal tail (19). Okwu
et al. (20) have recently presented data consistent with TP
phosphorylation in human platelets. Phosphorylation plays an important
role in regulating both homologous and heterologous desensitization of
GPCRs like the TPs. Additionally, other serine/threonine kinases such
as PKA, G protein-coupled receptor-dependent kinases, and
tyrosine kinases have been implicated in GPCR phosphorylation (38-40).
We have demonstrated that PKA may phosphorylate TP
to a minor degree
in vitro (19). Similarly, we demonstrate the capacity for
PKA to phosphorylate the receptor in the present study in
vivo. However, PKA appears to contribute trivially, if at all, to
rapid phosphorylation of the TPs induced by U46619. Although there is
no information on G protein-coupled receptor-dependent kinase-mediated phosphorylation of eicosanoid receptors, recent studies
have demonstrated their ability to phosphorylate angiotensin and
adrenoreceptor GPCRs (41, 42).
We also demonstrate that PKC appears to play a modest role in agonist-dependent phosphorylation of either TP isoform. Thus, two specific competitive inhibitors of PKC, GF 109203X and RO 31-8220, failed to modify the rate or extent of agonist-stimulated phosphorylation of either isoform. Similarly, down-regulation of PKC, by prolonged exposure to the phorbol ester, PMA, also failed to alter these phenomena. We demonstrate that homologous desensitization of agonist-evoked increases in IPs is similar for both isoforms. Again, the PKC inhibitor, GF 109203X, failed to modify homologous desensitization of this response evoked via either isoform. Although PKC appears largely irrelevant to the agonist-mediated desensitization response, activation of this enzyme may phosphorylate TPs. Thus, short term exposure to PMA will result in phosphorylation of both isoforms. However, treatment of the cells with thrombin (2 units/ml) did not induce phosphorylation of the TPs, although cells were activated, as assessed by [Ca2+]i mobilization. Transient co- expression of the human thrombin receptor and stimulation with thrombin did not result in the phosphorylation of the TP receptor isoforms. Similarly, we have recently shown that phosphorylation of the prostacyclin receptor (43) can be induced by thrombin in a PKC-dependent manner, confirming that the endogenous thrombin receptor in HEK-293 cells is sufficient to mediate PKC-dependent phosphorylation of a related GPCR.
In contrast to agonist-dependent phosphorylation, the PKC inhibitors appeared to diminish basal phosphorylation of both isoforms.
Similar to our findings with PKC, PKA appeared to be of trivial relevance to agonist-dependent phosphorylation of either isoform. Both FK and Bt2cAMP treatment of the stable transfectants resulted in phosphorylation of both isoforms under favorable conditions (i.e. pharmacological inhibition of phosphodiesterases), extending our previous in vitro observations (19). However, the PKA inhibitor, H-89, had no apparent effect on U46619-induced phosphorylation of either isoform. Although U46619 is a potent stimulus to phospholipase C activation, higher concentrations may also activate adenylate cyclase (10). However, these concentrations do not appear to result in TP phosphorylation by PKA in vivo. It is likely that, as with PKC, activation of this enzyme might assume more importance in TP phosphorylation during heterologous desensitization.
The use of our antibodies identified TPs as a broad band ranging from
55 to 65 kDa. This molecular mass is somewhat higher than that
previously observed by others in human platelets (44, 45). Indeed, we
identified a 45-50-kDa protein when we immunoprecipitate human
platelet TP receptors with Ab (Fig. 3B, lane
5). This may reflect different degrees of glycosylation or
palmitoylation (46) of TP isoforms in HEK-293 cells compared with
platelets. Interestingly, the molecular weight of the phosphorylated
protein is similar to that in the immunoblot analysis. The TP
antagonist prevents phosphorylation of that band, further confirming
its identity.
In conclusion, we have demonstrated that the rate and extent of agonist-dependent phosphorylation of the two cloned TP isoforms is similar, although their coupling to [Ca2+]i and IP formation is somewhat different. Neither PKC nor PKA plays a major role in such phosphorylation of either isoform, implicating the G protein-coupled receptor-dependent kinases in homologous desensitization of these receptors. However, both TP isoforms are substrates for PKC and PKA in vivo, and these enzymes may play a more important role in their heterologous desensitization or in receptor regulation in other cells. The availability of specific antibodies for TP isoforms is likely to facilitate investigation of their comparative distribution and biology.
We are grateful for the help of Dr. Yu-Min Shen in preparing the stable transfected cells and Maryline Lebret for a contribution in the development of antisera. We thank Dr. Marina Molino and Peter O'Brien for help in performing the Ca2+ experiments and the flow cytometry of the thrombin receptor and Drs. Ellen Van Obberghen-Schilling and Lawrence Brass for helpful advice.