From the Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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Thromboxane A2
(TXA2) potently stimulates platelet aggregation and smooth
muscle constriction and is thought to play a role in myocardial
infarction, atherosclerosis, and bronchial asthma. The TXA2
receptor (TXA2R) is a member of the G protein-coupled receptor family and is found as two alternatively spliced isoforms, Thromboxane A2
(TXA2)1 has a
variety of pharmacologic effects which modulate the physiological
responses of several cells and tissues (1). It is a product of the
sequential metabolism of arachidonic acid by the cyclooxygenases and
TXA2 synthase (2). TXA2 formation can result
from activation of various cell types, including platelets,
macrophages, and vascular smooth muscle cells (1). Binding of
TXA2 to its receptor (TXA2R) induces platelet aggregation, constriction of vascular and bronchiolar smooth muscle cells, as well as mitogenesis and hypertrophy of vascular smooth muscle
cells. TXA2 has been implicated in a wide variety of
cardiovascular diseases (1).
While pharmacological studies have suggested the existence of
TXA2R subtypes (3), the receptor appears to be encoded by a
single gene that can be alternatively spliced in the carboxyl-terminal tail (C-tail) leading to two variants, TXA2R For many G protein-coupled receptors (GPCRs), the presence of Ser and
Thr phosphorylation sites is critically important in receptor
desensitization and internalization (see Ref. 19, and references
therein). Homologous receptor desensitization appears to be primarily
initiated by phosphorylation of the agonist-activated GPCR by a family
of Ser/Thr kinases known as G protein-coupled receptor kinases (GRKs).
This leads to high affinity binding of a second class of proteins
referred to as arrestins (for review, see Refs. 19-21), resulting in
steric inhibition of G protein binding (22, 23). Recently, non-visual
arrestins were also found to promote receptor uptake into
clathrin-coated pits. The non-visual arrestins, arrestin-2 and
arrestin-3,2 function as
adaptor proteins to recruit receptors to coated pits by virtue of their
ability to bind to both activated, phosphorylated GPCRs and clathrin
(24). Studies of the m2 muscarinic acetylcholine (25, 26), angiotensin
II (27), We were interested in determining if the different C-tails of the
TXA2R Cell Culture and Transient Transfection--
Human embryonic
kidney cells (HEK293) and human epidermoid carcinoma cells (A431) were
maintained in Dulbecco's modified Eagle's medium (DMEM, Life
Technologies, Inc.) supplemented with 10% fetal bovine serum. MiaPACA
(human pancreatic carcinoma cells) were grown in the same medium also
containing 2.5% horse serum (Life Technologies, Inc.). Mouse lymphoid
neoplasm cells (P388D1) were maintained in RPMI supplemented with 15%
fetal bovine serum while CHO cells (Chinese hamster ovary cells) were
grown in Ham's F-12 with 10% fetal bovine serum. All cells were kept
in a humidified atmosphere of 95% air, 5% CO2 at
37 °C. Transfections were done with Fugene-6 (Boehringer Mannheim)
according to the manufacturer's recommendations.
Construction of Epitope-tagged TXA2 Receptors
and Mutants--
cDNAs for the TXA2R Radioligand Binding Assays--
Competition binding curves were
done on HEK293 cells expressing wild-type and mutant receptor species.
Cells were harvested and washed twice in Buffer A (10 mM
Hepes, pH 7.6, 129 mM NaCl, 8.9 mM
NaHCO3, 0.8 mM KH2PO4,
0.8 mM MgCl2, 5.6 mM dextrose,
0.38% sodium citrate, pH 7.4, 5 mM EDTA, and 5 mM EGTA). Binding reactions were carried out on 5 × 104 cells in a total volume of 0.25 ml in the same buffer
with 10 nM [3H]SQ29543 (a TXA2
antagonist) and increasing concentrations of nonradioactive SQ29543 for
2 h at room temperature. Reactions were stopped by centrifugation
at 14,000 rpm in a microcentrifuge for 1 min and the cell-associated
radioactivity was measured by liquid scintillation.
Internalization Assays--
For quantification of receptor
internalization, ELISA assays were performed essentially as described
by Daunt et al. (37). The cell lines were plated out at
6 × 105 cells per 60-mm dish, transfected with 6 µg
of DNA and split after 24 h into 6 wells of 24-well tissue culture
dishes previously coated with 0.1 mg/ml poly-L-lysine
(Sigma). After another 24 h, the cells were washed once with PBS
and incubated in DMEM at 37 °C for several minutes. Then the
TXA2 agonist U46619 was added at a concentration of 100 nM in prewarmed DMEM to the wells. The cells were then
incubated for various times at 37 °C and reactions were stopped by
removing the media and fixing the cells in 3.7% formaldehyde/TBS for 5 min at room temperature. The cells were then washed three times with
TBS and nonspecific binding blocked with TBS containing 1% BSA for 45 min at room temperature. The first antibody (monoclonal HA 101R, Babco)
was added at a dilution of 1:1000 in TBS/BSA for 1 h at room
temperature. Three washes with TBS followed, and cells were briefly
reblocked for 15 min at room temperature. Incubation with goat
anti-mouse conjugated alkaline phosphatase (Bio-Rad) diluted 1:1000 in
TBS/BSA was carried out for 1 h at room temperature. The cells
were washed three times with TBS and a colorimetric alkaline
phosphatase substrate was added. When the adequate color change was
reached, 100-µl samples were taken for colorimetric readings. Cells
transfected with pcDNA3 were studied concurrently to determine
background. All experiments were done in triplicate.
Inositol Phosphate Determination--
HEK293 cells were seeded
at a density of 80,000 cells per well of 12-well plates and transfected
as described above with the WT or mutant receptors and labeled the
following day for 18-24 h with
myo-[3H]inositol at 4 µCi/ml in DMEM (high
glucose, without inositol). After labeling, cells were washed once in
phosphate-buffered saline (PBS) and incubated in prewarmed DMEM (high
glucose, without inositol) containing 0.5% BSA, 20 mM
Hepes, pH 7.5, and 20 mM LiCl for 10 min. Cells were then
stimulated for 10 min with different concentrations of U46619. The
reactions were terminated by removing the stimulation media and by the
addition of 0.8 ml of 0.4 M perchloric acid. Samples were
harvested in Eppendorf tubes, and a 0.5 volume of 0.72 N
KOH, 0.6 M KHCO3 was added. Tubes were vortexed
and centrifuged for 5 min at 14,000 rpm in a microcentrifuge. Inositol
phoshates were separated on Dowex AG 1-X8 columns. Total labeled
inositol phosphates were then counted by liquid scintillation.
Immunofluorescence Microscopy--
Cells (HEK293, P388D1,
MiaPACA, A431, and CHO) were grown in 35-mm dishes on coverslips and
then transfected as described above with 2 µg of DNA/well. After
48 h, cells were incubated with Flag M1 antibody (1:500 dilution)
for 1 h at 4 °C in DMEM supplemented with 1% BSA and 1 mM CaCl2. Cells were washed twice with PBS
containing 1 mM CaCl2, then treated with 100 nM U46619 for 1 h at 37 °C in DMEM with 0.5% BSA,
20 mM Hepes, pH 7.4, 1 mM CaCl2.
The cells were then fixed with 3.7% formaldehyde/PBS for 15 min at
room temperature, washed with PBS/CaCl2, and permeabilized with 0.05% Triton X-100/PBS/CaCl2 for 10 min at room
temperature. Nonspecific binding was blocked with blotto (0.05% Triton
X-100/PBS/CaCl2 containing 5% nonfat dry milk) for 30 min
at 37 °C. Goat anti-mouse fluorescein isothiocyanate-conjugated
secondary antibody (Molecular Probes) was then added at a dilution of
1:150 in blotto for 1 h at 37 °C. The cells were then washed
six times with permeabilization buffer with the last wash left at
37 °C for 30 min. Finally, the cells were fixed with 3.7%
formaldehyde as described. Coverslips were mounted using Slow-Fade
mounting medium (Molecular Probes) and examined by microscopy on a
Nikon Eclipse E800 fluorescence microscope using a Plan Fluor 60×
objective. Cells expressing the lowest levels of transfected proteins,
but clearly above those of nonexpressing cells, were chosen for view.
Images were collected using QED Camera software and processed with
Adobe Photoshop v. 3.0.
A schematic representation of the carboxyl-terminal tails of the
two isoforms of the human TXA2 receptor is shown in Fig. 1. In order to investigate the
internalization of the TXA2R (343 residues) and
(407 residues), which share the first 328 residues. In the present report, we demonstrate by enzyme-linked immunosorbent assay and immunofluorescence microscopy that the TXA2R
, but not the TXA2R
, undergoes
agonist-induced internalization when expressed in HEK293 cells as well
as several other cell types. Various dominant negative mutants were
used to demonstrate that the internalization of the
TXA2R
is dynamin-, GRK-, and
arrestin-dependent in HEK293 cells, suggesting the
involvement of receptor phosphorylation and clathrin-coated pits in
this process. Interestingly, the agonist-stimulated internalization of
both the
and
isoforms, but not of a mutant truncated after
residue 328, can be promoted by overexpression of arrestin-3,
identifying the C-tails of both receptors as necessary in arrestin-3
interaction. Simultaneous mutation of two dileucine motifs in the
C-tail of TXA2R
did not affect agonist-promoted internalization. Analysis of various C-tail deletion mutants revealed that a region between residues 355 and 366 of the TXA2R
is essential for agonist-promoted internalization. These data
demonstrate that alternative splicing of the TXA2R plays a
critical role in regulating arrestin binding and subsequent receptor internalization.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and -
,
that share the first 328 amino acids. Complementary DNAs for the
343-amino acid TXA2R
were cloned from placental and
megakaryocytic sources (4), whereas a cDNA for the 407-amino acid
TXA2R
was isolated from a vascular endothelial library
(5). The TXA2Rs have been shown to couple to the G proteins
Gq, Gi2, G11, G12,
G13, G16, and an 85-kDa unidentified G protein,
explaining the multiplicity of TXA2R-mediated signal
transduction (6-11). While no isoform-specific biological functions
have been ascribed to either of the TXA2Rs, the different
C-tails are likely to play a role in G protein-coupling specificity
(12), and perhaps confer different desensitization characteristics. It
has been suggested that homologous desensitization of the
TXA2Rs in cells can be divided into two stages: the early stage involves uncoupling of receptors from G proteins, while a later
stage involves a loss of receptor sites from the plasma membrane
(13-16). Recently, Habib et al. (17) showed that the dose
and time dependence of agonist-induced receptor phosphorylation appeared similar for both isoforms in HEK293 cells stably
overexpressing the TXA2Rs. However, phorbol ester-promoted
activation of protein kinase C prevented the agonist-mediated
intracellular calcium rise in TXA2R
-, but not
TXA2R
-, stably expressing CHO cells (18).
2-adrenergic (24, 28-31), follitropin (32),
lutropin/choriogonadotropin (LH/CG) (33), and opioid (34) receptors
have implicated the GRKs and arrestins in GPCR internalization.
Similarly, the generation of dominant negative forms of these proteins
confirmed their role in GPCR internalization (25, 28, 35, 36).
and -
could bestow distinct internalization
characteristics to these receptor isoforms. Here, we report that the
TXA2R
, but not the TXA2R
, undergoes
agonist-induced internalization in several different cell types. The
internalization of TXA2R
is inhibited by dominant
negative forms of dynamin, GRK2, and arrestins, while internalization
of both isoforms can be promoted by overexpression of GRK2 and
arrestins. A small region which contains 3 serine residues in the
C-tail of TXA2R
was identified as being essential for
internalization. Taken together, these data suggest a role for receptor
phosphorylation in TXA2R
agonist-induced
internalization, in a process likely involving arrestins and
clathrin-coated pits. Our results indicate that the C-tail of these two
receptors is responsible for their different internalization properties.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
TXA2R
were obtained by reverse transcriptase-polymerase
chain reaction using a pool of total RNA from HeLa, A431, U937, and
MiaPACA cells. Primers used for TXA2R
were
5'-GGAATTCATGTGGCCCAACGGCAGTTCCCTG-3' (TXAF) and
5'-CCGCTCGAGTCTTCCAATGTCTGCATGCCC-3', while the primers TXAF and
5'-CCGCTCGAGCATTCAATCCTTTCTGGACAGAGC-3'(TXAB) were used for
TXA2R
. A pcDNA3 vector containing an HA epitope was
constructed by annealing the two primers
5'-AGCTTCGATCGTCGACATGTACCCATACGATGTTCCAGATTACGCTTCTAGAGGATCCCCGGGCGAGCTCG-3' and
5'-AATTCGAGCTCGCCCGGGGATCCTCTAGAAGCGTAATCTGGAACATCGTATGGGTACATGTCGACGATCGA-3' and ligating them in pcDNA3 digested with HindIII and
EcoRI. The same strategy was employed to generate a
pcDNA3FLAG vector using 5'-AGCTTGGGCACCATGAAGACGATCATCGCCCTGAGCTACATCTTCTGCCTGGTGTTCGCCGACTACAAGGACGATGATGACACCG-3' and
5'-AATTCGGTGTCATCATCGTCCTTGTAGTCGGCGAACACCAGGCAGAAGATGTAGCTCAGGGCGATGATCGTCTTCATGGTGCCCA-3' as primers. The receptors were then subcloned in these vectors, yielding in-frame constructs expressing epitope-tagged receptor polymerase chain reaction. Mutated receptors were constructed by
polymerase chain reaction using the Expand High Fidelity PCR System
(Boehringer Mannheim) according to the manufacturer's recommendations, with the TXA2R
and TXA2R
cDNAs as
templates. Mutants were created using the TXAF primer and the following
corresponding primers: S344Stop,
5'-GAGGAATTCCTACGAGATCGTGCCACTGTACTC-3'; C347Stop,
5'-CAGCTCGAGTCAGCAGTGAGCCGAGATCGTGCC-3'; L351Stop,
5'-CAGCTCGAGTCAGAGGCGGAGGTTGCAGTGAGC-3'; S355Stop,
5'-CAGCTCGAGTCAGCTTGAACCCGGGAGGCGGAG-3'; S362Stop,
5'-CAGCTCGAGTCAGGAGGCTGAGGCACGAGAATC-3'; G366Stop,
5'-CTCCTCGAGTCACCCAGCTGCTCGGGAGGCTGAG-3'; F380Stop,
5'-CTCCTCGAGTCAAAAGAGCATGCAAGGCGGGGC-3'; D385Stop,
5'-CTCCTCGAGTCAGTCAAATTCAGGGTCAAAGAG-3'; G389Stop,
5'-CAGCTCGAGTCACTGTACCCCAGCAAGTAGGTC-3'; F395Stop,
5'-CAGCTCGAGTCAAAAAGGAAGCAACTGTACCCCAGC-3'; and LLLLA,
5'-CAGCTCGAGTCAATCCTTTCTGGACAGAGCCTTCCCTGTTGGAGGTTCAAAAGGAGCCGCCTGTACCCCAGCAGCAGCGTCAAATTCAGGGTCAAAGAG-3'. All the mutants were subcloned in the HA-tag vector as mentioned above.
Mutations and the integrity of the coding sequence were confirmed
by dideoxy sequencing (Sidney Kimmel Nucleic Acid Facility).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and -
, epitope-tagged
TXA2Rs were transiently expressed in HEK293 cells. Similar
to previous reports (5, 12, 17), the TXA2R
and -
display similar binding affinities for the specific TXA2R antagonist SQ29543, with respective Kd values of
11.2 ± 1.4 and 12.4 ± 1.8 nM, indicating that
the C-tails do not affect the binding properties of these receptors.
These observations were confirmed by evaluation of ligand binding to a
mutant receptor truncated after residue 328 (R328Stop)
(Kd = 12.9 ± 1.7 nM). Addition of
an HA or Flag epitope tag at the NH2 terminus of the
receptors also did not alter ligand affinities, nor did it effect the
activation characteristics of the receptors as determined by their
respective EC50 values for inositol phosphate generation in
response to agonist (data not shown).
View larger version (27K):
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Fig. 1.
Schematic representation of the amino acid
sequence of the TXA2R and
TXA2R
carboxyl terminus.
Single lines indicate homology between the C-tail of
TXA2R
and residues 329-344 of TXA2R
.
Double lines illustrate the sites where truncation mutations
were made. Solid circles represent residues that were
mutated to alanines.
The TXA2R expressing HEK293 cells were next assessed for
agonist-promoted loss of cell surface TXA2Rs using an ELISA
assay. A time course analysis of receptor internalization following
stimulation with 100 nM of the agonist U46619 is
illustrated in Fig. 2. While the
TXA2R does not undergo agonist-stimulated
internalization, ~40% of the TXA2R
are internalized
after 2 h of agonist treatment, with a t1/2 of
~45 min. Stimulation of cells with U46619 concentrations ranging from
0.1 to 1000 nM all produced maximal effects which could be
blocked by the specific antagonist SQ29543, while even 1 µM U46619 treatment failed to induce TXA2R
internalization (data not shown). To confirm these results, we
performed an additional series of studies using immunofluorescence microscopy to directly visualize internalization of the Flag-tagged TXA2 receptors in HEK293 cells. Cells were incubated with
the Flag antibody prior to agonist exposure so that only the
trafficking of receptors initially present on the cell surface would be
detected. After agonist treatment, cells were permeabilized and the
Flag epitope was visualized with fluorescein-conjugated secondary
antibody. As shown in Fig. 3A,
the TXA2R
is found primarily at the cell surface even
after the cells have been stimulated with agonist for 1 h. In
contrast, agonist exposure of cells expressing the TXA2R
resulted in a striking redistribution of the receptors to intracellular
compartments. These observations support the results obtained by ELISA
analysis. Interestingly, there is also a significant amount of
TXA2R
redistribution when the cells are incubated at
37 °C in the absence of agonist compared with cells kept at 4 °C
(Fig. 3A). This suggests that the TXA2R
may
also undergo some tonic internalization in the absence of agonist.
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HEK293 cells may contain low levels of endogenous TXA2Rs
(38) and thus would be expected to possess the necessary
"cellular machinery" for TXA2R regulation. However,
we also wanted to test whether these observations could be extended to
other cell types. Thus, similar immunofluorescence microscopy studies
were performed in P388D1 and A431 cells, which contain endogenous
TXA2Rs, and MiaPACA and CHO cells, which lack endogenous
TXA2Rs (data not shown). As depicted in Fig. 3B,
the TXA2R but not TXA2R
undergoes agonist-induced internalization in A431 cells transiently transfected with the epitope-tagged receptors. Comparable results were obtained in
P388D1, MiaPACA, and CHO cells (data not shown). These results demonstrate that the differential internalization of
TXA2R
and -
is a property of the receptors and that
HEK293 cells are a good model for studying TXA2R trafficking.
We next addressed whether the inability of the TXA2R to
internalize was due to low levels of proteins involved in receptor trafficking or a low affinity of the TXA2R
for such
proteins. In recent studies, GRKs and arrestins have been shown to
promote GPCR internalization (24, 25, 27-30, 39, 40). Indeed, it has
been suggested that the extent of
2-adrenergic receptor
internalization correlates with the intracellular levels of GRKs and
arrestins (30). The ability of coexpressed GRK2, arrestin-2, and
arrestin-3 to promote TXA2R
internalization after U46619
stimulation was thus evaluated (Fig.
4A). Coexpression of GRK2
resulted in a small increase in internalization (from ~1% in the
absence to ~5% in the presence of GRK2), whereas co-transfection of
either arrestin-2 or arrestin-3 significantly increased
TXA2R
internalization (to ~22%). Coexpression of
GRK3, GRK5, or GRK6 also promoted only a modest increase in
TXA2R
internalization (data not shown), indicating that
GRKs are unlikely to be limiting in this process. Internalization of
the TXA2R
was also promoted by coexpression of GRK2 and
arrestins although the effects were modest (Fig. 4A). Time
course experiments of receptor internalization were then performed in
the presence of arrestin-3 coexpression (Fig. 4B). TXA2R
internalization in the presence of arrestin-3 was
~15% after 30 min of agonist exposure and reached a plateau of
20-24% after 60 min. The rate and extent of TXA2R
internalization was also enhanced by arrestin-3 coexpression and
reached levels of 50-55% with a t1/2 of ~30 min.
Since endogenous expression of arrestins in HEK293 cells was previously shown to be relatively high (30), our results suggest that the TXA2R
has a low affinity for arrestins. However, these
results do not exclude the possibility that the two receptor isoforms differ in their ability to bind other proteins involved in receptor trafficking that might also contribute to their distinct
internalization profiles.
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To further characterize the requirements for TXA2R
internalization, we examined the effects of dominant negative forms of arrestins, dynamin, and GRK2. As mentioned above, nonvisual arrestins are clathrin-binding proteins that act as adaptors, targeting GPCRs to
clathrin-coated pits (24). Two mutants of arrestin-2, arrestin-2-V53D
(29) and arrestin-2(319-418) (35), and several mutants of arrestin-3
(36) act as dominant negative mutants of arrestin-mediated GPCR
internalization. Arrestin-2(319-418) binds well to clathrin but
completely lacks receptor binding activity (35). Arrestin-3(1-320) and
arrestin-3(201-409) inhibit
2-adrenergic receptor
internalization, are unable to mediate redistribution of receptors to
clathrin-coated pits, and do not localize to coated pits in either the
presence or absence of receptor and agonist. Arrestin-3(284-409) and
arrestin-3(290-409) lack the receptor-binding domain, localize
constitutively to coated pits and also inhibit receptor internalization
(36). Dynamin is a GTPase involved in the fission of endocytic vesicles
from the plasma membrane (41). Dynamin mutants that are deficient in
GTP binding, such as dynamin-K44A, were reported to block coated
pit-mediated internalization of several receptors (27, 33, 41, 42).
GRK2-K220R binds to, but does not phosphorylate, GPCRs and thus can
function as a dominant negative mutant of GRK2, constituting a useful
tool to study the role of GPCR phosphorylation (43). GRK2-K220R was shown to decrease internalization of the m2 muscarinic acetylcholine and
2-adrenergic receptors (25, 28). The effects of
dominant negative arrestins, dynamin, and GRK2 on the agonist-induced
internalization of TXA2R
were determined in HEK293 cells
by ELISA analysis. As illustrated in Fig.
5, coexpression of the different dominant negative arrestins resulted in 35-55% inhibition of
TXA2R
internalization while GRK2-K220R also effectively
reduced internalization (55% inhibition). Coexpression of GRK2-K220R
and arrestin-3(201-409) further inhibited agonist-promoted
internalization of the TXA2R
(~75% inhibition), an
effect comparable to that seen for dynamin-K44A. Taken together, these
results demonstrate that the agonist-induced internalization of the
TXA2R
is GRK-, arrestin-, and
dynamin-dependent and suggest a clathrin-coated
pit-mediated mechanism of internalization.
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Since the C-tail of the TXA2R appears critical in the
internalization of the receptor, we next tried to determine whether this function could be ascribed to any particular region of the tail.
In addition to the 11 serine and 4 threonine residues serving as
potential phosphorylation sites involved in internalization, the C-tail
of the TXA2R
also contains other motifs that might contribute to this process. Among these are two dileucine motifs, Leu-386/387, and Leu-392/393. Dileucine motifs have been implicated in
internalization of several receptors, including the
2-adrenergic receptor (Ref. 44, and references therein).
To address the potential role of dileucine motifs we constructed a
mutant TXA2R
in which leucines 386, 387, 392, and 393 were simultaneously changed to alanines (LLLLA). In addition, 11 progressive deletion mutants of the TXA2R
C-tail were
also generated (Fig. 1). All constructs were transfected in HEK293
cells with transfection conditions (i.e. amount of DNA)
adjusted to give equivalent receptor expression (~1 pmol/mg of
protein). Truncation of the TXA2R
C-tail had no effect
on ligand binding (data not shown) and the R328Stop mutant had the same
EC50 for phosphatidylinositol turnover as the wild-type receptor (~70 nM). The ability of the various receptors
to internalize after 3 h of agonist exposure was then evaluated by
ELISA analysis (Fig. 6). The LLLLA
mutation had no apparent effect on U46619-induced internalization of
TXA2R
. Similarly, removal of up to 41 residues from the
C-tail (G366Stop) had no effect on agonist-promoted internalization. However, truncation to residue 362 significantly reduced
internalization while further deletion to residue 355 or beyond
completely abolished TXA2R
internalization. Thus, the
region found between residues 355 and 366 seems to play a critical role
in TXA2R
internalization. These results, together with
the potent inhibition of internalization caused by GRK2-K220R
coexpression (Fig. 5), suggest that the serine residues within this
domain may serve as GRK phosphorylation sites and prove critical in
arrestin binding and agonist-promoted receptor internalization.
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To further localize the arrestin-binding domain on
TXA2R, we assessed whether arrestin-3 coexpression would
enhance U46619-promoted internalization of the different truncation
mutants. Arrestin-3 promoted the internalization of wild-type
TXA2R
and -
, as well as the S344Stop, S355Stop,
G366Stop, and F380Stop mutants (Fig. 7).
In contrast, the R328Stop mutant was not internalized even in the
presence of arrestin-3. Since inclusion of various residues distal to
residue 328 restores TXA2R
internalization and some of
the arrestin-3 promotion, it seems likely that the C-tails of both
TXA2 receptor isoforms are required for the promotion of internalization by arrestin-3.
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DISCUSSION |
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TXA2 is a potent stimulator of platelet aggregation
and smooth muscle constriction and is regarded as a mediator of
myocardial infarction, atherosclerosis, and bronchial asthma (7).
TXA2 is synthesized by numerous cells in response to
various physiological and pathological stimuli (7, 45). It is then
rapidly secreted and acts as a local hormone in the immediate vicinity
of its site of production. Its wide spectrum of actions in the body are
mediated by a GPCR that can be alternatively spliced in its C-tail to
generate isoforms referred to as and
(5, 12). It would seem
likely that the formation and function of TXA2 would be
tightly regulated, given the critical role of this biological
mediator. The effects of TXA2 are regulated by its
hydrolysis to inactive thromboxane B2 as well as by
homologous desensitization of its membrane receptor-mediated responses
(17, 46, 47). The existence of agonist-induced receptor uncoupling and
internalization mechanisms for the TXA2 receptors might
seem surprising when the natural agonist has such a short half-life
(~30 s). However, TXA2 is released in large amounts over
prolonged periods of time during certain vascular disorders, and could
tonically stimulate target cells (16). Indeed, it has been postulated
that TXA2 levels sufficient to cause desensitization of the
receptor may be maintained for long periods in vascular beds in which
thrombosis and platelet activation are ongoing (16).
It was recently shown that both TXA2R and
TXA2R
undergo similar agonist-induced phosphorylation
and desensitization in HEK293 cells (6, 17). Desensitization of the
Ca2+ response after agonist stimulation was also similar
for both receptor isoforms when overexpressed in CHO cells (18).
However, PKC activation potently inhibited agonist-mediated
intracellular Ca2+ mobilization of TXA2R
,
but not TXA2R
, indicating that different mechanisms of
regulation of these receptor isoforms are likely to exist. Studies in
CHRF-288 megakaryocytic (15), 1321N1 human astrocytoma (14), and rat
glomerular mesangial (13) cells indicated that TXA2R
desensitization involves initial uncoupling from the G protein although
receptor internalization and degradation appear necessary for maximal desensitization.
The present study investigated the detailed mechanism for
agonist-induced internalization of the TXA2 receptors in
HEK293 cells. Using expression of epitope-tagged versions of each
receptor isoform in combination with ELISA analysis to measure the
proportion of receptor remaining at the cell surface after agonist
exposure, we have shown that the TXA2R does not
internalize even when incubated with high concentrations of U46619 for
3 h. In contrast, TXA2R
was observed to undergo
agonist-promoted internalization with a plateau of ~40% after
~2-3 h of stimulation. Immunofluorescence microscopy confirmed the
inability of TXA2R
to internalize, whereas the
TXA2R
redistributed to intracellular punctate
compartments, typical of early endosomes. Similar results were obtained
in several other cell types, including two that express endogenous
TXA2Rs (P388D1 and A431) and two that lack endogenous
TXA2Rs (MiaPACA and CHO). These results confirm our
observations in HEK293 cells and validate them as an appropriate model
to study TXA2R trafficking.
Our data are in accordance with results obtained in K562, CHRF-288 megakaryocytic and rat glomerular mesangial cells which displayed a 50-60% loss of endogenous TXA2R-binding sites after 3-6 h of agonist treatment (13, 15, 16). Internalized TXA2Rs were associated with light membrane fractions containing both microsomal and cytoplasmic enzyme markers (16), and treatment with endocytosis inhibitors prevented agonist-induced receptor internalization (15). Nonvisual arrestins can bind to agonist-activated phosphorylated GPCRs and promote their internalization by interacting not only with the GPCR but also with clathrin, the major protein component of the clathrin-based endocytic machinery (24).
Our data demonstrate that agonist-induced internalization of the
TXA2R, like that of the
2-adrenergic (24,
27), follitropin (32), LH/CG (33), and the opioid (34) receptors, is
mediated by both arrestin and clathrin-coated pits. Overexpression of
dominant negative forms of arrestin-2, arrestin-3, GRK2, and dynamin
inhibit the agonist-induced internalization of the
TXA2R
. While coexpression of wild-type GRK2 resulted in
a small increase in internalization of both TXA2R isoforms,
overexpression of arrestins significantly promoted internalization of
these receptors suggesting that the arrestins are limiting in this
process. Similar results were obtained for the LH/CG receptor where
overexpression of arrestin-3 enhanced its internalization about 2-fold
(33), whereas little or no effect on internalization of the
2-adrenergic receptor was seen by overexpressing
arrestins in HEK293 cells (27, 30, 35, 48). It was reported that the
LH/CG receptor had a slow rate of internalization
(t1/2 ~ 140 min), compared with that of the
2-adrenergic receptor (t1/2 < 30 min), suggesting that the LH/CG receptor might have a low affinity for
arrestins (33). While HEK293 cells express relatively high levels of
arrestins (30), the TXA2R
internalizes slowly with a
t1/2 ~ 45 min, whereas TXA2R
shows
no detectable internalization. Arrestin-2(319-418) (35), arrestin-3(1-320), and arrestin-3(201-409) each inhibited
2-adrenergic receptor internalization by ~40-50%,
while arrestin-3(284-409) and arrestin-3(290-409) inhibited
internalization by ~70 and ~30%, respectively (36).
Arrestin-2(319-418) inhibited the internalization of the LH/CG
receptor by ~30% (33). While our data for the TXA2R
generally corroborate these observations, we find greater inhibition of
TXA2R
internalization by overexpression of
arrestin-3(201-409) as compared with arrestin-3(284-409), in contrast
to the
2-adrenergic receptor results (36). Although the
mechanism of the dominant negative nature of arrestin-3(201-409)
remains unclear (36), these disparities suggest that differences may
emerge between the interaction of receptors with the different
components of the clathrin-coated pit-mediated internalization pathway.
The inability of dynamin-K44A or GRK2-K220R and arrestin-3(201-409) coexpression to completely inhibit agonist-induced internalization of
the TXA2R
may be a result of the fact that these studies
were performed using transient coexpression of receptor and dominant negative constructs. Alternatively, our results might suggest that
additional non-GRK/arrestin or non-clathrin-mediated mechanisms of
internalization may exist for TXA2R
, as was suggested
for other GPCRs (49), such as the m2 muscarinic (26), bradykinin B2 (50), and angiotensin II type 1A (27) receptors.
The role of intracellular domains of GPCRs in triggering
internalization has been studied by mutagenesis. Some segments in the
intracellular loops (51-53) as well as in the C-tail domain of the
yeast -factor, thyrotropin, gastrin, parathyroid hormone, platelet-activating factor, m2 muscarinic, LH/CG, neurotensin, cholecystokinin, somatostatin type 5, thrombin,
opioid, bradykinin B2, and angiotensin receptors were found to be specifically
involved in the internalization process (54-66), whereas deletion
mutants of the m1 muscarinic receptor were internalized to a similar
extent as the wild-type receptors (67). However, there is still a
paucity of data regarding motifs known to target GPCRs to intracellular compartments upon agonist activation. Given the fact that
internalization of the TXA2R in HEK293 cells seems largely
determined by the C-tail of the
isoform, we attempted to identify
specific residues responsible for this effect. One plausible motif
contained in the C-tail of the TXA2R
likely to be
involved in internalization was two dileucines found at residues
386/387 and 392/393. Dileucine motifs bind AP1 and AP2 clathrin adaptor
protein complexes (68) and have been implicated in internalization of
several receptors, including the
2-adrenergic receptor
(44). Our results indicate that the dileucine motifs are not required
for agonist-stimulated internalization of the TXA2R
.
However, this does not exclude the possibility that these sequences
might play important roles in other mechanisms of receptor regulation,
such as receptor down-regulation or recycling. Deletion mutants were
generated and a region corresponding to amino acids 355-366
(DSRASASRAAG) was demonstrated to be crucial in TXA2R
internalization. Significant receptor internalization was still present
when the tail was shortened to residue 362, suggesting that the DSRASAS
amino acid sequence contains essential determinants for this process.
Since dominant negative GRK2 strongly inhibits TXA2R
internalization, this suggests that GRK-promoted phosphorylation of the
receptor is important for its internalization. When taken together,
these data argue for a possible role of the 3 serines found between
residues 355 and 362 in receptor internalization, particularly Ser-357
which constitutes a good GRK phosphorylation site with an acidic
residue preceeding it (69). Evidence is now accumulating that
agonist-induced phos- phorylation is likely important for the
internalization of many GPCRs. A Ser/Thr-rich sequence was postulated
to play an important role in internalization of the m1, m2, and m3
muscarinic cholinergic receptors (70). Moreover, either truncation or
mutation of the Ser/Thr residues in the C-tail of the thrombin receptor
inhibited both its agonist-induced phosphorylation and internalization
(64). In addition, COOH-terminal deletions or point mutations of
Ser/Thr residues in the COOH terminus of the
-opioid receptor
significantly reduced agonist-induced internalization (65).
Overexpression of dominant negative GRK2 also inhibited internalization
of the
2-adrenergic and m2 muscarinic acetylcholine
receptors (25, 28). It will be interesting to examine the
desensitization of TXA2R mutants since it has been shown
for other GPCRs that mutation of different clusters of Ser/Thr delineate distinct mechanisms with unique structural requirements that
mediate receptor desensitization and internalization (71).
By observing the internalization of the TXA2R deletion
mutants, it can be hypothesized that some structural requirements need
to be met for proper interaction with the endocytic machinery. Thus,
the ability of each mutant to internalize in the presence of arrestin-3
was evaluated as a measure of interaction between the different
receptor constructs and arrestin-3. Both isoforms of the receptor
displayed enhanced internalization in the presence of coexpressed
arrestin-3, while the R328Stop mutant showed no such effect, revealing
the requirement of the different C-tails of both receptors to provide
some interaction with arrestin-3. Interestingly, despite significant
homology with the TXA2R
C-tail, agonist-induced
internalization of the TXA2R
S344Stop mutant was
promoted to a much lower extent by coexpression of arrestin-3, suggesting that residues specific to the TXA2R
C-tail
confer distinct interaction properties with arrestin-3. However, some promotion of internalization by arrestin-3 was seen with the S344Stop, S355Stop, G366Stop, and F380Stop mutants, demonstrating the importance of residues distal to 328 in the TXA2R
C-tail in the
interaction with arrestin-3. This region is likely to correspond to
only one of several essential receptor domains involved in arrestin
binding as suggested by the findings that interaction of arrestin-1
with rhodopsin, or arrestin-2 and arrestin-3 with the
2-adrenergic and m2 muscarinic receptors, involves
multiple contact sites that impart apparent positive cooperativity
(72-77). TXA2R
and TXA2R
constitute
potential models to study factors affecting the binding affinity of
arrestins to the receptor. In this regard, C-tail constructs of the two
receptor isoforms will be useful in characterizing detailed
interactions with arrestins, and to possibly identify additional
proteins involved in their differential regulation.
Internalization has been postulated to play an important role in
resensitization of GPCRs, enabling receptors to undergo
dephosphorylation and subsequent recycling back to the cell surface
(19). We show that the TXA2R do not internalize upon
agonist activation, contrary to the TXA2R
. Intriguingly,
it has been reported that the TXA2R
was subject to
down-regulation while the TXA2R
was up-regulated, when
CHO cells stably expressing TXA2Rs were stimulated for
24 h with agonist (18). It is tempting to speculate that
down-regulation of the TXA2R
is a mechanism to eliminate
a receptor that cannot be resensitized, and that replenishment of the
cell surface with functional TXA2R
would be achieved by
other means, such as synthesis of new receptors. It will be interesting
to evaluate these hypotheses in different cell types, and to correlate
their physiological implications with the specific biological responses
controlled by each receptor isoform as they become known.
In summary, our results demonstrate that the alternative splicing
of the C-tail of the TXA2R generates isoforms of the
receptor showing distinct internalization characteristics. The
TXA2R does not internalize after agonist stimulation,
whereas TXA2R
internalization is dynamin-, GRK-, and
arrestin-dependent. A region encompassing residues 355-366
of TXA2R
was shown to be essential for agonist-induced internalization, and possible receptor domains required for arrestin-3 interaction were mapped. We propose that agonist activation of the
TXA2R
results in GRK-promoted phosphorylation of the
receptor, increasing the affinity for arrestin binding, thereby
targeting the receptor for clathrin-coated pit-mediated endocytosis.
The TXA2R
and TXA2R
will constitute an
interesting model for studying interactions with proteins of the
endocytic machinery. Similar studies on other GPCRs with alternatively
spliced C-tails, like the prostanoid EP3 (78) and FP (79) receptors,
will be required to determine whether these findings also extend to
other members of this receptor family.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a postdoctoral fellowship from the Medical Research
Council of Canada.
§ Established investigator of the American Heart Association. To whom correspondence should be addressed: Thomas Jefferson University, 233 S. 10th St., Philadelphia, PA 19107. Tel.: 215-503-4608; Fax: 215-923-1098; E-mail: benovic{at}lac.jci.tju.edu.
2
While a variety of names have been used for the
various mammalian arrestins, in this article we use nomenclature based
on the order of discovery of the arrestins: arrestin-1 (visual
arrestin, S-antigen, 48-kDa protein); arrestin-2 (-arrestin,
-arrestin-1); arrestin-3 (
-arrestin-2, arrestin3, thy-X
arrestin); arrestin-4 (C-arrestin, X-arrestin).
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ABBREVIATIONS |
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The abbreviations used are: TXA2, thromboxane A2; TXA2R, TXA2 receptor; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; C-tail, carboxyl terminus; HEK, human embryonic kidney; CHO, Chinese hamster ovary; LH/CG, lutropin/choriogonadotropin; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin.
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REFERENCES |
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