From the Service de Rhumatologie, Faculté de Médecine and Centre de Recherche Clinique, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
Received for publication, October 8, 2002, and in revised form, January 22, 2003
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ABSTRACT |
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In the present report, we investigated the
effect of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50)
expression on the agonist-induced internalization of the thromboxane
A2 G protein-coupled receptor
(GPCR)1 signaling cascades
are regulated by different molecular mechanisms. Many proteins have
been shown to regulate the different GPCR-related signaling pathways through the direct regulation of the G protein activity (1). Following
agonist binding, many GPCRs undergo agonist-induced phosphorylation,
internalization, and down-regulation, resulting in a decrease of their
responsiveness (desensitization) (2). Our studies are mainly interested
in the mechanisms that regulate the signaling and the internalization
of the thromboxane A2 receptor (TP receptor). Two TP
receptor isoforms were identified which are generated by the
alternative splicing of a single gene, TP EBP50 (also known as NHERF1), a 55-kDa phosphoprotein, was first
identified as a cofactor essential for protein kinase A-mediated inhibition of Na+/H+ exchanger isoform 3 (9).
EBP50 contains two PDZ domains (PDZ1 and PDZ2) implicated in multiple
protein-protein interactions, and an ERM domain, which binds to the
actin-associated ERM proteins (ezrin, radixin, moesin, and merlin) (10,
11). EBP50 has been found to interact with a variety of proteins, and
these interactions are involved in a growing range of functions
including the assembly of signaling complexes, receptor recycling, and
transport of membrane proteins to the cell surface (12). In fact, Cao
et al. (13) have shown that EBP50 participates in GPCR
trafficking by demonstrating its involvement in the recycling back to
the cell surface of the It has long been assumed that ligand binding to GPCRs is the ultimate
control to their internalization and trafficking. However, recent
studies bring about a new concept of the tight regulation of GPCRs
internalization pathways by diverse signaling cascades (14). In fact,
many signaling molecules including the small G proteins Rho, Rab, and
ARF6, as well as different phosphoinositide molecules, have been shown
to regulate the endocytic pathways by either controlling the actin
cytoskeleton organization or by mediating clathrin-coated pit
formation, as reviewed by Cavalli et al. (14). One of the
questions often asked in GPCR regulation is whether GPCR signaling is
necessary for GPCR internalization. Evidence gathered over the years by
several groups suggests that there is no clear answer to that question
and that it may depend on the GPCR and the signaling pathways that are
studied. Several GPCR mutants unable to activate their cognate G
proteins could still internalize, whereas the opposite was also
observed, signaling-deficient GPCRs were unable to undergo
agonist-induced internalization (15). In most cases, however, effects
of the mutations on the receptor signaling were determined for only one
signaling pathway even though a given GPCR can sometimes couple to more
than one type of G In the present study, we demonstrate that G Materials--
G Cell Culture and Transfection--
HEK293 cells were maintained
in Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum at 37 °C in a humidified atmosphere containing 5%
CO2. Transient transfections of HEK293 cells grown to
75-90% confluence were performed using FuGENE 6TM
according to the instructions from the manufacturer. Empty pcDNA3 vector was added to keep the total DNA amount added per plate constant.
Cells grown on 60-mm plates were transfected using 6 µg of total DNA vectors.
Receptor Cell Surface Expression and Internalization
Assays--
Cell surface expression and internalization of CXCR4,
TP Immunofluorescence Microscopy--
TP Receptor Phosphorylation Assay--
PMA-induced phosphorylation
of the TP Inositol Phosphate Measurements in Cells--
Inositol phosphate
measurements were performed as described previously (24). HEK293 cells
(2 × 105) were grown overnight in 12-well
plates. The cells were then cotransfected as described above with the
indicated constructs. The cells were labeled the following day for
18-24 h with 4 µCi/ml myo-[3H]inositol in Dulbecco's
modified Eagle's medium (high glucose, without inositol). The cells
were washed once in PBS and incubated in prewarmed Dulbecco's modified
Eagle's medium (high glucose, without inositol) supplemented with
0.5% BSA, 20 mM Hepes, pH 7.5, and 20 mM LiCl
for 10 min. Cells were then stimulated for 30 min with 100 nM U46619 in the case of the TP receptor-transfected cells.
After stimulation, the medium was removed and the reactions were
terminated by addition of 0.8 ml of 0.4 M chilled
perchloric acid. Cells were then collected in Eppendorf tubes, and 0.5 volume of 0.72 N KOH, 0.6 M KOHCO3
was added. Tubes were mixed and centrifuged for 5 min at 14,000 rpm in
a microcentrifuge. Inositol phosphates were separated on Bio-Rad AG1-X8
columns. Total labeled inositol phosphates were then measured by liquid
scintillation counting.
Data Analysis--
Data are presented as means ± S.E. of
at least three independent experiments. Statistically significant
differences among groups were assessed by t test for paired
samples, with p values < 0.05 sufficient to reject the
null hypothesis.
EBP50 Inhibits TP
EBP50 has been shown to be involved in the control of the fate of
internalized membrane proteins such as the GRKs and Arrestins Cannot Overcome the Inhibition of
Internalization by EBP50--
Because we showed that the EBP50
inhibition mechanism of the TP G
To further demonstrate (and visualize) that G
Although the inhibition of G Protein Kinase C Activation Induces TP
The possible role of PLC G GPCR Specificity of the G
To further investigate the specificity of
G G Heterologous Internalization of G
In the present study, we provide new information to the question which
has been asked for a long time: is G
Another interesting finding presented here is that EBP50 strongly
inhibited the TP
Our work was performed in a system where proteins were overexpressed.
We have established the specificity of the interaction between
G receptor (TP
receptor). Interestingly, we
found that EBP50 almost completely blocked TP
receptor
internalization, which could not be reversed by overexpression of G
protein-coupled receptor (GPCR) kinases and arrestins. Because we
recently demonstrated that EBP50 can bind to and inhibit
G
q, we next studied whether G
q signaling could induce TP
receptor internalization, addressing the long standing question about the relationship between GPCR signaling and
their internalization. Expression of a constitutively active G
q mutant (G
q-R183C) resulted in a robust
internalization of the TP
receptor, which was unaffected by
expression of dominant negative mutants of arrestin-2 and -3, but
inhibited by expression of EBP50 or dynamin-K44A, a dominant negative
mutant of dynamin. Phospholipase C
and protein kinase C did not
appear to significantly contribute to internalization of the TP
receptor, suggesting that G
q induces receptor
internalization through a phospholipase C
- and protein kinase
C-independent pathway. Surprisingly, there appears to be specificity in
G
protein-mediated GPCR internalization. G
q-R183C
also induced the internalization of CXCR4 (G
q-coupled), whereas it failed to do so for the
2-adrenergic receptor
(G
s-coupled). Moreover, G
s-R201C, a
constitutively active form of G
s, had no effect on
internalization of the TP
, CXCR4, and
2-adrenergic receptors. Thus, we showed that G
protein signaling can lead to
internalization of GPCRs, with specificity in both the G
proteins and GPCRs that are involved. Furthermore, a new function has
been described for EBP50 in its capacity to inhibit receptor endocytosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(343 amino acids) and
TP
(407 amino acids), which share the first 328 amino acids (3, 4).
Previous experiments performed by Parent et al. (5)
demonstrated that only TP
, but not TP
, undergoes agonist-induced
and tonic (constitutive) internalizations, which are dictated by
distinct motifs in the C terminus of the TP
receptor. Different
experiments showed that agonist-induced production of the second
messenger inositol phosphate by the TP receptors results from the
activation of the Gq/11 family of the G
subunits (6).
G
q-mediated production of inositol phosphates involves
the stimulation of phospholipase C-
(PLC-
) isoforms (7). More
recently, we have shown that the ezrin-radixin-moesin-binding phosphoprotein EBP50 regulates the G
q signaling pathway
of the TP receptors (8). We showed that EBP50 binds preferentially to
the active form of G
q and thus diminishes the
G
q-induced inositol phosphates production on the one
hand by inhibiting the interaction of the activated G
q
with PLC
-1, and on the other hand by preventing the coupling of the
TP receptors to G
q (8).
2-adrenergic receptor following
its agonist-induced internalization. However, there is still no
evidence that EBP50 can regulate the internalization of GPCRs.
subunit. Moreover, different studies using
constitutively active GPCR mutants suggest a possible link between
receptor signaling and internalization. Indeed, many such mutations
seem to affect cellular distributions of various GPCRs by decreasing
their cell surface expression levels, resulting in their intracellular
localization (16, 17), which can be reversed by the addition of an
inverse agonist (17). Even if there is evidence of a link between the constitutive signaling activity of some GPCRs and their constitutive internalization, it is not yet clear whether this internalization is
the result of activation of the signaling cascades or simply of the
active conformation adopted by the constitutively active receptors. In
other words, it is not well understood whether GPCR conformational
changes triggered by the binding of the agonist are sufficient and
necessary to the internalization of different GPCRs or whether the
subsequent activation of the signaling cascades plays an important role
in initiating the internalization pathways. On the other hand, it has
been shown that heterologous activation of protein kinase C (PKC)
results in the phosphorylation and internalization of the CXCR4 (18)
and the
-opioid receptors (19). These studies suggested that
activation of intracellular signaling induces the agonist-independent
internalization of some GPCRs. However, there is still no or little
evidence for the direct involvement of G
protein signaling in the
control of internalization pathways.
protein signaling can
induce GPCR internalization. In fact, inhibition of G
q signaling almost completely blocked the agonist-induced internalization of the TP
receptor. Surprisingly, we show that there is specificity in this phenomenon. Indeed, G
q signaling leads to
G
q-, but not G
s-, coupled receptor
internalization, whereas G
s signaling does not trigger
internalization of the GPCRs that we studied. The
G
q-mediated internalization was determined to occur by
an arrestin-, PLC-, and PKC-independent, but
dynamin-dependent mechanism and was completely abrogated by
EBP50.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
q/11, G
s, and
PLC
1-specific polyclonal antibodies were from Santa Cruz
Biotechnology. Hemagglutinin (HA)-specific monoclonal antibody was from
Babco. ECL reagents were purchased from Amersham Biosciences.
Flag-specific monoclonal antibody was purchased from Sigma, whereas the
fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse and Texas
Red goat anti-rabbit antibodies were purchased from Molecular Probes.
The goat anti-mouse alkaline-phosphatase-conjugated antibody and the
alkaline phosphatase substrate kit were purchased from Bio-Rad.
, and
2AR were assessed by ELISA experiments using
transiently transfected HEK293 cells as we have described previously
(5). Briefly, 1.2 × 106 cells were grown
overnight in 60-mm plates. The cells were then transfected with empty
pcDNA3 vector or cotransfected with pcDNA3HA-TP
, pcDNA3-Flag-TP
, pcDNA3-Flag-TP
(Y339A), pcDNA3HACXCR4, pcDNA3-HA-
2AR, or pcDNA3-Flag-
2AR with
pcDNA3-HA-EBP50, pcDNA3-HA-EBP50(PDZ1-PDZ2), pcDNA3-dynamin-K44A,
pcDNA3G
q-R183C, pcDNA3-arrestin-3, pcDNA3-GRK2, pcDNA3-GRK5, pcDNA3-arrestin-3-(201-409) (DN), or
pcDNA3-arrestin-2-(319-418) (DN). Transfected cells were maintained as
described above for 24 h. Thereafter, 2 × 105
cells were transferred to 24-well plates precoated with 0.1 mg/ml poly-L-lysine and were maintained for an additional 24 h. To assess the agonist-induced or PMA-induced internalization, the
transfected cells were washed once with phosphate-buffered saline
followed by a 2-h stimulation at 37 °C with 100 nM
U46619 or 1 µM PMA in Dulbecco's modified Eagle's
medium, except for the cells that were cotransfected with the
receptors described above and pcDNA3-G
q-R183C. The
cells were then fixed with 3.7% formaldehyde plus 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 cells were then incubated with either a
HA-specific (Babco) or the Flag M1-specific (Sigma) monoclonal antibody
at a dilution of 1:1000 in TBS/BSA for 1 h at room temperature.
Three washes with TBS buffer followed, and cells were briefly reblocked
for 15 min at room temperature. Incubation with goat anti-mouse
conjugated alkaline phosphatase diluted 1:1000 was carried out for
1 h at room temperature. The cells were incubated with an alkaline
phosphatase-conjugated goat anti-mouse antibody for 1 h at room
temperature. The cells were washed three times with TBS, and a
colorimetric alkaline phosphatase substrate was added as specified by
the manufacturer. The resulting colorimetric reactions were measured
using a Titertek MultisKan MCC/340 spectrophotometer. Cells
transfected with pcDNA3 were studied concurrently to determine background. All experiments were done in triplicate.
(Y339A) (a receptor
mutant defective in constitutive internalization) internalization was
assessed with immunofluorescence microscopy. 1.2 × 106 HEK293 cells were grown overnight in 60-mm plates as
described above. The cells were then transfected with an empty
pcDNA3 vector or cotransfected with pcDNA3-HA-TP
(Y339A) (a
receptor mutant specifically deficient in constitutive internalization)
and either an empty pcDNA3 vector or
pcDNA3-G
q-R183C and maintained overnight as
described above. Then 2 × 105 cells were transferred
on coverslips and further grown overnight. Cells were incubated with an
HA-specific monoclonal antibody (1:500 dilution) for 1 h at
4 °C in Dulbecco's modified Eagle's medium supplemented with 1%
BSA, followed by a 2-h incubation at 37 °C. The cells were then
fixed with 3% paraformaldehyde plus PBS for 30 min at room
temperature, washed with PBS/CaCl2, and permeabilized with
0.1% Triton X-100 plus PBS for 10 min at room temperature. Nonspecific
binding was blocked with 0.1% Triton X-100 plus PBS containing 5%
nonfat dry milk for 30 min at room temperature. Goat anti-mouse
FITC-conjugated secondary antibody (Molecular Probes) was then added at
a dilution of 1:200 for 1 h at room temperature. The cells were
washed six times with permeabilization buffer, with the last wash left
at room temperature for 30 min. Finally, the cells were fixed with 3%
paraformaldehyde as described. Coverslips were mounted using
Vectashield mounting medium (Vector Laboratories) and examined by
immunofluorescence microscopy on a Zeiss Axioskop2 microscope using a
40× objective. Images were collected using QED Camera software and
processed with Adobe Photoshop.
receptor was assessed essentially as we described
previously (18). 3.5 × 105 HEK293 cells were grown
overnight in six-well plates as described above and transfected the
following day with an empty pcDNA3 vector or with
pcDNA3-Myc-TP
. Forty-eight hours after transfection, the cells
were washed once with TBS and incubated for 2 h in phosphate-free Dulbecco's modified Eagle's medium with 5 µCi/ml
32Pi at 37 °C (PerkinElmer Life Sciences).
The cells were then incubated in the presence or absence of 1 µM GF109203X (a PKC inhibitor) for 30 min at 37 °C,
followed by a 30-min stimulation with 1 µM PMA at
37 °C. The cells were then washed twice with cold TBS and harvested
in 800 µl of lysis buffer (150 mM NaCl, 50 mM
Tris, pH 8, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 10 mM Na2P2O7, 5 mM EDTA) supplemented with protease inhibitors (9 nM pepstatin, 9 nM antipain, 10 nM
leupeptin, and 10 nM chymostatin) (Sigma). After the cells
were incubated in lysis buffer for 60 min at 4 °C, the lysates were
clarified by centrifugation for 20 min at 14,000 rpm at 4 °C. Four
µg of Myc-specific monoclonal antibody were added to the supernatant.
After a 60-min incubation with rotation at 4 °C, 50 µl of 50%
protein A-agarose pre-equilibrated in lysis buffer was added, followed
by an overnight incubation at 4 °C with rotation. Samples were then
centrifuged for 1 min in a microcentrifuge and washed three times with
1 ml of ice-cold lysis buffer. Immunoprecipitated proteins were eluted
by addition of 50 µl of SDS sample buffer followed by a 30-min
incubation at room temperature. The immunoprecipitates were resolved on
10% SDS-PAGE gels. The gels were dried and subjected to
autoradiography at
80 °C.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Receptor Internalization--
We have already
shown that EBP50 inhibits TP
signaling through the G
q
pathway by both preventing the receptor coupling to G
q
and by sequestration of GTP-bound G
q blocking it from
binding and activating PLC
-1 (8). We then investigated the role of EBP50 in the agonist-induced internalization of an HA-tagged TP
receptor by the use of ELISA experiments, as we described previously (5). Surprisingly, we have found that EBP50 almost completely inhibited
the agonist-induced internalization of the TP
receptor, as shown in
Fig. 1A. In fact, EBP50
inhibited the internalization of the TP
receptor more efficiently
than the dominant negative mutant form of dynamin, dynamin-K44A, which
is deficient in GTP binding and known to block internalization of
several membrane proteins (20). We had already demonstrated that
agonist-induced internalization of the TP
receptor is
dynamin-dependent (5), and dynamin-K44A was thus included
as a control to compare with.
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Fig. 1.
Inhibition of TP
receptor internalization by EBP50. A, HEK293
cells were transiently transfected with pcDNA3-HATP
and
pcDNA3, pcDNA3-EBP50, pcDNA3-PDZ1-PDZ2, pcDNA3-PDZ1,
pcDNA3-PDZ2, or pcDNA3-dynamin-K44A constructs. The percentage of
cell surface receptor loss following a 2-h incubation with 100 nM U46619 was measured by ELISA as described under
"Experimental Procedures." B, HEK293 cells transiently
co-expressing the HA-tagged TP
receptor and either EBP50 or
dynamin-K44A were preincubated with monensin (50 µM) for
30 min followed by a 2-h incubation with 100 nM U46619. The
percentage of cell surface receptor loss was measured as above.
C, immunofluorescence analysis of TP
receptor
internalization inhibition by EBP50 expression in HEK293 cells. The
cells were transiently cotransfected with pcDNA3-HATP
and either
pcDNA3 or pcDNA3-EBP50myc. Cells were incubated with a
HA-specific monoclonal antibody at 4 °C for 1 h, followed by a
2-h incubation at 37 °C in the presence of 100 nM
U46619. The cells were then fixed and incubated with a Myc-specific
monoclonal antibody. Receptors and EBP50myc were visualized by
incubating the cells with FITC-conjugated anti-mouse and Texas Red goat
anti-rabbit secondary antibodies, respectively. The cells were then
processed for immunofluorescence detection as described under
"Experimental Procedures." Inhibition of receptor internalization
by EBP50 was observed for all the time points observed between 0 and
2 h of agonist stimulation.
2-adrenergic receptors (13) by promoting their recycling to the cell surface membrane. We were thus interested in determining whether the apparent lack of TP
receptor internalization in presence of EBP50 was not in
fact caused by an increase in receptor recycling to the cell surface.
Cao et al. (13) have shown that the ERM domain of EBP50 was
necessary for promoting the recycling of the
2-adrenergic receptor and that a dominant negative
mutant of EBP50 lacking the ERM domain was unable to enhance the
recycling of this receptor. Similarly, data later published showed that
the ERM domain of EBP50 was also necessary for the recycling of the
-opioid receptor (21). Thus, we used a PDZ1-PDZ2 construct (lacking
the ERM domain) of EBP50 to investigate its effect on TP
receptor
internalization. As seen in Fig. 1A, we have found that this
EBP50 mutant is still able to inhibit TP
receptor internalization,
strongly suggesting that the inhibition of TP
receptor
internalization by EBP50 could not be explained by an increase in the
recycling of the TP
receptor. We then verified the effect of EBP50
and its PDZ1-PDZ2 construct (lacking the ERM domain) on the
agonist-induced internalization of the
2AR receptor to
confirm that EBP50 was acting appropriately in our hands. The results
obtained showed that, in the presence of EBP50, the agonist-induced
internalization of the
2AR receptor decreased by 50%,
whereas cotransfection of the PDZ1-PDZ2 construct (lacking the ERM
domain) had no effect on
2AR receptor internalization (data not shown), indicating that the effect of EBP50 on the
agonist-induced internalization of
2AR results from an
increase in its recycling at the cell surface, as was elegantly shown
by Cao et al. (13). Moreover, as we illustrate in Fig.
1B, pretreatment of cells with monensin had no effect on
neither the EBP50 inhibition nor, as expected, on the dynamin-K44A
inhibition of the agonist-induced TP
receptor internalization.
Monensin is known to inhibit cell surface membrane recycling activity
(22), and we have shown it to inhibit TP
receptor recycling in our
laboratory.2 Data presented
so far strongly suggest that EBP50 inhibits the TP
receptor
internalization, rather than promoting its recycling back to the cell
surface. To further demonstrate that EBP50 acts at the level of TP
receptor internalization, we performed immunofluorescence microscopy
experiments. In Fig. 1C, cells transfected with
pcDNA3-HATP
alone or with pcDNA3HATP
and
pcDNA3-EBP50myc were labeled at 4 °C with an anti-HA antibody
prior to internalization experiments to allow for detection of only the
receptors that are trafficking from the cell surface, as we described
previously (5). After agonist stimulation, it can be seen that, in
absence of EBP50, the TP
receptors are internalized in intracellular
compartments (Fig. 1C, top left
panel). In contrast, receptor-associated fluorescence remained at the cell surface in presence of EBP50 following agonist treatment (Fig. 1C, bottom left
panel), and this, for all the time points observed between 0 and 2 h of receptor stimulation (data not shown). Fig.
1C further illustrates that EBP50 blocks TP
receptor
internalization. These observations were surprising in light of the
known role of EBP50 in receptor recycling and transport of membrane
proteins to the cell surface. Indeed, it has never been shown that
EBP50 can inhibit the internalization of a GPCR. Therefore, we were
then interested in investigating the mechanism involved in the
inhibition of the TP
receptor internalization by EBP50.
receptor internalization did not
involve the control of the TP
receptor recycling, we then
investigated the effect of EBP50 on the machinery known to participate
in GPCR internalization. We previously demonstrated that the
agonist-induced internalization of the TP
receptor is GRK- as well
as arrestin-dependent (5). Therefore, we performed an ELISA
experiment using HEK293 cells stably expressing HA-tagged TP
receptors transfected with pcDNA3, pcDNA3-EBP50,
pcDNA3-GRK2, pcDNA3-GRK5, pcDNA3arrestin-3, and both
pcDNA3-EBP50 and either pcDNA3GRK2, pcDNA3-GRK5 or
pcDNA3-arrestin-3. Fig. 2 shows that
40% of TP
receptors undergo agonist-induced internalization when
the cells are transfected with an empty pcDNA3 vector.
Overexpression of GRK2, GRK5, and arrestin-3 in the absence of EBP50
increased TP
receptor internalization to 50, 54, and 60%,
respectively. Similar results were obtained previously for GRK2 and
arrestin-3 (5). It is the first observation regarding regulation of the TP
receptor by GRK5, and thus
identifies the TP
receptor as a putative substrate for GRK5.
However, Fig. 2 also shows that EBP50 inhibition of TP
receptor
internalization could not be reversed by overexpression of GRK2, GRK5,
or arrestin-3. Overexpression of arrestin-2 also failed to reverse the
inhibition brought by EBP50 (data not shown). This result suggests that
EBP50 probably acts through a mechanism other than the GRK- and
arrestin-mediated internalization pathway.
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Fig. 2.
Analysis of the effect of overexpression of
GRK2, GRK5, and arrestin-3 on the inhibition of TP
receptor internalization by EBP50. HEK293 cells stably
expressing an HA-tagged TP
receptor were transfected with pcDNA3
or the different indicated constructs. The percentage of cell surface
receptor loss following a 2-h incubation with 100 nM U46619
was measured by ELISA as described under "Experimental
Procedures."
q Signaling Induces TP
Receptor
Internalization--
As mentioned above, we have recently shown that
EBP50 interferes with the TP
receptor signaling through the
G
q pathway (8). EBP50 mediates the interaction of
several membrane proteins with the cell cytoskeleton via its ERM domain
(11). However, the ERM domain is not involved in the regulation of
G
q signaling. Indeed, the PDZ1-PDZ2 peptide and the
individual PDZ1 and PDZ2 domains of EBP50 are sufficient for
G
q binding and the inhibition of
G
q-mediated signaling (8). Interestingly, the PDZ1-PDZ2 construct and the individual PDZ1 and PDZ2 domains of EBP50 are not
only capable of, but are sufficient for, the inhibition of TP
receptor internalization (Fig. 1A). These observations
prompted us to investigate the possible role of G
q
signaling in the regulation of the TP
receptor internalization.
Surprisingly, we have found that TP
receptor-expressing HEK293 cells
transfected with a constitutively active G
q mutant
(G
q-R183C) exhibited a very low level of TP
receptor
expression at the cell surface (Fig. 3).
In fact, more than 80% of TP
cell surface expression was lost when
G
q-R183C was expressed compared with TP
receptor-expressing cells transfected with pcDNA3. Moreover, loss
of TP
receptor expression at the cell surface was proportional to
the amount of transfected pcDNA3-G
q-R183C DNA (data
not shown). Interestingly, the loss of TP
receptor expression
induced by G
q-R183C was prevented by the co-expression of dynamin-K44A (Fig. 3). Dynamin-K44A inhibits the agonist-induced internalization of the TP
receptor (Fig. 1A), as well as
the internalization of several GPCRs (20). Thus, it appears that the
reduced surface expression of the TP
receptors in presence of
G
q-R183C is the result of an agonist-independent
internalization process. Because EBP50 binds G
q-R183C,
preventing it from activating G
q downstream signaling
(8), we investigated the effect of EBP50 on the
G
q-R183C-induced internalization of TP
receptors. We
observed a full recovery of cell surface expression of TP
receptors
in HEK293 cells cotransfected with pcDNA3-HA-TP
,
pcDNA3-G
q-R183C, and pcDNA3-EBP50 in comparison
to HEK293 cells cotransfected with pcDNA3HA-TP
,
pcDNA3-G
q-R183C, and pcDNA3 (Fig. 3). This
result indicates that EBP50 inhibits the
G
q-R183C-induced internalization of the TP
receptor.
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Fig. 3.
G q-R183C
expression induces TP
receptor
internalization. HEK293 cells were transiently cotransfected with
pcDNA3-HATP
and pcDNA3, pcDNA3-G
q-R183C
alone, or pcDNA3-G
q-R183C with either
pcDNA3-dynamin-K44A or pcDNA3-EBP50. Cell surface expression of
the TP
receptor was measured by ELISA as described under
"Experimental Procedures."
q-R183C
induces internalization of the TP
receptor, we performed
immunofluorescence microscopy experiments using HEK293 cells
cotransfected with pcDNA3-HA-TP
-Y339A and either
pcDNA3- G
q-R183C or an empty pcDNA3
vector. The wild-type TP
receptor exhibits constitutive
internalization, and its use would complicate the visual interpretation
of the results in this particular case. Instead, we are taking
advantage of the TP
-Y339A mutant, which is specifically deficient in
constitutive internalization (23). Receptors were labeled with an
anti-HA antibody at 4 °C for 1 h prior to internalization
experiments to follow the receptors trafficking from the cell surface,
as described for Fig. 1C. The cells were then transferred to
37 °C to allow internalization to take place, and the receptors
visualized by incubating with a goat anti-mouse FITC-conjugated
antibody. Fig. 4 shows that only the
cells that were cotransfected with both pcDNA3-HATP
-Y339A and
pcDNA3G
q-R183C exhibited intracellular
fluorescence, whereas the receptor-associated fluorescence remained at
the cell surface in cells cotransfected with pcDNA3-HATP
-Y339A
and pcDNA3. This result visually confirmed that
G
q-R183C induces an agonist-independent internalization
of cell surface TP
receptors, as was determined in Fig. 3 with the
ELISA data.
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Fig. 4.
Immunofluorescence analysis of
TP Y339A internalization induced by
G
q-R183C expression in HEK293
cells. HEK293 cells were transfected with pcDNA3-HATP
Y339A
and pcDNA3-G
q-R183C (A),
pcDNA3-HATP
Y339A and pcDNA3 (B), and pcDNA3 alone
(C). Cells were incubated with a HA-specific monoclonal
antibody at 4 °C for 1 h, followed by a 2-h incubation at
37 °C, and then processed for immunofluorescence detection as
described under "Experimental Procedures." Top and
bottom panels in A and B
show different cells from the same experiment. Results are
representative of three independent experiments.
q-R183C-induced
internalization of the TP
receptor by dynamin-K44A most probably
occurs at the level of the pinching off of forming endocytic
intracellular vesicules from the cell surface (20), the inhibition of
TP
receptor internalization by EBP50 is likely the result of its
capacity to interfere with G
q signaling. Taken together,
these results suggest that G
q-mediated signaling is
involved in TP
internalization, because on one hand G
q-R183C is able to induce TP
receptor
internalization and on the other hand TP
receptor agonist activation
results mainly in the activation of the G
q pathway (6).
Furthermore, EBP50, which efficiently inhibits the activation of the
G
q signaling pathway, is also able to inhibit both the
agonist-promoted and G
q-R183C-induced internalization of
TP
receptors, indicating that activation of G
q is
involved in triggering the internalization process.
Receptor
Internalization--
G
q activation induces inositol
phosphate production by PLC
isoforms leading to activation of PKC,
which mediates the phosphorylation of a broad range of cellular
substrates (24). We next studied the possible role of
G
q-mediated activation of PLC
and PKC in initiating
TP
receptor internalization. Interestingly, we have found that TP
receptors undergo agonist-independent internalization in HEK293 cells
cotransfected with pcDNA3-HATP
and pcDNA3 following PMA
treatment (Fig. 5A). As
expected, PMA-induced internalization of TP
receptors was inhibited
by the expression of dynamin-K44A (Fig. 5A). However,
expression of EBP50 did not prevent the PMA-induced TP
receptor
internalization (Fig. 5A). In fact, this result was expected
because we showed EBP50 to bind and inhibit G
q, which is
upstream of PKC activation, and should thus have no effect on direct
activation of PKC by PMA (8). The incapacity of EBP50 to block
PMA-induced internalization of TP
receptors supports our idea that
it does not interfere with the internalization machinery per
se. Therefore, our data strongly suggest that EBP50 inhibition of
agonist-induced internalization of TP
receptor is more likely a
result of its direct inhibition of G
q signaling. These
observations also indicate that receptor-, as well as
G
q-R183C-, induced activation of PKC could be involved
in internalization of the TP
receptor. PMA activation and
heterologous activation of PKC are already known to promote the
phosphorylation and internalization of both
-opioid and CXCR4
receptors (18, 19). To verify whether PKC activation is involved in
initiating agonist-promoted TP
receptor internalization, we looked
at the effect of the PKC-specific inhibitor GF109203X in this process.
Fig. 5B shows that TP
receptor agonist-induced internalization was partially inhibited following pretreatment of cells
with 1 µM GF109203X. The efficiency of PKC inhibition by
GF109203X was verified by carrying out a receptor phosphorylation assay. HEK293 cells transfected with pcDNA3 or pcDNA3-mycTP
were labeled with 32Pi and subjected to
immunoprecipitation experiments with an anti-Myc-specific monoclonal
antibody following a 1 µM PMA treatment for 30 min at
37 °C, in presence or absence of GF109203X (Fig. 5C). As
can be seen, PMA treatment of the receptor-transfected cells caused a
significant phosphorylation of TP
receptor, whereas pretreatment of
cells with GF109203X completely prevented the PKC-mediated phosphorylation of the receptor, showing that PKC was efficiently inhibited in our system. Thus, it seems that activation of PKC is not a
major player in the control of the agonist-induced internalization of
the TP
receptor. Moreover, G
q-R183C-induced
internalization was not prevented with PKC inhibitors (data not
shown).
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Fig. 5.
Role of PLC and PKC
in the regulation of TP
internalization.
A, HEK293 cells were transfected with pcDNA3-HATP
and
pcDNA3, pcDNA3-EBP50, or pcDNA3-dynamin-K44A. The
percentage of cell surface receptor loss following a 2-h incubation
with 1 µM PMA was measured by ELISA as described under
"Experimental Procedures." B, HEK293 cells transfected
with pcDNA3-HATP
were incubated for 1 h with either 1 µM GF109203X or 20 µM U73122 followed by a
2-h incubation with 100 nM U46619. Internalization of the
TP
was then evaluated by ELISA as described under "Experimental
Procedures." C, PKC-mediated phosphorylation of TP
receptor and its inhibition by the PKC-specific GF109203X inhibitor
were measured by a receptor phosphorylation assay. HEK293 cells
transfected with pcDNA3 or pcDNA3-Myc-TP
were incubated in
the presence or not of 1 µM GF109203X for 30 min at
37 °C followed by a 30-min stimulation with 1 µM PMA
at 37 °C. Immunoprecipitation of the receptor protein was then
performed using a Myc-specific monoclonal antibody and subjected to
SDS-PAGE and autoradiography as indicated under "Experimental
Procedures." D, inhibition of TP
-induced PLC
activation by the PLC
U73122 inhibitor. HEK293 cells transfected
with pcDNA-HATP
were incubated for 1 h with 20 µM U73122 followed by a 30-min incubation with 100 nM U46619. Inositol phosphate production was measured as
described under "Experimental Procedures."
activation in the regulation of the
agonist-promoted internalization of the TP
receptor was then evaluated. We have found that pretreatment of cells with U73122, a
PLC
-specific inhibitor, could not prevent the agonist-induced internalization of the TP
receptor (Fig. 5B), whereas it
reduced TP
receptor-mediated inositol phosphate production by 80%
(Fig. 5D). Thus, it appears that PLC
activation is also
not a major contributor in triggering the TP
receptor
internalization process. Taken together, our results suggest that
agonist-dependent internalization of TP
receptor is
strongly dependent on G
q signaling because direct
inhibition of G
q by EBP50 virtually abrogated completely both agonist-promoted and G
q-R183C-induced
internalization, but that there are mechanisms other than PLC
- and
PKC-associated pathways involved. In this regard, it is interesting to
note that G
q and G
q-coupled receptors
were recently shown to activate RhoA and its downstream signaling
events independently of second messengers production (25-27).
Furthermore, Rho signaling pathways are known to be involved in the
regulation of endocytic trafficking, as reviewed by Cavalli et
al. (14). This research avenue is currently pursued in our laboratory.
q but Not G
s Signaling Regulates the
Internalization of the TP
Receptor--
The results presented above
brought the first evidence for a direct regulation of
G
q-coupled receptor internalization by G
q
signaling. As a means to assess the specificity or generality of G
protein signaling in triggering GPCR internalization, we performed
ELISA experiments to investigate the effect of a constitutively active
G
s-R201C mutant on the cell surface expression of TP
receptors. Fig. 6A reveals
that co-expression of TP
receptor with G
s-R201C did
not affect the cell surface expression of TP
receptors in comparison
to the expression level observed in HEK293 cells expressing TP
receptor alone. Functionality of the G
s-R201C protein
was confirmed in our system by cAMP measurements (data not shown). Only
co-expression of G
q-R183C with the TP
receptor was
able to induce a decrease in its cell surface expression. This result
shows that, contrary to G
q signaling,
G
s-mediated pathways do not regulate the internalization
of the TP
receptor. Thus, a specificity in
G
q-mediated internalization of the TP
receptor seems
to exist, which is very interesting because this receptor is also known
to couple to G
s (Ref. 6 and data not shown).
View larger version (11K):
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Fig. 6.
Cell surface expression of the TP , CXCR4
and
2-adrenergic receptors in the presence of
G
q-R183C and G
s-R201C. HEK293 cells
were transfected with pcDNA3-HATP
plus pcDNA3,
pcDNA3-G
s-R201C, or pcDNA3-G
q-R183C
(A), pcDNA3-HACXCR4 receptor plus pcDNA3 or
pcDNA3-G
q-R183C (B), and
pcDNA3HA
2AR receptor plus pcDNA3,
pcDNA3-G
s-R201C, or pcDNA3-G
qR183C
(C). Receptor cell surface expression was measured by ELISA
as described under "Experimental Procedures."
q-R183C-induced
Internalization--
The effect of G
q signaling on the
regulation of the internalization of CXCR4 receptor, another
G
q-coupled receptor, was then studied. We performed
ELISA experiments using HEK293 cells cotransfected with
pcDNA3-HA-CXCR4 and either pcDNA3-G
q-R183C or an
empty pcDNA3 vector. Interestingly, we observed a significant decrease (85%) in the cell surface expression of HA-CXCR4 in HEK293 cells expressing G
q-R183C compared with the cells
cotransfected with pcDNA3 (Fig. 6B). These results show
that a direct activation of G
q signaling cascade by
expression of G
q-R183C in HEK293 cells promotes an
agonist-independent internalization of both TP
and CXCR4 receptors,
both G
q-coupled receptors. This is intriguing as it
suggests that heterologous activation of G
q signaling
could result in the internalization of perhaps several
G
q-coupled receptors (see below). However, because we
only used two G
q-coupled receptors in our studies, more
experiments will need to be done to examine how our observations will
apply to other G
q-coupled receptors.
q-R183C-induced internalization, we
performed ELISA experiments using HEK293 cells cotransfected with
pcDNA3-HA-
2AR and
pcDNA3-G
q-R183C, pcDNA3-G
s-R201C, or an empty pcDNA3 vector. Fig. 6C shows that
G
q-R183C co-expression did not affect the cell
surface expression of the
2AR in comparison to cells
cotransfected with pcDNA3-HA-
2AR and pcDNA3.
Because
2AR is a G
s-coupled receptor, we
looked at the effect of the constitutively active mutant
G
s-R201C on the cell surface expression of
2AR. The results shown in Fig. 6C demonstrate
that G
s-R201C expression did not induce internalization
of the
2AR, but that it actually slightly increased
2AR cell surface expression, for which we have no
explanation yet. These data suggest that neither G
q-R183C nor G
s-R201C induce
internalization of the
2AR. Therefore, G
q
signaling seems to promote the internalization of specific GPCRs,
possibly those which are coupled to G
q. The use of other GPCRs and active G
proteins to further characterize the specificity of G
-mediated internalization of GPCRs will constitute future interesting studies.
q-R183C-induced Internalization Is
Arrestin-independent--
We demonstrated that G
q-R183C
constitutive signaling induces internalization of both
G
q-coupled TP
and CXCR4 receptors. Because
agonist-dependent internalization of TP
is
arrestin-dependent (5), we investigated the effect of
arrestin-3-(201-409) and arrestin-2-(319-418) (28, 29) dominant
negative (DN) mutants on G
q-R183C-induced
internalization of the TP
receptor. ELISA experiments were performed
using HEK293 cells cotransfected with pcDNA3-HATP
and an empty
pcDNA3 vector, or with both pcDNA3-HATP
and
pcDNA3-G
q-R183C and pcDNA3-arrestin-3(DN),
pcDNA3arrestin-2(DN), or an empty pcDNA3 vector. Fig.
7 shows that the
G
q-R183C-induced internalization of the TP
receptor
could not be prevented by co-expression of both arrestin-2(DN) and
arrestin-3(DN) mutants. Together, results from Figs. 2 and 7 suggest
that internalization of the TP
receptor by G
q
signaling is an arrestin-independent mechanism. GPCR endocytosis
involves diverse molecular mechanisms that can be
arrestin-dependent or not (30). Our results also suggest
that both arrestin-dependent and arrestin-independent endocytosis pathways could be activated upon agonist stimulation of the
TP
receptor. This is interesting because we reported in a previous
study that the agonist-induced internalization of the TP
receptor
was only inhibited by roughly 50% by expression of arrestin dominant
negative mutants (5). We speculated at the time that there could be an
alternative route for TP
receptor endocytosis than the
arrestin-mediated pathway. In the present study, we provide part of the
answer to that question by showing that G
q signaling is
involved in the internalization of the TP
receptor in an
arrestin-independent manner.
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Fig. 7.
Effect of arrestin-2 and arrestin-3 dominant
negative mutants on
G q-R183C-induced internalization
of the TP
receptor. HEK293 cells were
cotransfected with pcDNA3-HATP
and pcDNA3,
pcDNA3-G
q-R183C, pcDNA3-G
q-R183C,
and pcDNA3arrestin-2(DN), or with
pcDNA3-G
q-R183C and pcDNA3-arrestin3(DN).
Arrestin(DN) constructs were transfected using two different amounts of
DNA. Cell surface expression of the TP
receptor was then measured by
ELISA as described under "Experimental Procedures."
q-coupled
Receptors--
Taken together, our results provide evidence of a new
mechanism consisting in G
q-mediated internalization of
some G
q-coupled receptors. It also raises the question
whether heterologous activation of the G
q signaling
pathway by any G
q-coupled receptor could activate the
agonist-independent internalization of another
G
q-coupled receptor present at the same cell surface. In
an attempt to answer this question, we performed an ELISA experiment
using HEK293 cells cotransfected with pcDNA3-HA-CXCR4 and either
pcDNA3-Flag-TP
or an empty pcDNA3 vector. We observed that
stimulation of the cells with the TP agonist U46619 results in a 70%
loss of cell surface CXCR4 receptors (Fig.
8). This loss was only observed in HEK293
cells expressing both TP
and CXCR4 receptors, but not in the cells
expressing CXCR4 alone. This experiment demonstrates that activation of
the TP
receptor results in an agonist-independent internalization of
the CXCR4 receptor. This result was interesting, but also expected, as
we have shown that the internalization of both the TP
and CXCR4
receptors could be induced by G
q-R183C. Interestingly,
consistent with our hypothesis, heterologous internalization of CXCR4
induced by stimulation of the TP
receptor was inhibited by
coexpression of EBP50 (Fig. 8). Thus activation of the
G
q signaling pathway by agonist stimulation of the TP
receptor can induce the internalization of the CXCR4 receptor, another
G
q-coupled receptor.
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Fig. 8.
Heterologous internalization of CXCR4 induced
by TP receptor activation. HEK293 cells
were transfected with pcDNA3-HACXCR4 and pcDNA3Flag-TP
,
pcDNA3-EBP50, or pcDNA3. The percentage of cell surface CXCR4 receptor
loss following a 2-h incubation with 100 nM TP
receptor-specific agonist U46619 was measured by ELISA as indicated
under "Experimental Procedures."
protein signaling involved in
internalization of GPCRs? Our results determined that G
protein
signaling can trigger GPCR trafficking. Indeed, we showed that
G
q signaling can induce internalization of GPCRs. There
is specificity in the G
protein that can promote GPCR
internalization, as illustrated by the fact that G
s
signaling could not induce internalization of the receptors included in
this report. We demonstrated that the G
q-mediated
internalization shows at least some specificity for
G
q-coupled receptors, because
2AR (a
G
s-coupled receptor) internalization was not induced by
G
q signaling. We presented evidence that the
G
q-induced internalization was PLC
-, PKC-, GRK-, and
arrestin-independent, therefore constituting a GPCR internalization
mechanism that has never been appreciated before. One possible link
connecting G
q signaling to GPCR internalization could be
the G
q-mediated activation of Rho-associated pathways (25-27), which are known to participate in the actin cytoskeleton rearrangement and to regulate endocytic processes (14). Interestingly, the TP receptor was recently shown to activate Rho (31). It is unclear
as to why in our system, where G
q signaling was
independent of ligand occupancy of the receptor, only specific GPCRs
were induced to internalize. This would suggest that receptor-specific sequences are implicated in the recognition by the machinery involved in the G
q-mediated internalization, which is the subject
of current work in our laboratory.
receptor internalization. Inhibition of receptor
endocytosis is a function that has never been described before for
EBP50. Overexpression of GRKs and arrestins could not overcome the
EBP50 inhibition of TP
receptor internalization, indicating that
EBP50 does not act at this level. EBP50 most likely inhibits TP
receptor internalization by binding to G
q. This is
supported by a few observations. First, we know that EBP50 binds
G
q and inhibits TP
receptor G
q
signaling pathways (8). Second, EBP50 strongly prevented the
G
q-R183C-mediated but not the PMA-induced
internalization of the TP
receptor. Finally, each of the individual
PDZ domains (which bind G
q) of EBP50 inhibited receptor
internalization, suggesting that the formation of a multiprotein complex by EBP50 is not necessary, but that sequestration of
G
q is sufficient for inhibition of TP
receptor
endocytosis. A schematic representation of our working model is
illustrated in Fig. 9.
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Fig. 9.
Schematic illustration of the internalization
mechanisms of the TP receptor. According
to our previous results (5) and to data presented here, the TP
receptor appears to undergo agonist-induced internalization through
various mechanisms. Upon agonist stimulation, internalization of the
receptor is promoted by GRKs and arrestins through a dynamin- and
clathrin-mediated pathway. However, dominant-negative mutants of GRKs
and arrestins were unable to completely inhibit TP
receptor
internalization (5). Our new data show that there is a GRK- and
arrestin-independent component in the internalization of the TP
receptor, which is mediated by G
q signaling. PLC
and
PKC activation do not appear to constitute major contributors in
G
q-mediated internalization of the TP
receptor, as
indicated by the thick arrow linking the
receptor, G
q activation, and an unknown pathway leading
to receptor internalization in a dynamin-dependent fashion.
EBP50, which we showed to bind to G
q and to inhibit its
signaling, efficiently blocked G
q-induced receptor
internalization. G
q activation promoted
G
q-, but not G
s-, coupled receptor
internalization, whereas G
s signaling failed to induce
receptor internalization (results not shown).
q and the PDZ domains of EBP50 previously (8). The fact
that EBP50 cannot block PMA-induced internalization of the receptor
supports the idea that EBP50 does not act nonspecifically in the
process of internalization. Furthermore, other PDZ domain-containing proteins that were tested, including NHERF2 (an isoform of EBP50) and
GIPC, do not bind G
q (8) and do not affect TP
receptor internalization (data not shown). The physiological
consequences and significance of our observations that
G
q signaling can lead to "heterologous" or
"cross-regulated" internalization of GPCRs is intriguing. It could
constitute another step in GPCRs heterologous desensitization, whereas
a cell could protect itself from overstimulation of particular
signaling pathways. Although we have no doubt that the heterologous
G
q-mediated internalization occurs in more physiological conditions, it is hard to imagine that all G
q-coupled
receptors expressed at a cell surface will undergo internalization once G
q is activated by a given GPCR. Mechanisms must exist
to regulate this phenomenon, such as cellular compartmentalization,
receptor-specific sequences, associated proteins conferring further
specificity, or a combination of some or all of these possibilities.
Clearly, much work remains to be done to fully appreciate the
mechanisms and consequences of G
protein-mediated internalization of GPCRs.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a grant (to J.-L. P.) from the Canadian Institutes of Health Research.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.
Supported by a doctoral fellowship from the "Fonds de la
Recherche en Santé du Québec."
§ Recipient of a New Investigator award from the Canadian Institutes of Health Research. To whom correspondence should be addressed: Service de Rhumatologie, Faculté de Médecine, Université de Sherbrooke, 3001, 12 Ave. Nord, Fleurimont, Québec J1H 5N4, Canada. Tel.: 819-564-5264; Fax: 819-564-5265; E-mail: jean-luc.parent@usherbrooke.ca.
Published, JBC Papers in Press, March 7, 2003, DOI 10.1074/jbc.M210319200
2 C. Thériault and J.-L. Parent, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GPCR, G protein-coupled receptor; EBP50, ezrin-radixin-moesin-binding phosphoprotein 50; ERM, ezrin-radixin-moesin; GRK, G protein-coupled receptor kinase; NHERF, NHE regulatory factor; PLC, phospholipase C; TP receptor, thromboxane A2 receptor; HA, hemagglutinin; PKC, protein kinase C; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; TBS, Tris-buffered saline; DN, dominant negative; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate.
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