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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 302:219–224, 2002
Vol. 302, No. 1
4837/990535
Printed in U.S.A.
Differential Internalization of the Prostaglandin F2␣ Receptor
Isoforms: Role of Protein Kinase C and Clathrin
DINESH SRINIVASAN, HIROMICHI FUJINO, and JOHN W. REGAN
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona
Received December 11, 2001; accepted March 8, 2002
This article is available online at http://jpet.aspetjournals.org
styl 13-acetate (PMA), an activator of protein kinase C (PKC).
Pretreatment of FPA-expressing cells with Gö 6976
[12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo5H-indolo[2,3-a]pyrrolo[3,4-c]carbozole], an inhibitor of PKC,
blocked both PGF2␣- and PMA-induced receptor internalization. However, Gö 6976 did not block constitutive internalization of the FPB isoform, suggesting that the mechanisms of
receptor internalization differ between the FPA and FPB isoforms. Furthermore, pretreatment with sucrose, an inhibitor of
clathrin-dependent internalization, blocked PGF2␣-induced internalization of the FPA isoform but did not block constitutive
internalization of the FPB isoform. In conclusion, the FPA receptor isoform shows an agonist-induced internalization involving PKC and clathrin, whereas the FPB isoform undergoes
agonist-independent internalization that does not involve PKC
or clathrin.
G-protein-coupled receptors (GPCRs) are heptahelical
transmembrane proteins (Palczewski et al., 2000) devoted to
cellular signal transduction. GPCRs are activated by numerous stimuli as varied as light, odorants, nucleotides, and
proteins. Intracellularly, they are coupled to G-proteins that
amplify the extracellular stimulus and convert it into an
intracellular response. GPCRs acting through G-proteins can
stimulate various second messenger systems, such as increase in intracellular Ca2⫹, activation of kinase cascades,
and induction of gene transcription. The regulation of GPCR
function and signaling is of paramount interest because
GPCRs are involved in numerous pathologies and are obvious targets for therapeutic intervention.
One of the ways that GPCR function is regulated is via
desensitization, which is an attenuated response of a GPCR
upon repeated or constant stimulation by its agonist. One
mechanism of desensitization is internalization, in which the
receptor is translocated from the cell surface membrane to an
intracellular compartment. The classical pathway for GPCR
internalization is exemplified by the agonist-induced inter-
nalization of the ␤2-adrenergic receptor (Lefkowitz, 1998).
Thus, upon agonist stimulation, the ␤2-adrenergic receptor is
phosphorylated by a GPCR kinase that recruits ␤-arrestin,
which in turn initiates clathrin-dependent internalization.
However, other mechanisms of GPCR internalization exist
that, for example, involve an initial phosphorylation by protein kinase C (PKC) instead of GPCR kinase (Ferrari et al.,
1999; Hipkin et al., 2000; Xiang et al., 2001). An important
development as it concerns receptor internalization is the
recognition that internalization is not the end of signaling.
The process of internalization can activate signaling pathways, such as that of the mitogen-activated protein kinase
(Pierce et al., 2000). Knowledge of the internalization of a
given receptor is, therefore, important toward understanding
its overall signaling potential.
The FP prostanoid receptors are GPCRs whose physiological agonist is prostaglandin F2␣ (PGF2␣). The FP receptors
regulate diverse physiological processes, including inflammation and luteolysis. FP receptors consist of two isoforms
called FPA and FPB, which were originally isolated and
ABBREVIATIONS: GPCR, G-protein-coupled receptors; PKC, protein kinase C; PGF2␣, prostaglandin F2␣; PMA, phorbol 12-myristyl 13-acetate;
Gö 6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]carbozole; HEK, human embryonic kidney; EGF,
epidermal growth factor; PY20, anti-phosphotyrosine antibodies; FP, receptor for PGF2␣; TP, receptor for thromboxane A2; TP␣ and TP␤, alternate
mRNA splice variants of TP receptor.
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ABSTRACT
FP prostanoid receptors are G-protein-coupled receptors that
mediate the actions of prostaglandin F2␣ (PGF2␣). Alternative
mRNA splicing gives rise to two isoforms, FPA and FPB, which
are identical except for their intracellular carboxyl termini. In this
study, we examined the internalization of recombinant FLAGepitope-tagged FPA and FPB receptors that were stably expressed in human embryonic kidney-293 cells. Cell surface
receptors on live cells were labeled with anti-FLAG antibodies
either in the presence or absence of PGF2␣ and were examined
by immunofluorescence microscopy. In the absence of PGF2␣,
FPA-expressing cells were labeled predominantly on the cell
surface; however, FPB-expressing cells were labeled on both
the cell surface and intracellularly, indicating constitutive internalization of the FPB isoform. After treatment with PGF2␣, FPAexpressing cells were labeled intracellularly, reflecting receptor
internalization, which could be mimicked with phorbol 12-myri-
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Experimental Procedures
Materials. Dulbecco’s modified Eagle’s medium, bovine serum
albumin, Opti-MEM, hygromycin B, geneticin, and gentamicin reagent solutions were obtained from Invitrogen (Carlsbad, CA). Gö
6976 and phorbol 12-myristyl 13-acetate (PMA) were purchased from
Calbiochem (San Diego, CA). Clathrin heavy-chain antibodies and
anti-phosphotyrosine (PY20) antibodies were obtained from BD Biosciences (San Jose, CA). Methanol, acetone, dimethyl sulfoxide, antiFLAG M2 antibodies, Fab-specific anti-mouse IgG conjugated to
fluorescein isothiocyanate, and sucrose were purchased from SigmaAldrich (St. Louis, MO). PGF2␣ was obtained from the Cayman
Chemical Company (Ann Arbor, MI). HEK-293 cells stably expressing either the FLAG-tagged FPA or FLAG-tagged FPB receptor isoforms were used in all the experiments (Fujino et al., 2000b). Cells
were maintained at 37°C with 5% CO2/95% air in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 250 ␮g/ml
geneticin, 200 ␮g/ml hygromycin B, and 100 ␮g/ml gentamicin.
Whole Cell Labeling. The protocol for whole cell labeling was
modified from that of Orsini and Benovic (1998). Approximately
100,000 cells expressing either the FLAG-tagged FPA receptor isoform or the FLAG-tagged FPB receptor isoform were split into tissue
culture plates containing glass coverslips. On day 3, prior to the start
of the experiments, the cells were examined by microscope to confirm
cell density (Fujino et al., 2000a). To evaluate agonist-dependent
internalization, the cells were treated either with a 1:500 dilution of
anti-FLAG M2 antibodies diluted in Opti-MEM or with a 1:500
dilution of anti-FLAG M2 antibodies concurrent with 1 ␮M PGF2␣
diluted in Opti-MEM for 10 min at 37°C. The tissue culture plates
were then placed on ice, and the medium was aspirated. The cells
were fixed and permeabilized with methanol/acetone (7:3) for 10 min
at ⫺20°C and blocked in BLOTTO (5% nonfat dry milk in Trisbuffered saline with 0.05% Triton X-100) at 37°C for 30 min. The
glass coverslips were removed and placed on stoppers in a covered
box, and 200 ␮l of a 1:500 dilution of secondary antibodies (Fabspecific anti-mouse IgG conjugated to fluorescein isothiocyanate) in
BLOTTO were applied to each coverslip. The box was then gently
shaken for 1 h at room temperature. The coverslips were then transferred to tissue culture plates, washed six times with antibody wash
buffer leaving 2 ml of the last wash in each well, and placed at 37°C
for 30 min. The coverslips were then mounted onto glass slides using
p-phenylenediamine and Cytoseal (ProSciTech, Kelso, QLD, Australia). Images were obtained using a Leica TCS-4D scanning confocal
microscope (Leica Microsystems, Inc., Deerfield, IL) using a 100⫻ oil
immersion objective and were processed using Adobe Photoshop
(Adobe Systems Inc., Mountain View, CA). Experiments with PMA
were done exactly as above using 10 ␮M PMA instead of PGF2␣.
Pretreatments with 100 nM Gö 6976 were done for 5 min prior to
treatment with either vehicle, PGF2␣, or PMA. Pretreatment with
0.4 M sucrose was for 15 min.
Immunoblot Analysis. FPA and FPB cells were cultured to 80%
confluency in 10-cm tissue culture dishes. After treatment with 1 ␮M
PGF2␣ or vehicle, the cells were scraped and sonicated in a lysis
buffer consisting of 20 mM Tris-HCl (pH 7.5), 10 mM EDTA, 2 mM
EGTA, 2 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml leupeptin,
and 2 mM sodium vanadate. Samples were centrifuged (16,000g) for
15 min at 4°C, the supernatant (cytosolic fraction) was removed, and
the pellet (particulate fraction) was solubilized with lysis buffer
containing 0.05% Triton X-100 and centrifuged again to remove
insoluble debris as previously described (Fujino and Regan, 2001).
Protein concentrations were determined using a Bio-Rad assay kit
(Bio-Rad Laboratories, Hercules, CA), and 30 ␮g of protein/sample
was separated on 7.5% SDS-polyacrylamide gels. The proteins were
then transferred to nitrocellulose membranes and blocked with 1.5%
nonfat dry milk in Tris-buffered saline containing 0.1% polyoxyethylenesorbitan monolaurate (TBST) overnight at 4°C. The membranes were then incubated with anti-phosphotyrosine antibodies
(1:1,000 dilution) for 1 h at room temperature with rotation. After
three 15-min washes each with TBST, the membranes were incubated with anti-mouse secondary antibodies conjugated to horseradish peroxidase (1:10,000 dilution) for 1 h at room temperature with
rotation. The membranes were washed and visualized by enhanced
chemiluminescence (SuperSignal; Pierce, Rockford, IL). To examine
the presence of clathrin heavy chain, the membranes were stripped
in a buffer containing 2% SDS, 62.5 mM Tris-HCl (pH 6.8), and 100
mM ␤-mercaptoethanol for 30 min at 65°C. The membranes were
washed, blocked overnight at 4°C, and incubated with the clathrin
heavy-chain antibodies (1:2,000 dilution) for 1 h at room temperature. The membranes were then washed, incubated with anti-mouse
secondary antibodies, and exposed to film as above.
Results
Agonist-Dependent Internalization of the FLAG-FPA
Contrasts with Constitutive Internalization of the
FLAG-FPB Isoform. Immunofluorescence microscopy was
used to examine the internalization of the FPA and FPB
isoforms in HEK-293 cells stably expressing the FLAGtagged constructs of these receptors. Initial labeling of cell
surface receptors was achieved according to a modified
method of Orsini and Benovic (1998), in which live cells were
exposed to either vehicle or 1 ␮M PGF2␣ concurrently with
anti-FLAG M2 antibodies for 10 min as detailed under Experimental Procedures. Figure 1 shows that, after treatment
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cloned from a sheep corpus luteum library (Pierce et al.,
1997). These two isoforms are generated by alternative
mRNA splicing that gives rise to differences in their intracellular carboxyl-terminal domain. Studies on these receptor
isoforms have demonstrated that upon stimulation with
PGF2␣ more than one signaling pathway can be activated.
Thus, stimulation of either FP receptor isoform by PGF2␣ has
been shown to activate both the G␣q and rho signaling pathways (Pierce et al., 1999). In addition, it has recently been
shown that the agonist stimulation of the FPB, but not the
FPA, isoform can activate transcription through a ␤-cateninsignaling pathway (Fujino and Regan, 2001). Additional differences between these isoforms have been shown with respect to their regulation by PKC in which the FPA, but not
the FPB, isoform is subject to negative feedback by PKC
(Fujino et al., 2000b). The mechanism of this negative feedback involved PKC-mediated phosphorylation of the carboxyl-terminal domain of the FPA isoform, leading to an inhibition of stimulated inositol phosphate formation.
Besides signaling differences, the FPA and FPB prostanoid
receptor isoforms may differ in their expression and localization, particularly in response to agonist exposure. In this
regard, it has recently been shown that there are significant
differences between the TP␣ and TP␤ thromboxane receptor
isoforms with respect to agonist-induced receptor internalization (Parent et al., 1999). These isoforms, like the FP
receptor isoforms, also represent carboxyl-terminal splice
variants, and TP␤, the longer of the two, undergoes clathrindependent internalization, whereas TP␣ does not. The
present study was conducted to determine whether similar
differences might exist between the FP receptor isoforms. We
now report that the FPA isoform undergoes a rapid agonistinduced internalization that requires PKC and involves
clathrin, whereas the FPB isoform undergoes a constitutive,
agonist-independent internalization that does not involve either PKC or clathrin.
Internalization of FP Receptor Isoforms
Fig. 1. Immunofluorescence localization of FLAG-tagged FPA and FPB
prostanoid receptor isoforms after treatment with either vehicle or 1 ␮M
PGF2␣ for 10 min at 37°C. HEK-293 cells stably expressing either the
FLAG-FPA or FLAG-FPB isoforms were labeled concurrently with antiFLAG M2 antibodies and examined by fluorescence microscopy as described under Experimental Procedures. Scale bars represent 10 ␮m. The
data shown are representative of three independent experiments.
are not associated with the outer membrane are likely to be
internal.
Inhibition of PKC by Gö 6976 Inhibits Agonist-Dependent Internalization of FLAG-FPA Receptors but
Has No Effect on the Localization of FLAG-FPB Receptors. Gö 6976, a specific inhibitor of PKC, was used to study
the role of PKC in the agonist-induced internalization of the
FLAG-FPA receptors and to determine whether PKC activity
was required for the constitutive internalization of the
FLAG-FPB receptors. Cells were pretreated with 100 nM Gö
6976 for 5 min at 37°C and incubated with either vehicle or
1 ␮M PGF2␣ concurrently with the anti-FLAG M2 antibodies
for 10 min. As shown in the top panels of Fig. 2, pretreatment
with Gö 6976 itself did not affect the localization of either the
FLAG-FPA or the FLAG-FPB receptors in the vehicle-treated
cells; i.e., the FLAG-FPA receptors are still localized predominantly on the cell surface, whereas the FLAG-FPB receptors
show punctate labeling on both the cell surface and intracellularly (compare with top panels of Fig. 1). On the other
hand, in cells that were treated with PGF2␣, Gö 6976 pretreatment blocked the internalization of the FLAG-FPA (Fig.
2, bottom left panel) but did not affect the localization of
FLAG-FPB receptors (Fig. 2, bottom right panel).
Activation of PKC by PMA Induces FLAG-FPA Receptor Internalization but Has No Effect on the Localization of FLAG-FPB Receptors. Since inhibition of
PKC by Gö 6976 blocked the PGF2␣-induced internalization of FLAG-FPA receptors, we decided to examine the
effects of PMA, a direct activator of PKC, on the localization of FLAG-FPA and FLAG-FPB receptors, both alone
and after pretreatment of the cells with Gö 6976. Cells
were pretreated with either vehicle or 100 nM Gö 6976 for
5 min at 37°C and were stimulated with 10 ␮M PMA for 10
min concurrently with the anti-FLAG M2 antibodies at
Fig. 2. The effect of Gö 6976 on immunofluorescence localization of
FLAG-FPA and FLAG-FPB prostanoid receptor isoforms after treatment
with either vehicle or 1 ␮M PGF2␣ for 10 min at 37°C. HEK-293 cells
stably expressing FLAG-FPA or FLAG-FPB isoforms were pretreated for
5 min with 100 nM Gö 6976, labeled concurrently with anti-FLAG M2
antibodies, and examined by fluorescence microscopy as described under
Experimental Procedures. Scale bars represent 10 ␮m. The data shown
are representative of three independent experiments.
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of cells with vehicle (top left panel), the FLAG-FPA receptors
are localized almost exclusively on the outer cell membrane,
whereas the FLAG-FPB receptors show a more punctate localization on both the outer membrane and intracellularly
(Fig. 1, top right panel). Thus, FLAG-FPB receptors undergo
constitutive internalization even in the absence of PGF2␣.
After treatment with PGF2␣, however, the localization of the
FLAG-FPA receptors shifts to a more punctate pattern with
an increase in intracellular labeling reflecting receptor internalization (Fig. 1, bottom left panel). Agonist-induced internalization of the FLAG-FPB receptors was difficult to assess
because of the constitutive internalization, and treatment of
FLAG-FPB-expressing cells with PGF2␣ does not produce any
obvious changes in receptor localization (Fig. 1, bottom right
panel).
Several lines of evidence support the view that the punctate labeling observed for FLAG-FPB-expressing cells represents internalized receptors and not aggregated receptors on
the cell surface. The first line of evidence comes from live cell
labeling experiments done after quickly lowering the temperature to 4°C, which effectively stops the internalization process (e.g., see Parent et al., 2001). Under these conditions, the
immunofluorescence labeling of both the FPA- and FPB-expressing cells is quite similar and appears exclusively on the
outer cell membrane (data not shown). Similar results were
obtained when FLAG-FPB-expressing cells were fixed, but
not permeabilized, and then labeled. Under these conditions,
the labeling of both FPA- and FPB-expressing cells was similar to the live cell labeling obtained at 4°C and consisted of
diffuse fluorescence on the outer cell membrane. A third line
of evidence comes from our use of confocal microscopy for
imaging. Thus, for the present study, images were taken with
a focal plane close to the midpoint of the cell so as to obtain
a cross-sectional view. Under these conditions, puncta that
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Fig. 3. Immunofluorescence localization of FLAG-tagged FPA and FPB
prostanoid receptor isoforms after treatment with either vehicle or 10 ␮M
PMA for 10 min at 37°C. HEK-293 cells stably expressing FLAG-FPA or
FLAG-FPB isoforms were pretreated for 5 min with 100 nM Gö 6976,
labeled concurrently with anti-FLAG M2 antibodies, and examined by
fluorescence microscopy as described under Experimental Procedures.
Scale bars represent 10 ␮m. The data shown are representative of three
independent experiments.
Fig. 4. The effect of sucrose on the immunofluorescence localization of
FLAG-tagged FPA and FPB prostanoid receptor isoforms after treatment
with either vehicle, 1 ␮M PGF2␣, or 10 ␮M PMA for 10 min at 37°C.
HEK-293 cells stably expressing FLAG-FPA or FLAG-FPB isoforms were
pretreated for 15 min with 0.4 M sucrose, labeled concurrently with
anti-FLAG M2 antibodies, and examined by fluorescence microscopy as
described under Experimental Procedures. Scale bars represent 10 ␮m.
The data shown are representative of three independent experiments.
PGF2␣ Stimulates Tyrosine Phosphorylation of a
p200 Protein in FLAG-FPA but Not in FLAG-FPB Cells.
Previous studies have shown that internalization of some
GPCRs by a clathrin-dependent mechanism results in the
activation of tyrosine kinases (Maudsley et al., 2000). To
examine the potential for tyrosine phosphorylation of intracellular proteins following the internalization of FP receptors, cells stably expressing either FLAG-FPA or FLAG-FPB
receptors were treated with 1 ␮M PGF2␣ and subjected to
immunoblotting with anti-phosphotyrosine antibodies as described under Experimental Procedures. As shown in Fig. 5, a
band of approximately 200 kDa was tyrosine-phosphorylated
in an agonist-dependent manner in FLAG-FPA-expressing
cells but not in the FLAG-FPB-expressing cells. The size of
this p200 tyrosine-phosphorylated protein is close to the
known size of clathrin heavy chain (⬃180 kDa) and, therefore, these experiments were repeated, immunoblotting first
with anti-phosphotyrosine antibodies, followed by stripping
and reprobing with anti-clathrin heavy-chain antibodies. As
shown in the top panel of Fig. 6, treatment of FLAG-FPA cells
with 1 ␮M PGF2␣ for 10 min again resulted in agonistinduced tyrosine phosphorylation of the p200 protein (Fig. 6,
arrow), which was not observed in FLAG-FPB-expressing
cells. Reprobing with anti-clathrin heavy-chain antibodies
(Fig. 6, lower panel) shows that both the FLAG-FPA cells and
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37°C. They were then fixed and examined by fluorescence
microscopy. As seen in the top panels of Fig. 3, treatment
with PMA alone induces internalization of FLAG-FPA receptors but does not influence the distribution of FLAGFPB receptors. However, when FLAG-FPA cells were pretreated with Gö 6976, PMA-mediated internalization was
blocked (Fig. 3, bottom left panel). Pretreatment of the
FLAG-FPB cells with Gö 6976, did not change the pattern
of receptor localization compared with treatment with
PMA alone (Fig. 3, bottom right panel).
Sucrose Blocks PGF2␣ and PMA-Stimulated Internalization of FLAG-FPA Receptors but Does Not Affect
the Constitutive Internalization of FLAG-FPB Receptors. Previous studies on GPCRs have indicated that agonist-dependent internalization is frequently a clathrin-dependent process. To test the role of clathrin in both the
agonist-induced internalization of FLAG-FPA receptors and
the constitutive internalization of FLAG-FPB receptors, we
examined the effects of sucrose, a known inhibitor of clathrin-dependent internalization, on the immunofluorescent localization of these receptors. Cells stably expressing either
FLAG-FPA or FLAG-FPB receptors were pretreated with 0.4
M sucrose for 15 min, followed by treatment with either
vehicle, 1 ␮M PGF2␣, or 10 ␮M PMA for 10 min concurrently
with the anti-FLAG M2 antibodies at 37°C. As shown in the
top panels of Fig. 4 sucrose pretreatment alone does not
affect the localization of either FLAG-FPA receptors or
FLAG-FPB receptors. Interestingly, however, sucrose pretreatment blocks internalization of FLAG-FPA receptors but
does not affect the localization of FLAG-FPB receptors after
stimulation with PGF2␣ (Fig. 4, middle panels). Similarly,
sucrose pretreatment blocks PMA-mediated internalization
of FLAG-FPA receptors but does not affect the localization of
FLAG-FPB receptors (Fig. 4, bottom panels).
Internalization of FP Receptor Isoforms
Fig. 5. Time course of tyrosine phosphorylation of a p200 protein (arrow)
in cells stably expressing either the FLAG-FPA or FLAG-FPB receptors
after treatment with PGF2␣. Cells were incubated with 1 ␮M PGF2␣ for
the indicated times, and particulate fractions were prepared and subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting
(IB) with PY20 as described under Experimental Procedures. Position of
the 118-kDa marker is indicated. The data shown are representative of
three independent experiments.
FLAG-FPB cells contained similar amounts of clathrin heavy
chain, which comigrated with the p200 tyrosine-phosphorylated protein shown in Fig. 6, top panel. To directly test
whether this p200 protein might be clathrin heavy chain, cell
lysates were immunoprecipitated with antibodies to clathrin
heavy chain and immunoblotted with anti-phosphotyrosine
antibodies. The results were inconclusive (data not shown)
and, thus, it was not possible to confirm the exact identity of
the p200 tyrosine-phosphorylated protein.
Discussion
The importance of the carboxyl terminus in receptor desensitization and internalization has been generally recognized for a number of GPCRs, although its role in the internalization of prostanoid receptors is not well established.
This is particularly true of the FP receptors, in which alternate mRNA splicing gives rise to two isoforms that differ only
in their carboxyl-terminal domains. These two isoforms,
termed FPA and FPB, differ from one another in that the FPB
is essentially truncated by 46 amino acids compared with the
FPA. Contained within these 46 amino acids are four consensus sites for PKC, and we have previously shown that the
FPA isoform, but not the FPB, is phosphorylated by PKC and
subject to a rapid negative feedback involving PKC (Fujino et
al., 2000b).
We have also shown in functional studies that the FPA and
FPB receptor isoforms undergo a similar degree of desensitization but that the FPA isoform resensitizes more rapidly
than the FPB (Fujino et al., 2000a). The potential role of
receptor internalization in this process is unknown, and until
now even the basic characteristics of the internalization of
these isoforms have never been described. To address this
issue, we FLAG-tagged the FPA and FPB isoforms and examined their localization by immunofluorescence microscopy
with or without PGF2␣ treatment. We found that even without PGF2␣ treatment, the FPB isoform undergoes a rapid
constitutive internalization, whereas the FPA isoform does
not. In the presence of PGF2␣, however, the FPA isoform was
internalized in a PKC- and clathrin-dependent manner. Because of constitutive internalization of the FPB receptor isoform, we could not determine whether there was additional
agonist-dependent internalization; however, we established
that the constitutive internalization of the FPB isoform is
neither PKC- nor clathrin-dependent.
As noted above, PGF2␣-induced internalization of the FPA
receptor was dependent upon PKC in that internalization
was blocked when cells were pretreated with Gö 6976, an
inhibitor of PKC. It would appear likely, therefore, that phosphorylation of the carboxyl terminus by PKC is required for
the internalization of the FPA isoform. This is further
strengthened by the fact that direct activation of PKC by
PMA could induce FPA receptor internalization even in the
absence of PGF2␣. Since activation of PKC by PMA leads to
internalization of the FPA receptor, cells expressing the FPA
receptors are potential candidates for heterologous desensitization similar to that shown for the somatostatin receptor
(Hipkin et al., 2000) and the opiate receptor (Xiang et al.,
2001). On the other hand, the constitutive internalization of
the FPB isoform clearly did not require PKC because it was
unaffected by treatments with either Gö 6976 or PMA. It
thus appears that PKC has a major role in the internalization of the FPA isoform, although it does not preclude a role
for a G-protein receptor kinase. Dileucine- and tyrosinebased motifs have also been shown to play a role in phosphorylation-dependent, clathrin-mediated, receptor internalization (Mukherjee et al., 1997; Parent et al., 2001). Inspection
of the unique carboxyl-terminal sequence of the FPA isoform
relative to the FPB isoform did not reveal any such motifs
that could explain the different internalization of these isoforms. Further mutagenesis studies, however, could shed
light on whether a functionally similar motif is present as
well as the specific PKC sites that are responsible for internalization.
Two lines of evidence indicate that the internalization of
the FPA receptor isoform involves a clathrin-dependent
mechanism. Thus, both PGF2␣- and PMA-induced internalizations of the FPA isoform were blocked by hypertonic sucrose. Hypertonic sucrose is a well known inhibitor of clathrin-dependent receptor internalization and has been shown
to inhibit the internalization of both GPCRs and tyrosine
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Fig. 6. Immunoblotting (IB) of cell lysates with PY20 (top panel) and with
anti-clathrin heavy-chain (CHC) antibodies (bottom panel) after treatment of FLAG-FPA or FLAG-FPB-expressing cells with 1 ␮M PGF2␣ for 10
min. Particulate fractions were prepared from cell lysates and subjected
to SDS-polyacrylamide gel electrophoresis and immunoblotting as described under Experimental Procedures. The membranes were first
probed with anti-phosphotyrosine antibodies and were stripped and reprobed with anti-clathrin heavy-chain antibodies. Position of the 118-kDa
marker is indicated. The data shown are representative of three independent experiments.
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Address correspondence to: Dr. John W. Regan, Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ
85721-0207. E-mail: [email protected]
Downloaded from jpet.aspetjournals.org at ASPET Journals on August 3, 2017
kinase receptors. Interestingly, the constitutive internalization of the FPB isoform was not blocked by pretreatment with
hypertonic sucrose, indicating that the internalization of the
FPB isoform does not involve a clathrin-dependent mechanism. A second line of evidence suggesting that clathrin is
involved in the internalization of the FPA isoform, but not the
FPB, is our observation of the possible tyrosine phosphorylation of clathrin heavy chain in FPA-expressing cells, but not
in FPB-expressing cells, after treatment with PGF2␣. Presently, we have not been able to confirm the tyrosine phosphorylation of clathrin heavy chain, and we do not know the
kinase or pathway involved. However, tyrosine phosphorylation of clathrin heavy chain has been shown to occur after the
agonist-induced internalization of the epidermal growth factor (EGF) receptor (Wilde et al., 1999). Furthermore, transactivation of the EGF receptor has been demonstrated following the activation of ␤2-adrenergic receptors (Maudsley et
al., 2000), which provides a potential mechanism linking
GPCR activation to an EGF receptor-mediated tyrosine phosphorylation of clathrin heavy chain. Recent studies have also
shown that the nonreceptor tyrosine kinase, c-src, is recruited to the ␤2-adrenergic receptor during agonist-induced
internalization (Miller et al., 2000), which could also mediate
a tyrosine phosphorylation of clathrin heavy chain. Further
work will hopefully provide answers to these possibilities.
In contrast to the FPA isoform, the FPB isoform showed a
remarkable degree of spontaneous receptor internalization
that occurred in the absence of agonist treatment. This constitutive internalization has also been observed for other
GPCRs, most notably for truncation mutants of ␮-opioid receptors and somatostatin-2 receptors (Segredo et al., 1997;
Schwartkop et al., 1999). Truncation per se, however, is
unlikely to be the basis for constitutive internalization as it
has been recently shown that TP␤, the longer of the two
carboxy-terminal splice variants of the thromboxane receptor, undergoes constitutive internalization, whereas TP␣, the
shorter isoform, does not (Parent et al., 2001). This constitutive internalization of the TP␤ isoform was associated with a
specific amino acid motif that is not present, however, in the
FPB isoform. Another significant difference between the
spontaneous internalization of the FPB and TP␤ receptor
isoforms is that internalization of the TP␤ isoform was
blocked by sucrose, whereas internalization of FPB isoform
was not. Despite these differences, it is interesting that for
both the TP and FP receptor subtypes there are two known
carboxyl-terminal splice variants that differ from one another in their degrees of constitutive receptor internalization.
Although the full biochemical consequences of the constitutive internalization of these receptor isoforms are presently
unknown, they are probably relevant to the physiological
functions of these isoforms.