Signal Transduction and Hormone-dependent
Internalization of the Thyrotropin-releasing Hormone Receptor in Cells
Lacking Gq and G11*
Run
Yu and
Patricia M.
Hinkle
From the Department of Pharmacology and Physiology and the Cancer
Center, University of Rochester School of Medicine and Dentistry,
Rochester, New York 14642
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ABSTRACT |
The thyrotropin-releasing hormone (TRH) receptor
was expressed in embryonic fibroblasts from mice lacking the
subunits of Gq and G11 (Fq/11 cells) to
determine whether G protein coupling is necessary for
agonist-dependent receptor internalization. Neither TRH nor
agonists acting on endogenous receptors increased intracellular calcium
unless the cells were co-transfected with the
subunit of
Gq. In contrast, temperature-dependent
internalization of [3H]MeTRH in Fq/11 cells was the same
whether Gq
was expressed or not. A rhodamine-labeled
TRH analog and fluorescein-labeled transferrin co-localized in
endocytic vesicles in Fq/11 cells, indicating that endocytosis took
place via the normal clathrin pathway. Cotransfection with
-arrestin
or V53D
-arrestin increased TRH-dependent receptor
sequestration. Fq/11 cells were co-transfected with the TRH receptor
and a green fluorescent protein (GFP)-
-arrestin conjugate.
GFP-
-arrestin was uniformly distributed in the cytoplasm of
untreated cells and quickly translocated to the periphery of the cells
when TRH was added. A truncated TRH receptor that lacks potential
phosphorylation sites in the cytoplasmic carboxyl terminus signaled but
did not internalize or cause membrane localization of GFP-
-arrestin.
These results prove that calcium signaling by the TRH receptor requires
coupling to a G protein in the Gq family, but
TRH-dependent binding of
-arrestin and sequestration do not.
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INTRODUCTION |
Many G protein-coupled receptors undergo ligand-driven
internalization through the clathrin pathway. Internalization or
sequestration appears to modulate various aspects of signal
transduction such as desensitization, resensitization, and activation
of mitogen-activated protein kinases (1-3). A fundamental question
regarding internalization of G protein-coupled receptors is how the
activated receptor is targeted to clathrin-coated pits in response to
agonist binding. Recently, this question has been answered with the
discovery that
-arrestin acts as an adaptor (4, 5), binding both to
receptors and to clathrin (6, 7). Because
-arrestin binds most
strongly to activated, phosphorylated receptors (5), and receptor
phosphorylation is promoted by agonist binding,
-arrestin can
discriminate between inactive and active receptors and target
agonist-occupied receptors to clathrin-coated pits and the endocytic pathway.
Most receptors coupled to G proteins in the Gq family are
sequestered following the binding of an agonist but not an antagonist (8). There are exceptions to this generalization, however, because some
Gq-coupled receptors do not internalize at all, others internalize only in some cell contexts (9), and a few internalize when
occupied by an antagonist (10, 11). It is uncertain if coupling to
Gq proteins is required for receptor phosphorylation,
-arrestin recognition of agonist-occupied receptor, or receptor internalization.
Internalization of the receptor for thyrotropin-releasing hormone
(TRH),1 which signals through
Gq and G11 to activate phospholipase C, has
been studied extensively (12-18). The TRH receptor internalizes rapidly (t1/2 ~ 2.5 min) through the clathrin
pathway upon agonist binding (13). When its cytoplasmic, carboxyl-terminal tail is deleted, the TRH receptor binds ligand with
high affinity and stimulates phospholipase C but does not internalize,
showing that coupling to Gq is not sufficient to cause
internalization (15, 17). It is not clear whether coupling to
Gq is necessary for receptor sequestration. Several
investigators have approached this question by characterizing the
internalization of mutant receptors using antibodies rather than
hormones to promote internalization and blocking signal pathways
pharmacologically (15, 17). They have reached contradictory conclusions
about whether receptor-G protein coupling is necessary for TRH receptor internalization.
In this report, we have taken advantage of a fibroblast cell line
that lacks Gq and G11 to
examine directly the role of these G proteins in the internalization of
TRH receptor (19, 20). The Fq/11 cells were derived from the embryos of
mice in which the
subunits of both Gq and
G11 had been knocked out by targeted gene disruption. Our
results show that Gq or G11 is absolutely required for calcium signaling but not for coupling between the activated TRH receptor and
-arrestin or for receptor internalization.
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EXPERIMENTAL PROCEDURES |
Materials--
LipofectAMINE PLUS was from Life
Technologies, Inc. Plasmids encoding the mouse TRH receptor
(pCDM8mTRHR) and a truncated internalization-defective TRH receptor
(pAd/CMVmTRHRC335STOP) were gifts from Dr. Marvin C. Gershengorn
(Cornell University, New York, NY). A plasmid-encoding mouse
Gq
subunit (pCMVGq) was a gift from Dr. Dianqing Wu
(University of Rochester, Rochester, NY). The plasmid pRSVCAT encoding
chloramphenicol acetyltransferase was from the American Type Culture
Collection (Manassas, VA). Plasmids encoding wild type rat
-arrestin
(pCMV5
arr1),
-arrestin V53D (pcDNA1
arr1V53D), and a
conjugate protein of
-arrestin 2 and green fluorescent protein
(p
arr2GFP) (21) were generous gifts from Dr. Marc Caron (Duke
University, Durham, NC).
Fq/11 cells are fibroblasts derived from
Gq
/G11
knockout mice and were generously
provided by Dr. Melvin Simon (California Institute of Technology,
Pasadena, CA) (19, 20). Fq/11 cells were grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum and were
passaged by trypsinization. Transfection of Fq/11 cells was achieved
with LipofectAMINE PLUS according to the manufacturer's suggestions.
In cotransfection assays, 1 µg of pCDM8mTRHR and 2 µg of pCMVGq or
pRSVCAT were used. Primary mouse embryonic fibroblasts were a gift from
Dr. Dianqing Wu (University of Rochester, Rochester, NY) and were cultured and transfected similarly.
Methods--
Ca2+ imaging was carried out as
described (22, 23). To measure radioligand binding, cells in 35-mm
dishes were incubated in Hank's buffered saline solution containing 15 mM HEPES, pH 7.4 containing 5 nM
[3H]MeTRH with or without a 1000-fold molar excess of
unlabeled hormone. Dishes were then washed and incubated in ice-cold
0.5 M NaCl, 0.2 M acetic acid (pH 2.8) for 1 min, and radioactivity in the acid wash (surface) and the cells
(internalized) was quantified (13). Binding data shown are typical of
results in 2 to 4 independent experiments.
Staining with Rhod-TRH and fluorescein-labeled transferrin
(FITC-transferrin) was carried out as described (12). In colocalization experiments, cells were incubated in Hepes-buffered saline solution with 170 nM Rhod-TRH and 5 µg/ml fluorescein-labeled
transferrin (FITC-transferrin) at 37 °C for 1 h. Control
experiments, in which cells were stained with either Rhod-TRH or
FITC-transferrin alone, showed that bleed-through was negligible.
To examine translocation of the
-arrestin-green fluorescent protein
(GFP) conjugate (
arr2GFP), Fq/11 cells were transfected with 2 µg
of pCDM8mTRHR or pAd/CMVmTRHRC335STOP and 0.4 µg of p
arr2GFP/dish.
About 24 h after transfection, cells were washed and incubated
with Hepes-buffered saline solution at 37 °C. Cells were stimulated
with 1 µM TRH for up to 20 min. Localization of
arr2GFP in live cells was followed microscopically with a 40× objective and a fluorescein filter. Microscopic results are typical of
data obtained in 3 to 10 independent experiments.
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RESULTS |
We transfected Fq/11 cells with cDNA encoding the TRH receptor
and a 2-fold higher concentration of either a control plasmid or
plasmid encoding Gq
and then measured the ability of
various agonists to increase the concentration of cytoplasmic free
Ca2+, [Ca2+]i, in individual cells
loaded with the Ca2+ reporter Fura-2. Following
transfection with the TRH receptor alone, Fq/11 cells failed to respond
to TRH or to a mixture of agonists acting on endogenous receptors
(thrombin, endothelin, bombesin, and bradykinin) (Figs.
1 and 2 and
Table I). When transfected with the TRH
receptor and the
subunit of Gq, a significant number of
cells responded to TRH (15%) and to the mixture (11%) (Figs. 1 and 2
and Table I). Subsequent experiments showed that bradykinin was
responsible for most of the activity in the agonist mixture (Table I).
TRH caused a consistently larger increase in
[Ca2+]i than the mixture. Based on
[3H]MeTRH binding, the number of TRH receptors expressed
was within 50% of the same in cultures transfected with a control
plasmid or plasmid encoding Gq
(data not shown). To
determine whether intracellular Ca2+ stores were adequate
in the nonresponsive cells, we added the Ca2+ chelator
BAPTA to reduce extracellular Ca2+ to ~4 nM
and then added a low concentration of ionomycin to release Ca2+ from intracellular stores. All of the Fq/11 cells
tested responded to ionomycin with a large increase in Ca2+
except those cells that had previously responded to TRH, which had
predictably smaller responses because their Ca2+ stores had
been partially depleted.

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Fig. 1.
Single-cell Ca2+ responses to
agonists in Fq/11 cells. Fq/11 cells were transfected with
plasmids encoding the mouse TRH receptor (mTRHR, 1 µg/dish) and control plasmid, pRSVCAT (2 µg/dish), or the TRH
receptor (1 µg/dish) and Gq (2 µg/dish). After
24 h, cells were loaded with Fura-2, and single-cell
[Ca2+]i responses were recorded. Drugs were added
as noted: TRH, 1 µM; BAPTA, 1.5 mM; ionomycin
(Iono), 500 nM; cocktail-thrombin, 10 units/ml;
bradykinin, 1 µM; endothelin, 100 nM; and
bombesin, 1 µM. Solid traces show the average
of 2 to 5 responsive cells, and dashed traces show the
average of 9 to 28 unresponsive cells.
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Fig. 2.
Single-cell Ca2+ responses to
agonists in Fq/11 cells. Fq/11 cells were transfected with either
TRH receptor and control plasmid (A-D) or TRH receptor and
Gq (E-H) as described for Fig. 1. Shown are
340/380 fluorescence ratios in pseudocolor before treatment
(A and E) and immediately after the addition of 1 µM TRH (B and F), 1.5 mM BAPTA (C and G), and 500 nM ionomycin (D and H).
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Table I
Summary of [Ca2+]i responses to agonists in
transfected Fq/11 cells
Fq/11 cells were transfected as described in the legend of Fig. 1. Data
were derived from 4-6 experiments done in 2 days. ND, not
determined.
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To determine whether internalization of the TRH receptor also requires
G protein coupling, we measured sequestration of
[3H]MeTRH to an acid/salt-resistant compartment in Fq/11
cells with and without transfection of Gq
. When Fq/11
cells were transfected with only the TRH receptor and then incubated
with [3H]MeTRH at 37 °C, 32% of specifically bound
hormone was acid/salt resistant, in comparison to only 12% when
binding was performed at 0 °C (Fig.
3). When cells were transfected with the
receptor and Gq
, the extent of internalization was the
same (Fig. 3). Internalization of [3H]MeTRH was
unaffected by treatment with pertussis toxin, indicating that
Gi and Go are not involved. We also transfected
embryonic fibroblast cultures obtained from wild type mice of the same
strain as the knockout mice (MEF cells) with the TRH receptor cDNA
and followed [3H]MeTRH internalization. The wild type
fibroblasts expressed TRH receptors at the same level as the Fq/11
cells, and they also internalized [3H]MeTRH by a
pertussis toxin-insensitive pathway.

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Fig. 3.
Internalization of [3H]MeTRH in
Fq/11 cells and in normal primary mouse fibroblasts.
Left, Fq/11 cells were transfected with plasmids encoding
TRH receptor (1 µg/dish) and control plasmid (2 µg/dish)
(Con) or TRH receptor (1 µg/dish) and Gq (2 µg/dish) (Gq ). Right, mouse
embryonic fibroblasts (MEF) from wild type animals and Fq/11
cells were transfected with TRH receptor alone. Cells were incubated
for 60 min at 0 or 37 °C with 5 nM
[3H]MeTRH, and the percent of specifically bound hormone
internalized was determined. [3H]MeTRH binding averaged
1150 ± 134 cpm/dish in Fq/11 cells and 1517 ± 68 cpm/dish
for mouse embryonic fibroblasts cultures. Cells transfected with
Gq bound 141% as much [3H]MeTRH as those
transfected with receptor alone. Some cells were treated with pertussis
toxin (PTx, 100 ng/ml) overnight.
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Because the TRH receptor normally internalizes in a
clathrin-dependent manner, we compared internalization of
FITC-transferrin, a classical marker for the coated pit pathway, and a
bioactive, rhodamine-labeled TRH analog, Rhod-TRH, in Fq/11 cells.
Rhod-TRH fluorescence in Fq/11 cells was dim, consistent with the low
level of [3H]MeTRH binding. When Fq/11 cells were
transfected with only the TRH receptor and incubated with Rhod-TRH at
0 °C, fluorescence was on the cell surface (Fig.
4). Incubation with excess TRH blocked the membrane fluorescence, indicating that it was because of
receptor-bound Rhod-TRH. When these cells were incubated with Rhod-TRH
at 37 °C, Rhod-TRH was concentrated inside the cell, indicating that internalization of the liganded receptor had taken place. The bright
specks that can be seen in all panels were associated with transfection reagents and were also seen in mock-transfected cells (data not shown). Internalization of FITC-transferrin was rapid and
extensive, with nearly all the label in an endocytic compartment by 5 min (data not shown). Cells were also incubated simultaneously with
Rhod-TRH (red) and FITC-transferrin (green) at
37 °C. There was extensive colocalization of the two labels, shown
in yellow and orange (Fig.
5). These findings all indicate that TRH
drives internalization of its receptor via the normal coated pit
pathway without G protein interaction.

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Fig. 4.
Internalization of rhodamine-labeled TRH in
Fq/11 cells. Fq/11 cells were transfected with the TRH receptor
and incubated for 1 h with Rhod-TRH at 0 °C (A),
Rhod-TRH plus 1 µM TRH at 0 °C (B),
Rhod-TRH at 37 °C (C), and Rhod-TRH plus 1 µM TRH at 37 °C (D). Cells not expressing
the TRH receptor are barely visible to the right of those expressing
the receptor.
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Fig. 5.
Colocalization of rhodamine-labeled TRH and
FITC-labeled transferrin. Fq/11 cells were transfected with TRH
receptor and incubated with Rhod-TRH and FITC-transferrin at 37 °C
for 1 h. Panels show Rhod-TRH (red) (A),
FITC-transferrin (green) (B), an optical overlay
in which regions containing both rhodamine and fluorescein are shown as
yellow and orange (C), and bright
field image (D).
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To establish whether overexpression of
-arrestin would increase
sequestration, we transfected Fq/11 cells with the TRH receptor with or
without
-arrestin. Cells transfected with the receptor and
-arrestin internalized more [3H]MeTRH (56%) than
cells transfected with receptor alone (32%) (Fig.
6), suggesting that
-arrestin was
functioning as an adaptor in the absence of G protein coupling. A
mutant
-arrestin, V53D, has been shown to act in a dominant negative
manner in some settings but not in others. V53D
-arrestin increased
internalization of [3H]MeTRH as effectively as wild type
arrestin (Fig. 6).

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Fig. 6.
Internalization of [3H]Me TRH
in Fq/11 cells cotransfected with
-arrestin. Fq/11 cells were cotransfected with
plasmids encoding the TRH receptor (1 µg/dish) and either control
plasmid (2 µg/dish), -arrestin (2 µg/dish), or V53D -arrestin
(2 µg/dish). Binding assays were done on ice or at 37 °C for 60 min, and internalization of receptor-bound [3H]MeTRH was
measured. [3H]MeTRH binding averaged 348 ± 46 cpm/dish, and dishes transfected with wild type or mutant -arrestin
bound 93 and 86% as much [3H]MeTRH, respectively, as
controls transfected only with receptor.
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To determine whether G protein coupling is needed for
-arrestin
binding, we transfected Fq/11 cells with the TRH receptor and a
GFP-
-arrestin conjugate and followed the localization of GFP-
-arrestin before and after activating the receptor.
GFP-
-arrestin was uniformly distributed in the cytoplasm of
untreated cells (Fig. 7). Following the
addition of TRH, GFP-
-arrestin quickly translocated to the periphery
of the cells, suggesting that it bound to the agonist-occupied receptor
in the absence of G protein interaction. Once translocated to the
plasma membrane, much of the GFP-
-arrestin remained there for up to
1 h in the continued presence of TRH (data not shown). TRH did not
cause translocation of GFP-
-arrestin in cells expressing a
carboxyl-terminal-truncated TRH receptor (Fig. 7, also see below). Even
at a concentration of 10 nM, TRH rapidly and effectively
translocated GFP-
-arrestin (Fig. 8).
The cells shown in Figs. 7 and 8 were fairly dim. In cells with bright
GFP fluorescence, translocation was not as evident, presumably because
the bright cells expressed more GFP-
-arrestin, and the proportion
translocated in response to TRH was small. We never observed
translocation of GFP-
-arrestin to the membrane following activation
of the endogenous bradykinin receptor (Fig. 8). We have found that
agonist mixtures activating endogenous receptors in transfected 293 cells or GH3 cells do not cause translocation of GFP-
-arrestin,
although they cause [Ca2+]i responses comparable
with those evoked by TRH.2
These results suggest that the TRH receptor is more effective than most
in translocating GFP-
-arrestin.

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Fig. 7.
Translocation of
GFP- -arrestin. Fq/11 cells were
transfected with plasmid encoding a GFP- -arrestin conjugate (0.4 µg/ml) and 2 µg/ml plasmid encoding either wild type (A
and C) or truncated C335STOP (B and D)
TRH receptor. Panels A and B show cells
expressing GFP- -arrestin before stimulation, and panels C
and D show the same cells 5 min after the addition of 1 µM TRH at 37 °C.
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Fig. 8.
Effect of bradykinin and low dose TRH on
GFP- -arrestin localization. Fq/11 cells
were transfected with GFP- -arrestin conjugate and TRH receptor.
Shown are images of transfected cells before treatment (A),
1 min after the addition of 1 µM bradykinin
(B), and 1 min after the subsequent addition of 10 nM TRH (C). GFP- -arrestin translocation was
not observed in any cell after bradykinin treatment. GFP- -arrestin
translocation was observed in independent experiments where 10 nM TRH was added alone.
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We also evaluated the effect of a TRH antagonist, chlordiazepoxide, on
GFP-
-arrestin localization. Chlordiazepoxide (10 µM) did not cause translocation of GFP-
-arrestin by itself but blocked GFP-
-arrestin translocation initiated by a low dose of TRH, 10 nM (Fig. 9). When added at 10 µM, TRH overcame the effect of the antagonist and caused
GFP-
-arrestin to move to the membrane. The Kd
values of TRH and chlordiazepoxide for the TRH receptor are ~10
nM and 3 µM, respectively.
-Arrestin is
believed to bind more tightly to phosphorylated receptors (6, 7), and
although the phosphorylation sites on the TRH receptor have not been
mapped, most potential phosphorylation sites are found in the
intracellular carboxyl-terminal tail after residue 341. We therefore
transfected cells with GFP-
-arrestin and a truncated form of the TRH
receptor (C335 STOP) that lacks these potential phosphorylation sites.
The truncated receptor was expressed and capable of transducing a
signal, because TRH increased [Ca2+]i if
Gq
was cotransfected with the C335STOP receptor (Table
I). Activation of the truncated TRH receptor with TRH did not cause
GFP-
-arrestin to move to the membrane, however (Fig. 7).

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Fig. 9.
Effect of the TRH antagonist chlordiazepoxide
on GFP- -arrestin localization. Fq/11
cells were transfected with GFP- -arrestin conjugate and TRH
receptor. The following drugs were added in sequence to transfected
cells: 10 µM chlordiazepoxide, 10 nM TRH, and
10 µM TRH at 37 °C. Shown are two cells before
treatment (A), 20 min after the addition of 10 µM chlordiazepoxide (B), 1 min after 10 nM TRH (C), and 1 min after 10 µM
TRH (D). Note that the two cells moved and changed shapes
slightly during the 20-min chlordiazepoxide treatment, as do control
cells, but the localization of GFP- -arrestin did not change.
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Because it has been suggested that a receptor-
-arrestin complex can
signal directly to the mitogen-activated protein kinase pathway (3), we
asked whether TRH can activate mitogen-activated protein kinase in
Fq/11 cells expressing the TRH receptor with and without
Gq
. We did not detect significant TRH effects on the
abundance of phosphorylated mitogen-activated protein kinases on
Western blots using cells that had been serum-starved for 24 h and
treated with 1 µM TRH for up to 20 min, regardless of
expression of Gq
. Because background mitogen-activated
protein kinase activity was high and TRH receptor levels were low,
these results do not rule out the possibility that TRH activates the
mitogen-activated protein kinase pathway weakly in transfected Fq/11
cells, but any response must be very small.
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DISCUSSION |
Previous studies have led to contradictory conclusions concerning
the requirement for G proteins in the sequestration of G protein-coupled receptors coupled to the Gq family (15, 17, 24, 25). By using cells lacking the
subunits of Gq and
G11, we have obtained definitive evidence that the TRH
receptor undergoes agonist-dependent sequestration in the
absence of its normal signal transducers, Gq and
G11. It is improbable that the receptor internalized as a
result of coupling to some other G protein. Although the TRH receptor
has been reported to exert effects on ion channels via Gi
and Go (26-28), pertussis toxin did not alter
agonist-dependent internalization in Fq/11 cells. The TRH
receptor has been reported to couple to phospholipase C via
Gs in oocytes (29), but neither TRH nor bradykinin evoked
any calcium response in Fq/11 cells unless the cells were transfected
with Gq
. The TRH receptor was not overexpressed in mouse
fibroblasts. Assuming that 15% of Fq/11 cells expressed receptors,
because 15% of the cells exhibited a calcium response to TRH when
Gq
was co-transfected, we calculate that there were no
more than 30,000 TRH binding sites/cell. This is below the level in
pituitary cells (at least 50,000) and well below the levels in other
transfected cell models (HEK, COS, HeLa, Chinese hamster ovary, C6)
(16, 30, 31).
Petrou et al. (15) suggested that TRH receptor
internalization involves a Gq-independent pathway requiring
the CNC sequence in the carboxyl-terminal tail and perhaps an
additional, Gq-dependent pathway, based on the
study of mutant receptors and the finding that antibody to an
extracellular, amino-terminal epitope tag induced internalization but
not signaling. They also suggested that Gq
and the TRH
receptor internalize in the same vesicles. Milligan and co-workers (32)
find that TRH down-regulates Gq
and causes an increased
accumulation of Gq
in endocytic vesicles, but they do
not think that the receptor and Gq cointernalize because of
large discrepancies in the half-times for receptor and
Gq
internalization. Our results prove that
Gq/11 is not needed for internalization but do not
establish whether the receptor co-internalizes with Gq/11
when the appropriate G protein is available. Gershengorn and co-workers
(18) suggest that G protein coupling and phospholipase C activation are
both necessary for TRH receptor sequestration, because the
phospholipase C inhibitor U73122 inhibits sequestration and several
mutant receptors that bind TRH with high affinity but do not activate
phospholipase C fail to internalize (18). An alternative interpretation
is that mutants unable to activate phospholipase C do not internalize
because they do not assume the activated conformation necessary for
interaction with receptor kinases or
-arrestin as well as G protein.
U73122 exerts multiple effects on calcium homeostasis and other
processes (33-35) and may have impaired TRH-driven internalization by
a nonspecific action. Uncertainty regarding the requirement for
Gq coupling is not limited to the TRH receptor, because
U73122 inhibits the internalization of muscarinic receptors (25), and
sequestration of bombesin receptors has been reported to require G
protein interaction (24).
It was not previously known whether receptors needed to couple to
Gq either to promote an activated receptor conformation or
to promote phosphorylation by receptor kinases to bind
-arrestin. Our results, which show that GFP-
-arrestin moves to the membrane in
response to TRH in Fq/11 cells, confirm the role of
-arrestin in TRH
receptor sequestration and prove that the process does not require G
protein interaction. By demonstrating that GFP-
-arrestin does not
bind to the carboxyl-terminal-truncated C335STOP receptor, we have
established the molecular basis for its failure to internalize. These
results suggest that phosphorylation sites between Thr-342 and Ser-391
are important for
-arrestin binding and receptor sequestration.
Although
-arrestin appears to be involved in the internalization of
most G protein-coupled receptors, there are exceptions, including the
Gq-coupled M1 and M3 muscarinic receptors (36).
The maximal extent of TRH receptor sequestration was relatively low in
the Fq/11 cells and in wild type mouse fibroblasts, below 40%, whereas
the extent of internalization is above 80% when the receptor is
expressed in HEK293, HeLa, or C6 cells (16, 18). Furthermore, TRH
receptor sequestration in Fq/11 cells was increased by overexpression
of
-arrestin. Because the extent of internalization correlates with
the level of expression of
-arrestin and receptor kinases in a
number of cell lines (37), it seems likely that
-arrestin was
expressed at low levels in the Fq/11 cells. The failure of the V53D
mutant
-arrestin to act as a dominant negative is not completely
surprising, because the dominant negative effect is only partial for
the
2-adrenergic receptor (5, 7).
Previous work had shown that internalization of Gs- and
Gi/Go-coupled receptors can occur without G
protein involvement. The
2-adrenergic receptor can internalize in
cyc
and unc S49 cells (with absent or nonfunctional
Gs
, respectively) (38, 39), and at least some
Gi/Go-coupled receptors can internalize normally in pertussis toxin-treated cells (40). We have now shown that
-arrestin binding and internalization of Gq-coupled receptors can also occur in the absence of G protein coupling.
-Arrestin apparently discriminates between the agonist-activated and
the inactive TRH receptor either by recognizing the active conformation
of the receptor or by recognizing a protein that interacts with the
activated receptor. Further work will be required to establish the
molecular basis of this process.
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FOOTNOTES |
*
This work was supported by National Institutes of Health
Research Grant DK19974, by Cancer Center Core Research Grant CA 11098, and by a Wilmot Fellowship (to R. Y.).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.
To whom correspondence should be addressed: Dept. of Pharmacology
and Physiology, Box 711, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-4933; Fax:
716-461-0397; E-mail: hinklep{at}pharmacol.rochester.edu.
2
R. Yu and P. M. Hinkle, unpublished observations.
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ABBREVIATIONS |
The abbreviations used are:
TRH, thyrotropin-releasing
hormone;
Rhod-TRH, rhodamine TRH;
FITC, fluorescein isothiocyanate;
GFP, green fluorescent protein;
BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N',-tetraacetic
acid.
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