Agonist-Induced Endocytosis and Recycling of the Gonadotropin-Releasing Hormone Receptor: Effect of ß-Arrestin on Internalization Kinetics
Milka Vrecl,
Lorraine Anderson,
Aylin Hanyaloglu,
Alison M. McGregor,
Alex D. Groarke,
Graeme Milligan,
Philip L. Taylor and
Karin A. Eidne
MRC Reproductive Biology Unit (M.V., L.A., A.H., A.M.M., P.L.T.,
K.A.E.) Centre for Reproductive Biology Edinburgh, EH3 9EW,
United Kingdom
Molecular Pharmacology Group (A.D.G.,
G.M.) Division of Biochemistry and Molecular Biology Institute
of Biomedical Life Sciences University of Glasgow Glasgow G12
QQ, United Kingdom
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ABSTRACT
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This study examined the dynamics of endocytotic
and recycling events associated with the GnRH receptor, a unique G
protein-coupled receptor (GPCR) without the intracellular
carboxyl-terminal tail, after agonist stimulation, and investigated the
role of ß-arrestin in this process. Subcellular location of
fluorescently labeled epitope-tagged GnRH receptors stably expressed in
HEK 293 cells was monitored by confocal microscopy, and the
receptor/ligand internalization process was quantified using
radioligand binding and ELISA. Agonist stimulation resulted in
reversible receptor redistribution from the plasma membrane into the
cytoplasmic compartment, and colocalization of internalized GnRH
receptors with transferrin receptors was observed. Internalization
experiments for the GnRH receptor and another GPCR possessing a
carboxy-terminal tail, the TRH receptor, showed that the rate of
internalization for the GnRH receptor was much slower than for the TRH
receptor when expressed in both HEK 293 and COS-7 cells. TRH receptor
internalization could be substantially increased by coexpression with
ß-arrestin in COS-7 cells, while GnRH receptor internalization was
not affected by coexpression with ß-arrestin in either cell type.
Coexpression of the GnRH receptor with the dominant negative
ß-arrestin (319418) mutant did not affect its ability to
internalize, and activated GnRH receptors did not induce time-dependent
redistribution of ß-arrestin/green fluorescent protein to the
plasma membrane. However, the ß-arrestin mutant impaired the
internalization of the TRH receptor, and activated TRH receptors
induced the ß-arrestin/green fluorescent protein translocation. This
study demonstrates that, despite having no intracellular
carboxy-terminal tail, the GnRH receptor undergoes agonist-stimulated
internalization displaying distinctive characteristics described for
other GPCRs that internalize via a clathrin-dependent mechanism and
recycle through an acidified endosomal compartment. However, our data
indicate that the GnRH receptor may utilize a ß-arrestin-independent
endocytotic pathway.
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INTRODUCTION
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GnRH and its receptor are key mediators in the regulation of the
reproductive process. After its pulsatile release from the
hypothalamus, GnRH reaches the anterior pituitary gland where it
interacts with specific high- affinity GnRH receptors causing the
release of the gonadotropins, LH and FSH. Of the G protein-coupled
receptors (GPCRs) cloned to date, the mammalian GnRH receptor is unique
as it is the only receptor in which the functionally important
intracellular cytoplasmic C-terminal tail is completely absent. It is
truncated at the cell membrane immediately after the seventh
transmembrane domain (1). The GnRH receptor is preferentially coupled
to phosphoinositidase C via the Gq/G11 family
of G proteins, and its stimulation by GnRH induces the production of
inositol phosphates (IPs) and diacylglycerol, which results in the
elevation of cytosolic calcium and the activation of protein kinase C
(2). Agonist-induced receptor activation leads to a complex set of
intracellular events including desensitization of GPCRs by receptor-G
protein uncoupling followed by receptor internalization and recycling,
which is now thought to be essential for the reestablishment of
receptor responsiveness (3, 4, 5).
Experiments measuring endocytotic events at the level of the receptor
have been performed for the ß2-adrenergic receptor
(6, 7, 8), TRH (9), and muscarinic (10) and angiotensin II type 1A
(AT1A) receptors (11). After agonist activation these
receptors internalize into intracellular endosomes via clathrin-coated
pits and subsequently recycle back to the plasma membrane as functional
receptors. The binding of ß-arrestins to GPCRs has been shown to be a
convergent step of GPCR signaling with ß-arrestins acting as
adaptor-like proteins regulating the rate and specificity of receptor
internalization (12, 13). GPCR phosphorylation appears to be a
prerequisite for arrestin-receptor interaction (14), and it is proposed
that ß-arrestin binding to ligand-activated GPCRs specifically
directs the phosphorylated receptors into clathrin-coated pits due to
its ability to interact with both the receptor and clathrin.
Mutagenesis studies have revealed that the N-terminal half of nonvisual
arrestins has the ability to recognize the agonist-activated GPCRs
while the clathrin-binding domain is located in the COOH terminus of
the molecule (15, 16). The role of ß-arrestin in promoting GPCRs
internalization has been further confirmed using ß-arrestin dominant
negative mutants to impair receptor internalization (17). Recently, the
jellyfish (Aqueora victoria) green fluorescent
protein/ß-arrestin fusion protein has been introduced as a sensitive
tool for real time visualization of the receptor-mediated translocation
of ß-arrestin in living cells (18). Such a process has been
demonstrated for a number of ligand-activated GPCRs (19).
Sites located within the intracellular receptor loops, and in
particular the intracellular C-terminal region, have been shown to be
important in GPCR internalization, as receptor mutants with truncated
C-terminal tails or lacking putative G protein-coupled receptor kinase
phosphorylation sites displayed impaired internalization (13, 20, 21).
We have demonstrated that the addition of a functional intracellular
C-terminal tail to the GnRH receptor significantly increased
internalization rates (22).
The aim of this study was to examine endocytotic and recycling
events of the GnRH receptor. Previous studies on internalization events
connected with the GnRH receptor have employed radiolabeled ligand
binding (23, 24, 25, 26, 27, 28), which is based on the assumption that ligand and
receptor sort similarly through the endocytotic pathway. To test this
assumption, we employed the receptor tagging methodology to determine
the GnRH receptor endocytotic trafficking cycle at the level of the
receptor. The role of ß-arrestin in promoting internalization in the
GnRH receptor and in a GPCR possessing a C-terminal tail, the TRH
receptor, was also determined.
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RESULTS
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Characteristics of Hemagglutinin (HA)-Tagged GnRH Receptors
Insertion of the HA tag into the amino terminus of the rat GnRH
receptor did not perturb either receptor-ligand binding or second
messenger activation. Membranes prepared from HEK 293-B5 cells, which
stably express HA-tagged GnRH receptors, bound the GnRH receptor
agonist, [D-Trp6]-GnRH, with a dissociation
constant (Kd) of 0.27 nM and a Bmax
of 3.0 pmol/mg protein. These values were comparable to those obtained
in a HEK 293 cell line stably expressing wild-type (WT) GnRH receptors
(HEK 293-A2) with a Kd of 0.20 nM and a
Bmax of 3.1 pmol/mg protein (29). EC50 values
of 2.8 and 3.6 nM were obtained for GnRH-stimulated total
IP production in HEK 293-B5 and HEK 293-A2 cell lines,
respectively.
Visualization and Cellular Localization of GnRH Receptors
GnRH receptor distribution was examined in HEK 293 cells
stably expressing HA-tagged GnRH receptors using indirect
immunofluorescent staining. Fluorescently labeled HA-tagged GnRH
receptors were predominantly distributed around the cell circumference
in unstimulated HEK 293-B5 cells. Permeabilized, nontreated cells (Fig. 1a
) showed similar fluorescent
distribution consistent with localization of the receptor to the cell
surface. GnRH agonist treatment (1 or 2 h at 37 C) elicited a
redistribution of cellular immunostaining indicative of GnRH receptor
internalization (Fig. 1b
). The redistributed cytoplasmic signal showed
a vesicular pattern, and in some cells positive staining was observed
in the perinuclear region. TRH, another hypothalamic releasing hormone,
had no effect on GnRH receptor distribution (Fig. 1c
). The same was
also observed for the GnRH receptor antagonist
[Ac-3,4-dehydro-Pro1,
D-p-F-Phe2,
D-Trp3,6]-GnRH(GnRH-antag) (Fig. 1d
),
and pretreatment of cells with GnRH-antag prevented agonist-induced
internalization (Fig. 1e
). No specific staining was observed in
untransfected HEK 293 cells (Fig. 1f
).

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Figure 1. Visualization of GnRH Receptor Internalization by
Confocal Microscopy
a, Untreated HEK 293-B5 cells stably expressing HA-tagged GnRH
receptors; b, cells incubated with GnRH agonist,
[D-Trp6]-GnRH; c, TRH; d, GnRH-antag ([Ac-3,
4-dehydro-Pro1, D-p-F-Phe2,
D-Trp3, 6]-GnRH); e, GnRH-antag and GnRH; f,
untransfected HEK 293 cells. All peptides were at a concentration of 1
µM and were incubated for 2 h at 37 C. Scale
bar, 5 µm.
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Colocalization studies using redistributed HA-tagged GnRH
receptors with transferrin receptors were also carried out. Cells were
first incubated with GnRH (1 µM for 1 h at 37 C),
then fixed and incubated with both anti-HA and antitransferrin receptor
antibodies. The receptors were detected using
fluorescein-isothiocyanate (FITC) and Texas Red-conjugated secondary
antibodies, respectively. Agonist-treated redistributed GnRH receptors
(Fig. 2a
) showed a similar distribution
pattern with transferrin receptor (Fig. 2b
), and colocalization is
shown on Fig. 2c
.

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Figure 2. Colocalization of Internalized GnRH Receptors with
Transferrin Receptor
HEK 293-B5 cells were first treated with GnRH (1 µM for
1 h at 37 C), fixed, and then incubated with rabbit polyclonal
anti-HA antibody (a) and mouse monoclonal antitransferrin receptor
antibody (b). Agonist-induced redistributed GnRH receptors showed
distribution patterns similar to those of transferrin receptors (c).
Scale bar, 5 µm.
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Time-Dependent GnRH Receptor/Ligand Internalization as Quantified
by Radioligand Binding and ELISA
The quantitative radioligand assay is based on the assumption that
receptor and ligand sort similarly and that the intracellular
receptor/ligand complex can be determined by measuring intracellular
ligand. ELISA measurements allow us to monitor the changes in
surface-expressed HA-tagged receptor, and comparisons can be made with
results obtained from the radioligand internalization kinetic studies.
Figure 3
shows a comparison between
GnRH-induced internalization as quantified by radioligand binding and
ELISA. The two methods gave comparable results regarding
receptor/ligand internalization kinetics at 37 C in the presence or
absence of hypertonic sucrose medium. The agonist-induced
internalization process was almost completely suppressed at 4 C, and no
internalization was observed for GnRH-antag as determined by
radioligand assays (data not shown). ELISA measurements also
demonstrated that the level of surface-expressed receptor after
GnRH-antag treatment was comparable with the levels observed in
untreated cells (Fig. 4
).

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Figure 3. Comparison between GnRH Internalization Time Course
as Quantified by Radioligand Binding and ELISA Techniques
HEK 293-B5 cells were incubated at 37 C either with
125I-labeled GnRH agonist or GnRH (1 µM) for
the indicated time intervals. The amount of internalized receptor was
then expressed either as a percentage of the total binding at that time
interval or, in the case of ELISAs, calculated from decrease in the
level of surface-expressed receptor after agonist treatment compared
with untreated, control cells. The two assays were also performed in
the presence of 0.4 M sucrose. Circles and
squares represent ELISA and radioligand binding results
at 37 C in the absence (closed symbols) and presence of
0.4 M sucrose (open symbols) respectively.
All results are the mean ± SE from three independent
experiments performed in triplicate.
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Figure 4. ELISA Measurement of Surface-Expressed GnRH
Receptors after GnRH and GnRH Antagonist Treatment
The levels of surface-expressed receptors in HEK 293-B5 cells were
monitored after treatment with either GnRH or GnRH-antag (1
µM for 1 or 2 h at 37 C). The results (mean ±
SE) are expressed as a percentage of the value obtained in
untreated cells from three independent experiments performed in
triplicate.
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Recycling of GnRH Receptors
To study the recycling of the internalized GnRH receptor, cells
were initially treated with GnRH (1 µM for 2 h) and
then allowed to recover after ligand removal and replacement with fresh
medium. Receptor recycling was observed (by ELISA) within 1530 min
after ligand removal, and by 1 h almost all of the internalized
receptor reappeared at the cell surface (Table 1
). Coincubation of cells with the
protein synthesis inhibitor, cycloheximide (10 µg/ml), inhibited
neither agonist-induced internalization nor receptor recycling (Table 1
), showing that during the course of the experiment no substantial
de novo synthesis of the receptor occurred.
To test whether intracellular receptors represented a static pool or a
fraction of dynamically endocytosed and recycling receptors, we
performed recycling experiments in the continuous presence of agonist.
In addition, we tested whether constitutive recycling proceeds through
the acidified endosomal compartment by assessing the effect of
NH4Cl, which interferes with the acidic pH found in
endosomal vesicles (4). First, receptor internalization was induced by
incubating the cells with 125I-labeled GnRH agonist (1 h at
37 C) to obtain a substantial intracellular receptor pool followed by
an acid wash to remove surface-bound ligand. The absence of
extracellular label during recycling ensured that no additional labeled
intracellular receptors were generated, and the presence of a
saturating concentration of unlabeled GnRH agonist prevented rebinding
of any label that dissociates after recycling. Upon removal of labeled
GnRH agonist and incubation of cells in normal medium, GnRH receptor
recycled back to the plasma membrane. Under these conditions, the
intracellular pool of receptors decreased by around 22% in 5 min (from
27.28 ± 0.66 to 21.24 ± 0.51%), and by 1 h the
reduction was about 68% (from 27.28 ± 0.66 to 8.6 ±
1.33%). However, the addition of the 25 mM
NH4Cl to the medium substantially inhibited the recycling
of the GnRH receptor as evidenced by the reduction of the intracellular
receptor pool by only 42% (from 27.28 ± 0.66 to 15.71 ±
0.64%) by 1 h.
Effect of ß-Arrestin on Internalization Kinetics of the GnRH and
TRH Receptors
To address the role of ß-arrestin on the internalization
kinetics of GnRH and TRH receptors we 1) performed internalization
experiments in HEK 293 and COS-7 cells that express different levels of
endogenous ß-arrestins, 2) studied the effect of both WT
ß-arrestin and ß-arrestin dominant negative mutant [ß-arrestin
(319418)] coexpression on internalization of GnRH and TRH receptors
in HEK 293 and COS-7 cells, respectively, and 3) followed the
agonist-induced receptor-mediated redistribution of ß-arrestin/green
fluorescent protein (GFP) conjugate to the plasma membrane. Studies
performed using HEK 293 cells stably expressing either the GnRH or TRH
receptors showed a striking difference between internalization kinetics
of the two receptor types. A rapid decrease of surface-bound ligand was
observed for TRH receptor with an estimated half-time
(t1/2) of 2.2 min after TRH treatment. Internalization
reached a steady state within the first 510 min after agonist
exposure, and after this period the proportion of the internalized
receptors was about 70% (data not shown). In contrast, the
disappearance of GnRH receptors from the cell surface was delayed, with
an estimated t1/2 of 20 min. Equilibrium between surface
and intracellular receptors was reached only after 1 h of
continuous agonist exposure with the estimated intracellular pool of
receptor being around 30% (Fig. 3
). The effect of different endogenous
levels of ß-arrestin on receptor internalization rate was evaluated
by performing experiments in COS-7 cells, which express about 70% less
total ß-arrestins/mg protein than HEK 293 cells (30). Under
conditions of low endogenous ß-arrestin expression, TRH receptor
internalization was significantly slower in COS-7 (t1/2 of
5.7 min) than in HEK 293 cells. However, the rate was significantly
increased by coexpression of ß-arrestin in COS-7 cells
(t1/2 of 3.1 min) (Fig. 5
).
In contrast, coexpression of GnRH receptor with ß-arrestin in COS-7
cells had no effect on internalization kinetics (Fig. 5
). Coexpression
with ß-arrestin in HEK 293 cells did not promote internalization in
either receptor (data not shown). We next assessed the ability of a
dominant negative ß-arrestin mutant (ß-arrestin (319418)), which
has been previously characterized to retain clathrin binding but lack
the receptor binding activity (17), to impair receptor internalization.
COS-7 cells were transfected either with GnRH or TRH receptor alone or
together with different ß-arrestin constructs and the intracellular
receptor pool estimated after agonist treatment (15 min at 37 C). The
coexpression of ß-arrestin (319418) substantially reduced the
effect of ß-arrestin on TRH receptor internalization in COS-7 cells
(Fig. 6
). The coexpression of
ß-arrestin (319418) only slightly decreased the basal
agonist-induced internalization in COS-7 cells, whereas a significant
decrease was observed in HEK 293 cells stably expressing the TRH
receptor (Fig. 7
). No significant effect
of ß-arrestin construct coexpression on GnRH receptor internalization
was observed either in COS-7 or in HEK 293 cells (Figs. 6
and 7
). In
addition, the ability of coexpressed ß-arrestin/GFP conjugate in
COS-7 cells to enhance the internalization of TRH, but not the GnRH
receptor, was also confirmed (Fig. 6
).

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Figure 5. Effect of ß-Arrestin on Internalization of GnRH
and TRH Receptors Transiently Expressed in COS-7 Cells
Receptors were coexpressed with a pcDNA3 expression vector together
with 5 µg of empty pcDNA3 vector or pcDNA3 ß-arrestin. The
time-dependent loss of the surface GnRH and TRH receptors was measured
by changes in radioligand binding. Each point is the average ±
SE of three independent experiments performed in
triplicate. The amount of the receptors on the cell surface as a
function of time was fitted in the four-compartment model (31 ).
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Figure 6. Effect of ß-Arrestin (319418) Mutant on
ß-Arrestin-Induced Internalization of GnRH and TRH Receptors
Transiently Expressed in COS-7 Cells
Receptors (5 µg cDNA in pcDNA3/100 mm dish) were coexpressed with a
pcDNA3 expression vector together with 5 µg of empty pcDNA3 vector or
1 µg pcDNA3 ß-arrestin, 1 µg pcDNA3 ß-arrestin/GFP, 5 µg
pcDNA3 ß-arrestin (319418), 1 µg pcDNA3 ß-arrestin, and 4
pcDNA3 ß-arrestin (319418). The percentage of internalized
receptors after 15 min agonist exposure at 37 C was determined by
radioligand binding. Solid and hatched bars represent
the data for the GnRH and TRH receptor, respectively. Each point is the
average ± SE of three independent experiments
performed in triplicate.
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Figure 7. Effect of ß-Arrestin (319418) Mutant on the
Internalization of GnRH and TRH Receptors Stably Expressed in HEK 293
Cells
HEK 293 cells expressing either GnRH or TRH receptors were coexpressed
with 10 µg of empty pcDNA3 vector or 10 µg ß-arrestin (319418)
pcDNA3, and the percentage of internalized receptors was determined
after 15 min agonist exposure at 37 C by radioligand binding.
Solid and hatched bars represent the data for the GnRH
and TRH receptor, respectively. Each point is the average ±
SE of three independent experiments performed in
triplicate.
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Effect of ß-Arrestin on GnRH- and TRH Receptor Endocytosis and
Recycling Rate Constants
To acquire further information on the effect of ß-arrestin on
internalization kinetics for GnRH and TRH receptors, the
four-compartment model described by Koenig and Edwardson (31) was
applied in an attempt to model receptor intracellular trafficking. This
model predicts four major pathways of receptor movement within the
cells: 1) receptor endocytosis from the cells surface into endosomes,
2) recycling from endosomes back to plasma membrane, 3) receptor
movement from endosomes to lysosomes for degradation, and 4) the
delivery of newly synthesized receptor to the plasma membrane.
Utilizing a numerical parameter-fitting routine, we calculated a
complete set of rate constants for the receptor endocytosis
(ke), recycling (kr), synthesis
(ks), and degradation (kx), which are
summarized in Table 2
. Comparable rate
constants derived from data using either quantitative radioligand
binding or ELISA measurements were obtained for HA-tagged GnRH receptor
in HEK 293-B5 cells [ke = 0.013 ± 0.001;
kr = 0.032 ± 0.001 min-1; ks
= 0; kx= 0.010 ± 0.001 min-1
(radioligand binding); and ke = 0.013 ± 0.001;
kr = 0.028 ± 0.002; ks = 0;
kx = 0.018 ± 0.001 min-1 (ELISA
measurements)], confirming the use of radioligand-binding assays for
assessing receptor intracellular trafficking. Similar rate constants
(ke = 0.012 ± 0.001; kr = 0.033 ±
0.003; ks = 0; kx = 0.008 ± 0.001
min-1) were also obtained for WT GnRH receptor stably
expressed in HEK 293-A2 cells. The GnRH receptor internalization rates
were similar in both cell types (HEK 293 and COS-7) and also unaffected
by different ß-arrestin expression levels (Table 2
A). The
ke for TRH receptor in COS-7 cells was nearly 4-fold lower
than the corresponding value in HEK 293 cells. However, ß-arrestin
coexpression in COS-7 cells resulted in an almost 2-fold increase in
ke for TRH receptor (Table 2
B). The obtained kx
values were also substantially higher for TRH- than GnRH receptor in
both cell types (Table 2
, a and b). In addition, internalization rates
obtained with
T3 cells (ke = 0.014 ± 0.002;
kr = 0.032 ± 0.005) and GH3
(ke = 0.181 ± 0.025; kr = 0.078 ±
0.006) cells, which endogenously express GnRH and TRH receptors,
respectively, were comparable to those observed for the respective
receptor types stably expressed in HEK 293 cells. In all calculations,
the fitted value of ks was not significantly different from
zero, confirming the absence of synthesis of either receptor during the
course of the experiment (data not shown).
Cellular Distribution of ß-Arrestin/GFP Fusion Protein in HEK 293
Cells
To further study the role of ß-arrestin in GnRH and TRH receptor
internalization, GFP has been coupled to ß-arrestin (ß-arrestin/GFP
fusion protein), thus allowing us to visualize the agonist-induced
translocation of ß-arrestin in cells expressing GPCRs.
ß-Arrestin/GFP retained its biological activity with respect to its
ability to enhance the TRH receptor internalization in COS-7 cells
(Fig. 6
). Confocal microscopy showed cytosolic distribution of
ß-arrestin/GFP in untreated HEK 293 cells expressing either TRH (Fig. 8a
) or GnRH receptor (Fig. 8c
). After
agonist stimulation, a time-dependent TRH-mediated redistribution of
ß-arrestin/GFP from the cytosol to the cell membrane was observed
(Fig. 8b
), while no increase in membrane-associated fluorescence
indicative of ß-arrestin/GFP translocation was observed in GnRH
receptor-expressing cells even after prolonged exposure to GnRH (Fig. 8d
). Similar results were obtained in COS-7 cells transiently
expressing either TRH or GnRH receptor (not shown).

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Figure 8. Confocal Microscopy of ß-Arrestin/GFP Cellular
Distribution in HEK 293 Cells Stably Expressing Either GnRH or TRH
Receptors
HEK-293 cells stably expressing either TRH (a and b) or GnRH receptors
(c or d) were transfected with cDNA for ß-arrestin/GFP.
ß-Arrestin/GFP distribution is initially cytosolic in untreated cells
(a and c). After agonist stimulation (TRH, 1.5 min; GnRH, 10 min),
TRH-mediated redistribution of ß-arrestin/GFP from the cytosol
to the membrane is shown (b), while no redistribution of
ß-arrestin/GFP is observed for GnRH receptor expressing HEK 293 cells
(d). Scale bar, 5 µm.
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DISCUSSION
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Previous studies on the GnRH receptor have not measured actual
receptor internalization, but rather the internalization of labeled
ligand. Using immunocytochemical methodology and a cell line stably
expressing epitope-tagged GnRH receptors, this study reports direct
visualization of GnRH receptor internalization. In unstimulated cells
expressing HA-tagged GnRH receptor, the signal was confined mainly to
the cell membrane. After treatment with agonist, GnRH receptors were
internalized in a time-dependent manner with internalization being
demonstrable after 20 min but pronounced after 12 h. The signal
corresponding to the internalized receptor also showed a high degree of
spatial correlation with transferrin receptor.
The agonist-induced internalization time course for GnRH receptors was
further quantified by two different methods; radioligand binding and
ELISA, of which the latter is not influenced by the state of receptor/G
protein coupling (32). Relatively low internalization rates were
confirmed using both methods. Radioligand binding studies in both
primary (23) and immortalized
T31 gonadotrope cells (24), as well
as in GH3 lactotropes stably transfected with the GnRH
receptor (25), also demonstrate a surface GnRH receptor loss over a
similar time frame.
Endocytosis by a clathrin-mediated pathway is temperature dependent and
can also be inhibited by hypertonic sucrose (33), which induces
abnormal clathrin polymerization and subsequently reduces the number of
clathrin-coated pits. Another well established marker for endocytosis
via clathrin-coated pits is the transferrin receptor (34). We observed
that GnRH-induced internalization was inhibited both at 4 C and in
the presence of sucrose. The location of redistributed GnRH receptors
also overlapped substantially with that of the transferrin receptors.
Therefore, our results suggest that agonist-triggered endocytosis of
the GnRH receptor probably occurs via a clathrin-dependent
mechanism.
After internalization, receptors can either be sorted into endosomes
for recycling back to the cell surface or, alternatively, may undergo
degradation within lysozomes (31). After agonist removal, GnRH
receptors reappeared at the cell surface, a process unaltered by
cycloheximide, suggesting that GnRH receptor recycling from endosomes,
rather than de novo receptor synthesis, was involved.
However, if recycling had occurred only after agonist removal, we would
have anticipated a linear increase in the intracellular pool of
receptor. Our observations show that this is not the case, and the
recycling data confirmed that in the continuous presence of agonist, a
dynamic equilibrium between endocytosed and recycling receptors was
reached. GPCR internalization is now thought to be associated primarily
with receptor resensitization, which is achieved through endosomal
sorting and recycling of functional receptor back to the plasma
membrane (4, 35). Recycling of the GnRH receptor was inhibited by
NH4Cl, indicating the involvement of acidified endosomal
compartments in receptor trafficking, as has been previously shown for
ß2-adrenergic (4) and angiotensin receptors (11).
The efficiency of GPCRs endocytosis is not only receptor- but also cell
type-dependent and should reflect differences in endogenous levels of
certain intracellular proteins. HEK 293 and COS-7 cells express high
and low levels of ß-arrestins, respectively (30), and thus provide us
with an opportunity for examining the effect of different endogenous
ß-arrestin expression levels on GPCR internalization dynamics in a
natural cellular environment. Therefore, we determined internalization
kinetics for two functionally distinct members of the GPCR family; the
GnRH receptor and also the TRH receptor, which possesses a functional
carboxy-terminal tail. Additional reasons to choose TRH receptor were
that its endocytosis displays characteristics of the clathrin-mediated
pathway (9), and the importance of the carboxy-terminal tail in
agonist-induced internalization was previously confirmed using this
receptor (20).
The ke and kr values were similar for the WT
and epitope-tagged GnRH receptor stably expressed in HEK 293 cells and
for WT GnRH receptor transiently expressed in COS-7 cells, and this
ke is also comparable with a reported value for WT GnRH
receptor in COS-7 cells (28). Considering the reported differences in
endogenous ß-arrestin levels between the two cell types, a higher
GnRH internalization rate in HEK 293 than in COS-7 cells was expected,
which was not the case. The rate constants for the GnRH receptor in
both cell types were much lower than those obtained for TRH receptor
and also those reported for some other GPCRs (8, 36); however, they are
still within the range of internalization rates reported for
clathrin-mediated endocytosis in nonneuronal cells (37). TRH receptor
internalization rates were cell type dependent: lower in COS-7 than in
HEK 293 cells; however, overexpression of ß-arrestin in COS-7 cells
enhanced TRH receptor kinetics to levels approaching those in HEK 293
cells. The receptor degradation can increase in the presence of agonist
(36); therefore, it is necessary to take degradation into account in
any attempt to model intracellular trafficking. The rate of TRH
receptor degradation (kx) was higher than kx
for GnRH receptor in both cell types, whereas the synthesis rate
constant (ks) was negligible for either receptor type. A
loss of approximately 10% of surface GnRH receptor was observed during
the recycling experiments in the absence or presence of the protein
synthesis inhibitor cycloheximide. This result is in close agreement
with the kx and ks values for GnRH receptor
obtained from the four-compartment model, which predicts that 3040%
of receptor in the endosomal compartment is destined for degradation,
and no new synthesis of receptor occurs during the course of the
experiment.
The GnRH receptor internalization kinetics in the two cell types were
similar, and no obvious role of ß-arrestin in this process was
observed. This observation was further supported by results obtained
with ß-arrestin dominant negative mutant [ß-arrestin (319418)],
which efficiently impaired agonist-induced internalization of the TRH
but not of the GnRH receptor. The applicability of this construct for
studying agonist-induced GPCRs internalization in different cell lines
has been previously determined (17). In addition, we constructed the
ß-arrestin/GFP fusion protein, which enabled us to assess
translocation of ß-arrestin/GFP complex to the plasma membrane in
response to GPCR activation. The construct was fully functional with
respect to its ability to promote TRH receptor internalization, and the
time- and agonist-dependent redistribution of ß-arrestin/GFP to the
plasma membrane was easily observed by confocal microscopy. However, no
evident redistribution of ß-arrestin/GFP was observed in cells
expressing the GnRH receptor. The results obtained with ß-arrestin
constructs could suggest either that GnRH receptor internalization is
not ß-arrestin-dependent or that ß-arrestin, which preferentially
binds phosphorylated receptor (14), has a low affinity for GnRH
receptor due to the lack of a tail and therefore of potential
phosphorylation sites. Truncation of the carboxy-terminal tail of the
ß2-adrenergic receptor has been shown to reduce
ligand-induced internalization by 50%, an effect that is reversed when
the receptor is coexpressed with ß-arrestin, and coexpression of the
WT receptor with inactive mutants of ß-arrestin reduced
internalization by 70% (13). Similarly, the removal of the
phosphorylation sites in the carboxyl tail of the
ß2-adrenergic receptor reduces receptor internalization
to levels comparable to those observed for the GnRH receptor (30). This
observation indicates that GPCR kinase-mediated phosphorylation is not
absolutely required as a signal initiating internalization, but only as
a factor determining its rate.
The obvious question is can clathrin-dependent endocytosis be
ß-arrestin independent, since ß-arrestins specifically target
activated receptor into clathrin-coated vesicles? In HEK 293 cells
expressing AT1A receptor, agonist-promoted sequestration of
this receptor was shown to be ß-arrestin independent, although it can
be recruited to this pathway by overexpression of ß-arrestin (38).
Furthermore, it has been suggested that AT1A receptor
internalization is clathrin-mediated, since colocalization with
transferrin receptor in the same cell line was observed (11).
Similarly, we showed that the GnRH receptor undergoes agonist-induced
receptor internalization (albeit at a slower rate than other GPCRs)
probably via a clathrin-dependent mechanism and provided evidence for
the dynamic nature of this process. However, it seems likely that the
GnRH receptor endocytotic pathway is ß-arrestin and cell type
independent as overexpression of ß-arrestin in different cell types
does not affect internalization kinetics.
 |
MATERIALS AND METHODS
|
---|
Materials
Chamber slides, inositol-free DMEM, penicillin, and streptomycin
were obtained from GIBCO (Paisley, UK). Transfectam was obtained from
Promega (Southampton, UK). All other tissue culture reagents and media
were supplied by Sigma Cell Culture (Dorset, UK). Anti-HA mouse
monoclonal antibody (clone 12CA5) and anti-HA rabbit polyclonal
antibody were obtained from Boehringer Mannheim (East Sussex, UK) and
Babco (Richmond, CA), respectively. Antitransferrin receptor
mouse monoclonal antibody (clone Ber-T9) was obtained from Dako A/S
(Glostrup, Denmark); mouse/goat serum from the Scottish Antibody
Production Unit (Carluke, UK), and Citifluor from Chem Lab (Canterbury,
UK). The Bio-Rad protein assay kit and Dowex resin were obtained from
Bio-Rad (Herts, UK). 3,3',5,5'-Tetramethylbenzidine liquid
substrate system was obtained from Sigma (Dorset, UK).
TRH-(3-Met-His2)-[3H] (250 mCi) was from NEN
Life Science Product (Hertfordshire, UK). All other compounds and
reagents were obtained from either Sigma (Dorset, UK), Calbiochem
(Nottingham, UK), or Amersham (Buckinghamshire, UK). HEK 293 and COS-7
cells were obtained from the European Collection of Animal Cell
Cultures, Centre for Applied Microbiology and Research (Salisbury,
UK).
Cell Culture
Cell lines were routinely grown in complete DMEM containing 10%
(vol/vol) heat inactivated FCS, glutamine (0.3 mg/ml), penicillin (100
IU/ml), and streptomycin (100 µg/ml) and incubated at 37 C in a
humidified atmosphere of 5% (vol/vol) CO2 in air.
Derivation of Epitope-Tagged GnRH Receptor-Expressing Cell
Lines
A double-stranded oligonucleotide fragment corresponding to the
sequence of the HA tag (YPYDVPDYA) was synthesized and ligated in
frame, into the amino terminus of the rat GnRH receptor in the vector
pcDNA3. Linearized HA-tagged GnRH receptor plasmid DNA was then stably
transfected into human embryonal 293 cells (HEK 293) cells using
Transfectam. Receptor-containing clones were then selected after the
treatment of cell cultures with geneticin (1 mg/ml), expanded, and
maintained in complete DMEM containing geneticin (500 µg/ml).
HA-tagged GnRH receptor-containing clones were subsequently identified
using previously described GnRH receptor ligand-binding assays (39) and
total inositol phosphate assays (40). The clone HEK 293-B5, expressing
the HA-tagged GnRH receptor, was chosen for further study.
Detection and Visualization of HA-Tagged GnRH Receptors
HA-tagged GnRH receptors were detected using indirect
immunocytochemistry on cell monolayers. Trypsinized cells were plated
into eight-well chamber slides at a density of 2.5 x
104 cells per well in complete DMEM. After 2 days, cells
were washed twice with 0.01 M PBS, pH 7.4, and treated as
required in HEPES-modified DMEM with 0.1% BSA, pH 7.4, before fixing
with freshly prepared 4% paraformaldehyde for 30 min at room
temperature. To reduce the nonspecific binding, cells were incubated in
blocking solution (PBS containing 1% BSA and 10% normal serum, pH
7.4) for 30 min. If permeabilized cells were required, to enable the
visualization of internalized HA-tagged GnRH receptors, nonionic
detergent (Nonidet P-40) was added to the blocking solution to a final
concentration of 0.2%. Subsequently, cells were washed with PBS (three
times) before incubating with primary antibody in blocking solution
overnight at 4 C. After washing (four times in PBS) cells were
incubated with goat antimouse FITC-conjugated secondary antibody (20
µg/ml) for 60 min at room temperature. Slides were then washed (four
times in PBS), mounted in Citifluor, and sealed with coverslips. Cells
were examined under an oil immersion objective (x60) using a Zeiss LMS
510 confocal laser microscope and a filter selective for FITC
fluorescence. Optical sections (0.45 µm) were taken, and
representative sections corresponding to the middle of the cells were
presented. After indirect immunofluorescent staining, no specific
fluorescence was observed in untransfected HEK 293 cells, or in HEK
293-B5 cells treated with secondary FITC-linked goat antimouse IgG
antibody only.
Colocalization of HA-Tagged GnRH Receptors with Transferrin
Receptor
For colocalization of epitope-tagged receptor with transferrin
receptor, the cells were first treated (1 µM GnRH for
1 h at 37 C), fixed, and incubated with both rabbit polyclonal
anti-HA antibody and mouse antihuman transferrin receptor antibody
overnight at 4 C. The epitope-tagged receptors and the transferrin
receptors were detected using antirabbit FITC and antimouse Texas
Red-conjugated secondary antibody (as described previously). Cells were
then viewed as previously described using filters selective for either
rhodamine or fluorescein fluorescence.
Total IP Assays
Assays were performed in 24-well plates containing 1.5 x
105 cells per well, and total IPs were extracted and
separated as described previously (40).
Iodination of GnRH Agonist and Antagonist
Iodinated radiolabeled GnRH analogs were prepared using the
glucose oxidase/lactoperoxidase method and purified by chromatography
on a Sephadex G-25 column in 0.01 M acetic acid/0.1% BSA.
The specific activities of the
125I-des-Gly10,[D-Trp6]-GnRH
(GnRH agonist) and 125I-[Ac-3,4-dehydro-Pro1,
D-p-F-Phe2,
D-Trp3,6]-GnRH (GnRH-antag) tracers were 53
µCi/µg and 28.2 µCi/µg, respectively, and were calculated from
self-displacement assays using either rat pituitary homogenates or
COS-7 cells transiently transfected with the WT GnRH receptor cDNA.
Assays were incubated for 2 h at 4 C before filtration through a
cell harvester using Whatman GFB filter paper.
Receptor Internalization and Recycling Assays
Receptor internalization and recycling assays were based on
protocols described by Lauffenburger and Linderman (41). Briefly, HEK
B5293 cells were plated at a density of 1.5 x 105
cells per well in 24-well plates. After 2 days, cells were washed once
with assay medium (HEPES-modified DMEM with 0.1% BSA, pH 7.4) before
being incubated with 125I-labeled GnRH agonist or
antagonist (100,000 cpm/well) in 0.5 ml assay medium for time intervals
ranging from 5 min to 2 h at either 4 C or 37 C. To test the
effect of hypertonic medium, assays were also performed in the presence
of sucrose; in this case, the cells were pretreated with assay medium
containing 0.4 M sucrose for 20 min at 37 C, the sucrose
concentration being maintained during the ligand treatment. At
appropriate times, cells were transferred onto ice and washed twice
with ice-cold PBS. Subsequently, the extracellular receptor-associated
ligand was removed by washing once with 1 ml of acid solution (50
mM acetic acid and 150 mM NaCl, pH 2.8) for 12
min. The acid wash was collected to determine the surface-bound
radioactivity, and the internalized radioactivity was determined after
solubilizing the cells in 0.2 M NaOH and 1% SDS (NaOH/SDS)
solution. Nonspecific binding for each time point was determined under
the same conditions in the presence of 10 µM unlabeled
agonist or antagonist. After subtraction of nonspecific binding, the
internalized radioactivity was expressed as a percentage of the total
binding at that time interval. All time points were performed in
triplicate for at least three separate experiments. To compare the
agonist-induced internalization kinetics of the GnRH receptor with
another member of the GPCR superfamily, HEK E2293 cells, which stably
express WT TRH receptor (42), were also assayed under identical
conditions using 3H-labeled TRH agonist. To evaluate the
effect of ß-arrestin on internalization, COS-7 cells and HEK 293
cells stably expressing either GnRH or TRH receptor (2 x
106/100 mm dish) were transiently transfected with various
constructs using Transfectam. Cells were then split into 24-well
plates, and internalization assays were performed with the appropriate
labeled agonist as described above.
For the recycling experiment, HEK 293-B5 cells were preincubated with
125I-labeled GnRH agonist (1 h at 37 C) to get a
substantial intracellular pool, followed by an acid wash to remove
surface-bound ligand; cells were then incubated in medium containing
saturating concentrations of unlabeled agonist for varying periods of
time (560 min at 37 C) to monitor the decrease in the intracellular
pool due to recycling of the ligand. To test whether the recycling
proceeded through acidified endosomal compartments, 25 mM
NH4Cl was added to the incubation medium during the
recycling period, and the decrease in the intracellular receptor pool
was monitored as described above. From the data obtained, the
endocytosis (ke), recycling (kr), synthesis
(ks), and degradation (kx) rate constants were
calculated using the four-compartment model described by Koenig and
Edwardson (31).
ELISA
ELISA assays for the measurement of surface-expressed HA-tagged
GnRH receptors and quantification of receptor internalization were
based on the method described previously (43). Cells were plated out at
a density of 5 x 104 cells per well in 48-well
plates. After 2 days, cells were treated as required in either
HEPES-modified DMEM with 0.1% BSA, pH 7.4, or hypertonic sucrose
medium for varying periods of time at 37 C before fixing with freshly
prepared 4% paraformaldehyde for 10 min at room temperature. Cells
were then washed three times in PBS, blocked (PBS containing 10%
normal serum, pH 7.4), and incubated with a 1:200 dilution of primary
anti-HA monoclonal antibody in blocking buffer overnight at 4 C.
Subsequently, cells were washed with PBS (three times) and incubated
for 1 h at 37 C in a 1:2000 dilution of a horseradish
peroxidase-conjugated sheep antimouse IgG. After final washes in PBS
(six times) and 0.9% NaCl (once), the reaction was developed using the
3,3',5,5'-tetramethylbenzidine liquid substrate system. The enzymatic
reaction was stopped after 30 min at room temperature with 0.5 N
H2SO4, and a 100 µl sample was taken for
colorimetric measurement at 450 nm using a Labsystem multiscan MCC/340
reader. For the recycling experiment, a preliminary treatment (1
µM GnRH for 2 h at 37 C) was followed by an acid
wash (50 mM acetic acid and 150 mM NaCl, pH
2.8) for 5 min to remove surface-bound ligand, and the cells were
incubated in serum-free HEPES-modified DMEM/BSA medium at 37 C for
varying periods of time (5 min to 1 h) to monitor the recovery of
surface-expressed receptors. Cycloheximide (10 µg/ml), when used, was
present throughout the whole experiment. Untransfected HEK 293 cells
were assayed concurrently to determine background. All experiments were
done in triplicate.
Construction of the ß-Arrestin-GFP Expression Construct
Production and subcloning of the ß-arrestin/GFP fusion protein
were performed in two separate steps. In the first step, the coding
sequence of a modified form of GFP (44) was altered by PCR
amplification. Using the amino-terminal primer
5'-CCGCTCGAGAGTAAAGGAGAAGAACTTTTCAC-3' a XhoI
restriction site (underlined) was introduced adjacent to the
sequence of codon 2 of GFP. The ATG initiator codon was removed. Using
the carboxyl-terminal primer
5'-TGCTCTAGATTATTTGTATAGTTCATCCATGCC-3' an XbaI
restriction site (underlined) was introduced after the stop
codon. The amplified fragment of GFP digested with XhoI and
XbaI was ligated into the pcDNA3 expression vector
(Invitrogen, San Diego, CA) digested with XhoI and
XbaI. To obtain the ß-arrestin/GFP fusion protein, the
coding sequence of ß-arrestin (45) was amplified by PCR. Using the
amino-terminal primer
5'-AAAAAAGCTTTCTACCATGGGCGACAAAGGGACAC-3', a
HindIII restriction site (underlined) and a
partial Kozak site were introduced in front of the initiator Met of
ß-arrestin. Using the carboxyl-terminal primer
5'-AACTCGAGTCTGTTGTTGAGGTGTGGAGAGC-3' a XhoI
restriction site (underlined) was introduced just in front
of the stop codon of ß-arrestin. Finally, the GFP construct in pcDNA3
was digested with XhoI and HindIII and was
ligated together with the PCR product of the ß-arrestin amplification
that was digested with HindIII and XhoI. The open
reading frame so produced represents the coding sequence of
ß-arrestin/GFP. This construct was fully sequenced before its
expression and analysis. HEK 293 cells (1.5 x 106/60
mm dish) stably expressing either GnRH (B5 cells) or TRH (E2 cells)
receptor were transfected with 2.5 µg of ß-arrestin/GFP cDNA using
Transfectam. After 24 h, cells were plated into eight-well chamber
slides, and treatments were carried out 4872 h after transfection.
The cells were then fixed with 4% paraformaldehyde, mounted, and
sealed with coverslips. Confocal microscopy was performed as described
previously.
 |
ACKNOWLEDGMENTS
|
---|
The authors would like to thank Professor J. F. Benovic
(Jefferson Medical College, Philadelphia) who kindly provided us with
cDNA for ß-arrestin and ß-arrestin (319418) dominant negative
mutant, Professor S. V. Bavdek for support and encouragement, Dr.
B. Byrne, Mr. R. Sellar, and Ms. S. Nettleship for expert technical
assistance, and Dr. H. Rahe for critical evaluation of the
manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. K. A. Eidne, Medical Research Council Reproductive Biology Unit, 37 Chalmers Street, Centre for Reproductive Biology, EH3 9EW Edinburgh, United Kingdom. E-mail: keidne{at}hgmp.mrc.ac.uk
M. Vrecl is financially supported by the Ministry of Science and
Technology of the Republic of Slovenia.
Received for publication July 10, 1998.
Accepted for publication August 18, 1998.
 |
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