From the 7TM Receptor Laboratory, Western Australian
Institute for Medical Research, § Keogh Institute for
Medical Research, Sir Charles Gairdner Hospital, and ¶ Animal
Sciences, University of Western Australia, Perth, Western Australia
6009, Australia, ** Veterinary Faculty, University of Ljubljana, 1000 Ljubljana Slovenia, and
Baker
Medical Research Institute, Melbourne 8008, Australia
Received for publication, October 11, 2000, and in revised form, March 8, 2001
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ABSTRACT |
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We have previously shown that the mammalian
gonadotropin-releasing hormone receptor (GnRHR), a unique
G-protein-coupled receptor (GPCR) lacking an intracellular carboxyl
tail (C-tail), does not follow a Although the receptors for the hypothalamic releasing factors,
gonadotropin-releasing hormone
(GnRH)1 and
thyrotropin-releasing hormone (TRH), belong to the rhodopsin family of
G-protein-coupled receptors (GPCRs), the mammalian GnRH receptor is
distinct from other members of the GPCR family, including nonmammalian
GnRH receptors, in that it lacks the functionally important C-terminal
domain. This structural difference raises questions about the role of
the functional domains of these receptors in desensitization,
internalization, and down-regulation. Chimeric GnRH and TRH receptor
constructs have provided us with a useful tool for dissecting molecular
interactions involved in determining the phosphorylation status of
these receptors and the resultant effects on receptor function and
trafficking. We have previously shown that the absence of a C-terminal
tail (C-tail) is responsible for the slow internalization kinetics of
the mammalian GnRHR and its inability to undergo acute desensitization
(1, 2). The GnRHR is not phosphorylated upon activation and is
The current model for regulating agonist-activated GPCRs involves
recruitment of arrestins, which cause rapid desensitization by
uncoupling the receptor from its cognate G-protein to attenuate signaling (5) and to target the receptor into clathrin-coated vesicles
for its internalization into endosomes (6). Receptor phosphorylation is
a prerequisite step in A large number of GPCRs contain multiple consensus sites for casein
kinase II (CKII). CKII is a highly conserved serine/threonine kinase
expressed ubiquitously in all eukaryotic organisms (19). This
tetrameric kinase is composed of two We observed that chimeric GnRH receptors with extended C-tails
containing CKII consensus sites gained Materials--
[D-Trp6,Pro9,N-Et]GnRH,
staurosporine, and apigenin were supplied by Sigma. GnRH agonist
(leuprolide) was obtained from Abbott Australasia, and TRH was obtained
from Peninsula Laboratories Europe Ltd. (Merseyside, United Kingdom).
3H-Labeled [Me-His2]TRH was supplied by
PerkinElmer Life Sciences.
Cell Culture--
HEK 293 and COS-1 cells (ATCC) were maintained
in Dulbecco's modified Eagle's medium containing 10% fetal calf
serum, glutamine (0.3 mg/ml), and penicillin-streptomycin (100 units/ml) (Life Technologies, Inc.) at 37 °C in a humidified
atmosphere of 5% CO2 in air. Prior to transfection, cells
were plated in either 100- or 60-mm dishes at 60-80% confluency and
transfected with 10 or 5 µg of total cDNA, respectively, using
Superfect (Qiagen). Cells were utilized 48-72 h post-transfection.
Expression Constructs and Mutagenesis--
The rat GnRHR/rat
TRHR C-tail chimera (GnRHR/TRHR tail), rat GnRHR/catfish GnRHR C-tail
chimera (GnRHR/cf tail), and the GFP/ Iodination of GnRH Agonist--
Iodinated
[D-Trp6,Pro9,N-Et]GnRH
was prepared using the lactoperoxidase method and purified by
chromatography on a Sephadex G-25 column in 0.01 M acetic
acid, 0.1% bovine serum albumin. The specific activity was 47 µCi/µg and was calculated as described previously (23).
Total Inositol Phosphate Assays--
Total IPs were extracted
and separated as described previously (24). Briefly, 24 h after
transfection, cells were plated into 24-well plates with 0.5 ml of
inositol-free Dulbecco's modified Eagle's medium containing 1%
dialyzed fetal calf serum and incubated for 24 h with
[3H]myoinositol (2 µCi/ml; Amersham Pharmacia Biotech).
48 h posttransfection, medium was removed, cells were washed twice
with buffer A (1 mg/ml fatty acid-free bovine serum albumin, 140 mM NaCl, 20 mM Hepes, 4 mM KCl, 8 mM D-glucose, 1 mM MgCl, 1 mM CaCl2) followed by incubation for 10 min
with buffer A containing 10 mM LiCl with or without the
addition of either TRH or GnRH agonist (10 Receptor Phosphorylation, Immunoprecipitation, and
Immunoblotting--
Methods for the phosphorylation and
immunoprecipitation of epitope-tagged receptors were as described
previously (25). In brief, COS-1 cells were transiently transfected
with epitope-tagged receptors or vector in 12-well plates. Cells were
serum-starved for 16 h, loaded with
[32P]Pi (200 µCi/ml), and stimulated for 20 min at 37 °C with either 100 nM GnRH agonist for the
GnRH receptors or 100 nM TRH for the TRH receptors. After
stimulation, the plates were placed on ice and washed twice with 1 ml/well Hank's buffered salt solution (4 °C), and the cells were
solubilized with 300 µl of a lysis buffer containing phosphatase
inhibitors (25). Cell lysates were centrifuged (14,000 × g, 15 min) and precleared by adding bovine serum albumin and
protein A-agarose for 1 h at 4 °C. The epitope-tagged receptors
were immunoprecipitated from the precleared lysates by adding 2 µg of
affinity-purified 12CA5 monoclonal antibody and 20 µl of protein
A-agarose. Following overnight agitation at 4 °C, the
immunoprecipitates were washed five times, resuspended in 55 µl of a
urea-based SDS sample buffer, heated at 60 °C for 15 min, and
resolved by 10% SDS-polyacrylamide gel electrophoresis. Gels were
Western blotted to polyvinylidene difluoride membrane, and
phosphorylated bands were detected and quantified by PhosphorImaging. To compare the level of receptor expression for the various constructs, the membranes were subsequently probed with a rat monoclonal anti-HA high affinity antibody (3F10; Roche Molecular Biochemicals) and an
anti-rat IgG-horseradish peroxidase complex (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by enhanced chemiluminescence.
Receptor Internalization Assays--
Receptor internalization
assays were done as previously described (1). Briefly, cells in 24-well
plates were incubated with labeled agonist for time intervals ranging
from 5 min to 2 h at 37 °C. Surface-bound radioactivity
was removed by washing with acid solution (50 mM acetic
acid, 150 mM NaCl, pH 2.8). Internalized radioactivity was
determined after solubilizing cells in 0.2 M NaOH,
1% SDS. Nonspecific binding for each time point was determined under
the same conditions in the presence of 1 µM unlabeled
agonist. After subtraction of nonspecific radioactivity, internalized
radioactivity was expressed as a percentage of the total binding. All
time point measurements were performed in duplicate in at least
three separate experiments.
Visualization of GFP/ Statistical Analysis--
Statistical significance was
determined using Student's t test. Differences are
considered significant at p < 0.05.
The Addition of a Series of CKII Sites into the GnRHR/cf Tail
Chimera Results in
The effect of sequential addition of CKII sites into the cytoplasmic
C-tail of the chimeric GnRHR/cf tail was evaluated in the absence or
presence of Mutation of CKII Sites in the TRHR Results in Loss of
Visualization of the Effect of Receptor C-tail CKII Sites on
To confirm our findings in another cell line, internalization
experiments with GnRHR/cf tail and TRHRs were carried out in HEK 293 cells. These cells express high endogenous levels of A Consequences of the C-tail CKII Sites on Receptor
Phosphorylation--
The capacity of
The phosphorylation status and expression of WT TRHR and TRHR A Specific CKII Inhibitor, Apigenin, Inhibits
The role of The C-tails of two The finding that the GnRHR/cf tail chimera was The hypothesis that CKII sites are required for Sequential mutation of CKII sites in the TRHR C-tail resulted in a
receptor that was insensitive to The loss of From the site-directed mutagenesis studies, we propose that the kinase
involved in targeting the TRHR and GnRH/cf tail +3CKII to the
Numerous GPCRs have been shown to require phosphorylation by a member
of the GRK family for recruitment of To conclude, we present evidence that in order to confer
-arrestin-dependent
internalization pathway. However, internalization of a chimeric GnRHR
with the thyrotropin-releasing hormone receptor (TRHR) C-tail does
utilize
-arrestin. Here, we have investigated the sites within the
intracellular C-tail domain that are important for conferring
-arrestin-dependent internalization. In contrast to the
chimeric GnRHR with a TRHR C-tail, a chimeric GnRHR with the catfish
GnRHR C-tail is not
-arrestin-dependent. Sequence
comparisons between these chimeric receptors show three consensus
phosphorylation sites for casein kinase II (CKII) in the TRHR C-tail
but none in the catfish GnRHR C-tail. We thus investigated a role for
CKII sites in determining GPCR internalization via
-arrestin.
Sequential introduction of three CKII sites into the chimera with the
catfish C-tail (H354D,A366E,G371D) resulted in a change in the
pattern of receptor phosphorylation and
-arrestin-dependence, which
only occurred when all three sites were introduced. Conversely,
mutation of the putative CKII sites (T365A,T371A,S383A) in the C-tail
of a
-arrestin-sensitive GPCR, the TRHR, resulted in decreased
receptor phosphorylation and a loss of
-arrestin-dependence.
Mutation of all three CKII sites was necessary before a loss of
-arrestin-dependence was observed. Visualization of
-arrestin/GFP
redistribution confirmed a loss or gain of
-arrestin sensitivity for
receptor mutants. Internalization of receptors without C-tail CKII
sites was promoted by a phosphorylation-independent
-arrestin mutant
(R169E), suggesting that these receptors do not contain the necessary
phosphorylation sites required for
-arrestin-dependent
internalization. Apigenin, a specific CKII inhibitor, blocked the
increase in receptor internalization by
-arrestin, thus providing
further support for the involvement of CKII. This study presents
evidence of a novel role for C-tail CKII consensus sites in targeting
these GPCRs to the
-arrestin-dependent pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin-independent in comparison with the TRHR that does undergo
phosphorylation and is internalized in a
-arrestin-dependent manner (1, 3). The addition of the
C-tail from the TRHR to the carboxyl-terminal end of the GnRHR results
in a chimeric receptor that is phosphorylated (3) and undergoes
-arrestin-dependent internalization (4).
-arrestin binding (7), although a few GPCRs
can internalize via
-arrestin in a phosphorylation-independent manner (8, 9). G-protein receptor kinases (GRKs) have been identified
as the kinases involved in GPCR phosphorylation, although consensus GRK
phosphorylation sites on GPCRs have not yet been clearly defined. In
some cases, GPCR internalization via the
-arrestin-dependent pathway is not regulated by the GRKs
(10); however, the kinases involved remain uncertain. Other kinases
implicated in GPCR internalization are the second messenger kinases,
protein kinase C (PKC) and protein kinase A. These kinases have been
shown to be involved in internalization of somatostatin receptor type
2A (11), secretin receptor (12), gastrin-releasing peptide receptor
(13), and the
-arrestin-2 dependence of the parathyroid hormone
receptor 1 (14). Phosphorylation by casein kinase 1
is involved in
the regulation of M1 and M3 muscarinic receptors (15-17) and is also
involved in the endocytosis of the yeast GPCR Ste3p (18).
and two
subunits, and it
phosphorylates and interacts with a myriad of proteins involved
in diverse cellular functions such as signal transduction, growth, and
proliferation. CKII also plays a role in the internalization of
single transmembrane receptors such as CD5 (20) and the transferrin receptor (21). To date, the only evidence for a role of this kinase in
GPCR function has been in the regulation of the "frizzled" receptor
Wnt signaling pathway (22).
-arrestin-dependence, whereas
GnRH receptors with C-tails lacking these sites remained
-arrestin-insensitive. This prompted us to investigate the role of
CKII sites in GPCR regulation. This study provides evidence for a novel
role of C-terminally located CKII sites in determining the sensitivity
of a GPCR to internalize via the
-arrestin-dependent pathway.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin-1 fusion protein have
been described previously (3, 4). Introduction and elimination of CK II
sites was performed using the QuickChange site-directed mutagenesis kit
(Stratagene, Sydney, Australia) according to the manufacturer's
instructions.
-Arrestin-1 cDNA was a gift from Prof. L. J. Benovic (Jefferson Medical College, Philadelphia, PA). The
phosphorylation-independent
-arrestin-1 mutant (R169E) was kindly
provided by Prof. V. V. Gurevich (Sun Health Research Institute,
Sun City, AZ). To examine receptor phosphorylation, an epitope tag
sequence was incorporated into the receptor to allow
immunoprecipitation. HA-tagged TRHR and GnRHR/cf tail have been
previously validated and used for phosphorylation studies (3).
Introduction of CKII sites was carried out in the HA-tagged GnRHR/cf
tail. To HA-tag the TRHR
3CKII, a restriction site 3'
of the coding region of HA TRHR, located in the multiple coding
region of pcDNA3 (ApaI), and an internal restriction
site 3' of the HA tag TRHR (EcoRV) were chosen in
order to subclone the TRHR
3CKII digested with the equivalent
restriction sites. Sequences of all cDNA clones were verified using
Dye Deoxy sequencing and an ABI 373 sequencer (PE Applied Biosystems).
11
to 10
5 M) at 37 °C for 60 min.
The assay buffer was removed, and cells were incubated at 4 °C for
30 min with 10 mM formic acid and subsequently transferred
to tubes containing Dowex (AG 108) anion exchange resin (Bio-Rad).
Total IPs were then eluted, and the amount of radioactivity was
counted. All treatments were performed in triplicate in at least three
separate experiments.
-Arrestin--
Transfected HEK 293 cells
were plated onto poly-L-lysine-coated eight-well chamber
slides. Treatments were carried out 48 h post-transfection, and
cells were fixed in 4% paraformaldehyde, mounted in FluoroGuard
(Bio-Rad), and sealed with coverslips. Cells were examined under an oil
immersion objective (× 60) using a Bio-Rad confocal laser microscope
with a filter selective for fluorescein isothiocyanate. Optical
sections (1.0 µm) were taken, and representative sections
corresponding to the middle of the cells are presented.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Arrestin-independent Internalization of the GnRHR/cf Tail
Chimera--
We have previously shown that internalization of the rat
GnRHR is not affected by overexpression of
-arrestin in COS cells or
by the high endogenous levels of
-arrestin when expressed in HEK 293 cells (1). The addition of the TRHR C-tail to the carboxyl terminus of
the GnRHR (GnRHR/TRHR tail) created a chimera that displayed
-arrestin-dependent internalization (4). Treatment of
transfected COS-1 cells with GnRH agonist for 60 min caused a
significant increase in the levels of GnRHR/TRHR internalized in the
presence of
-arrestin (p < 0.01) (Fig.
1), confirming previous results (4). We
extended this study to investigate the effect of
-arrestin on the
internalization of the GnRHR with another GPCR C-tail, (the
nonmammalian cfGnRHR C-tail). Interestingly, we found no increase in
the levels of receptor internalized with the GnRHR/cf tail chimera in
the presence of
-arrestin (Fig. 1). This suggested that other
factor(s), in addition to the presence of a C-tail, were involved in
targeting the GnRHR to the
-arrestin-dependent pathway.
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Fig. 1.
Effect of -arrestin
on agonist-induced internalization of wild type rat GnRHR and GnRHRs
with added C-tails from either TRHR or cfGnRHR. COS-1 cells were
transfected either with receptor and pcDNA3 (white
bars) or with receptor and
-arrestin (black
bars). Internalization assays were carried out in duplicate
samples following agonist treatment (60 min). The data represent values
of at least three independent experiments. **, p < 0.01 as compared with receptor transfected with pcDNA3.
-Arrestin-dependent
Internalization--
Given that the TRHR and cfGnRHR tails differed in
their ability to confer
-arrestin-dependence to the GnRHR, we
compared the consensus phosphorylation sites present in the C-tails of these receptors. Both the cfGnRHR and TRHR C-tails contain consensus phosphorylation sites for PKC; however, the wild type (WT) cfGnRHR C-tail contains no consensus CKII sites (Fig.
2A), while the TRHR C-tail
contains three CKII sites (Fig. 2B). We employed
site-directed mutagenesis to sequentially introduce one, two, and three
CKII sites, utilizing existing serines, into the C-tail of the GnRHR/cf tail chimera at amino acid positions A366E (+1CKII), G371D (+2CKII), and H354D (+3CKII). These amino acids were chosen in order to create
CKII consensus sites with minimal change to the C-tail sequence. Each
mutated construct bound 125I-labeled GnRH agonist
comparable with that of wild type (data not shown).
Agonist-dependent total IP accumulation was measured for the WT and +3CKII construct under a range of ligand concentrations (10
5 to 10
11 M) (Fig.
3). Although the maximal stimulation was
similar for each receptor, the GnRHR/cf tail with three added CKII
sites had slightly higher basal signaling than the GnRHR/cf tail
receptor. The IP dose-response curves showed an increased
EC50 value for the GnRHR/cf tail +3CKII compared with the
WT receptor (0.667 ± 0.224 nM for WT and 4.55 ± 0.082 nM for +3CKII).
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Fig. 2.
Structure of the C-terminal domains of the
cfGnRHR and the TRHR, with sequential addition or removal of CKII
consensus sites, respectively. The cytoplasmic C-tails of the WT
and mutated forms of rat GnRHR with an added cfGnRHR C-tail (GnRHR/cf
tail) (A) and TRHR (B) are shown. Sequential CKII
sites have been either added (GnRHR/cf tail) or removed (TRHR) by
alanine substitution, and numbers refer to the relevant
amino acid positions in the wild type receptor sequences. The CKII
motifs ((S/T)XX(D/E)) are shaded, the PKC motifs
((S/T)X(R/K)) are underlined, and mutated
residues are in boldface type.
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Fig. 3.
Agonist mediated total IP production of wild
type and mutated GnRHR/cf tail and TRHRs. Agonist-induced total IP
accumulation was measured in COS-1 cells transiently expressing WT
GnRHR/cf tail, GnRHR/cf tail +3CKII, TRHR, and TRHR 3CKII. Total IPs
were extracted and measured after treatment with either GnRH or TRH
(10
11 to 10
5 M, 45 min) as
described under "Experimental Procedures." Results shown are the
mean ± S.E. of triplicate observations from a single
representative experiment. Nonlinear regression analyses and curve
fitting were performed with PRISM software (GraphPad Software,
Inc.).
-arrestin (Fig. 4). COS-1
cells were transfected with either receptor and pcDNA3 vector or
with receptor and
-arrestin, and the levels of internalized receptor
were measured following agonist treatment (5-120 min). In the absence
of
-arrestin, all mutated receptors internalized at the same rate as
WT receptor (Fig. 4). Co-expression of
-arrestin had no effect on
the internalization rate of WT GnRHR/cfGnRHR, +1CKII, or +2CKII
chimeras. However, three CKII sites within the GnRHR/cfGnRHR C-tail
(+3CKII) resulted in a significant increase in the internalization rate
in the presence of
-arrestin at all time points (Fig.
4D). To determine whether the third CKII site alone was
responsible for the
-arrestin-dependent increase of the
+3CKII construct, a single mutation at residue 354 (H354D) was
introduced into the GnRH-R/cf tail, and the effect of
-arrestin on
the internalization of GnRH-R/cf tail H354D was assessed. Co-expression
of
-arrestin with H354D receptor did not enhance GnRH-promoted
internalization (Fig. 4E). Thus, we hypothesize that at
least three CKII consensus sites are required for internalization of
the GnRHR/cf tail via the
-arrestin-dependent pathway.
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Fig. 4.
Influence of
-arrestin on internalization of GnRHR/cf tail
chimeras with sequentially added C-tail CKII consensus sites.
COS-1 cells were co-transfected either with receptor and pcDNA3
(
) or with receptor and
-arrestin (
). Transfected cells were
plated into 24-well plates and internalization assays were carried out
at different time points (5 min to 2 h) following agonist
treatment. A, WT GnRHR/cf tail; B, GnRHR/cf
+1CKII (A366E); C, GnRHR/cf +2CKII (A366E,G371D);
D, GnRHR/cf +3CKII (A366E,G371D,H354D); E,
GnRHR/cf H354D. H354D refers to the introduction of the third CKII site
alone (see Fig. 2). Assays were carried out in duplicate at least three
times, and the results shown are representative of a single
experiment.
- Arrestin-dependent Internalization--
To test our
hypothesis that the presence of CKII consensus sites plays a role in
-arrestin-dependent internalization of the TRHR, the
three CKII sites in the TRHR C-tail were sequentially mutated by
substitution of the threonine or serine in the CKII consensus site to
alanine (T365A,T371A,S383A) (Fig. 2B). Each mutant TRHR
construct displayed levels of ligand binding similar to the WT receptor
(data not shown). Measurement of intracellular signaling under a range
of agonist concentrations (10
5 to 10
11
M) revealed that the magnitude of agonist-induced signaling
at maximal concentrations was similar between the WT TRHR and TRHR
3CKII, although removal of all three CKII sites resulted in
significantly lower levels of basal signaling and thus decreased levels
of IP production at lower TRH doses (Fig. 3). However, the
EC50 values for each receptor were similar (7.95 ± 0.189 nM for WT and 8.83 ± 0.055 nM for
3CKII). The effect of
-arrestin on internalization of the TRHR and
the mutated TRHRs lacking either one (TRHR
1CKII), two (TRHR
2CKII), or three (TRHR
3CKII) CKII sites was determined in COS-1
cells (Fig. 4). In the absence of
-arrestin, all mutated receptors
internalized at the same rate as WT TRHR (Fig.
5). In the presence of
-arrestin, TRHR
1CKII and TRHR
2CKII behaved similarly to WT TRHR. Mutation of all
three CKII sites from the TRHR C-tail was necessary before a total loss
in
-arrestin-dependent internalization was observed
(Fig. 5D). To determine if this effect was due to loss of
the third CKII site alone, a TRHR with only the third mutation (S383A)
was examined for its effect on
-arrestin-dependent internalization. The S383A receptor displayed a
-arrestin-dependent promotion in internalization to
similar levels as exhibited by WT TRHR (Fig. 5E). These
results suggest that at least one CKII consensus site in the TRHR
C-tail is required to confer
-arrestin-dependent internalization.
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Fig. 5.
Influence of
-arrestin on internalization of TRHRs with
sequentially mutated C-tail CKII consensus sites. The three CKII
consensus sites in the C-tail of the TRHR were sequentially removed by
alanine mutagenesis (see Fig. 2). COS-1 cells were co-transfected
either with receptor and pcDNA3 (
) or with receptor and
-arrestin (
). Transfected cells were plated into 24-well plates,
and internalization assays were carried out at different time points (5 min to 2 h) following agonist treatment. A, WT TRHR;
B, TRHR
1CKII; C, TRHR
2CKII; D,
TRHR
3CKII; E, TRHR S383A. S383A refers to the mutation of
the third CKII site alone (see Fig. 2). Assays were carried out in
duplicate at least three times, and the results shown are
representative of a single experiment.
-Arrestin/GFP Trafficking--
The effect of C-tail CKII sites on
-arrestin sensitivity of the receptor constructs was confirmed by
visualization of the cellular distribution of GFP-tagged
-arrestin
using confocal microscopy. HEK 293 cells co-expressing GFP/
-arrestin
with either WT or mutated receptors were analyzed for
agonist-dependent redistribution of
-arrestin from the
cytoplasm to the plasma membrane. The distribution of
-arrestin/GFP
was mainly cytoplasmic in untreated cells expressing all receptors
studied (Fig. 6, A,
C, E, and G). After agonist treatment,
cells expressing the GnRHR/cf tail chimera (Fig. 6B), +1CKII, or +2CKII mutants (results not shown) showed no change in
-arrestin distribution. However, in cells expressing the GnRHR/cf tail +3CKII, there was a redistribution of GFP-
-arrestin to the plasma membrane within 3 min of agonist stimulation, thus confirming the
-arrestin dependence of this receptor (Fig. 6D).
Similar experiments were carried out with the WT TRHR (Fig. 6,
E and F) and the TRHR
3CKII (Fig. 6,
G and H). The WT TRHR showed a rapid
-arrestin
translocation within 90 s of agonist stimulation (Fig. 6F), as shown previously (1, 4). However, the TRHR with mutated C-tail CKII sites showed no
-arrestin redistribution (Fig.
6H) even following 10 min of agonist stimulation (data not shown), thus confirming that loss of CKII sites results in
-arrestin insensitivity. Table I summarizes the
receptors used in this study, illustrating the presence or absence of
CKII consensus sites and their corresponding
-arrestin
dependence.
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Fig. 6.
Visualization of the effect of receptor
C-tail CKII sites on -arrestin/GFP
trafficking. Transiently transfected HEK 293 cells co-expressing
-arrestin/GFP and WT GnRHR/cf tail (A and B),
GnRHR/cf tail +3CKII sites (C and D), WT TRHR
(E and F), and TRHR
3CKII sites (G
and H). Transfected cells were plated onto
poly-L-lysine-coated chamber slides and treated over
periods of 0-10 min, fixed, and analyzed with confocal microscopy.
B and D (GnRHR/cf tail constructs) show agonist
treatment for 3 min. F and H show cells treated
for 1.5 min (TRHR constructs). Untreated cells are shown in
A, C, E, and G.
Summary of the -arrestin dependence of all receptors used in
this study illustrating the presence or absence of C-tail CKII
consensus sites
-arrestin dependence. 1CKII, 2CKII, and 3CKII
refer to CKII sites positioned 5' to 3' in the receptor C-tail
sequence.
-arrestin when
compared with COS cells (26). Internalization of the WT GnRHR/cf tail,
GnRHR/cf tail +1CKII, GnRHR/cf tail +2CKII, and GnRHR/cf tail H354D
were not significantly different from each other (Fig.
7A). However, the GnRHR/cf
tail +3CKII did show a significant increase in internalization compared
with WT GnRHR/cf tail (Fig. 7A; p < 0.05).
Likewise, the TRHR
1CKII and TRHR
2CKII internalized similarly to
WT TRHR (Fig. 7B), while the TRHR
3CKII showed a
significant decrease in levels of receptor internalized (p < 0.01; Fig. 7B), thus supporting our
finding in COS-1 cells.
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Fig. 7.
Internalization of wild type and mutated
GnRHR/cf tail and TRHR in HEK 293 cells. HEK 293 cells were
transfected with receptor cDNA, and internalization assays were
carried out in duplicate samples following agonist treatment (60 min).
A, GnRHR/cf tail WT and mutated constructs; B,
TRHR WT and mutated constructs. The data represent values of four
independent experiments. *, p < 0.05; **,
p < 0.01 as compared with WT receptor.
-Arrestin Phosphorylation-independent Mutant (R169E) Can
Promote Internalization of Receptors without C-tail CKII Sites--
A
mutant form of
-arrestin (R169E) that prevents the protein from
discriminating the phosphorylation state of the receptor (27) was used
to determine whether the mutant receptors can still use
-arrestin in
the absence of C-tail CKII sites. In transfected COS-1 cells, R169E
-arrestin significantly promoted internalization of the WT GnRHR/cf
tail when compared with WT
-arrestin (p < 0.05)
(Fig. 8, top). A
significant increase (p < 0.01) with R169E was also
observed for the TRHR with no CKII sites (TRHR
3CKII) (Fig. 8,
bottom). The GnRHR/cf tail +3CKII and the WT TRHR showed
-arrestin-dependent internalization in the presence of
both WT and R169E
-arrestins. The increase in internalization by
R169E
-arrestin was similar to that produced by WT
-arrestin,
suggesting the receptor kinases required for
-arrestin sensitivity
of these receptors are not rate-limiting in COS-1 cells. Results
obtained with R169E
-arrestin demonstrate that receptors with no
CKII sites in the C-tail are able to undergo
phosphorylation-independent
-arrestin internalization. This suggests
that the mutations do not affect
-arrestin-dependent
internalization in a nonspecific manner, but rather the phosphorylation
status of the receptor affects their
-arrestin insensitivity.
View larger version (42K):
[in a new window]
Fig. 8.
Effect of phosphorylation-independent
-arrestin mutant (R169E) on receptor
internalization. COS-1 cells were transfected with receptor and
pcDNA3 (white bars), receptor and WT
-arrestin (black bars), or receptor and R169E
-arrestin (lined bars). Top, WT
GnRHR/cf tail or GnRHR/cf tail +3CKII; bottom, WT TRHR or
TRHR
3CKII. Internalization assays were carried out in duplicate
samples following agonist treatment (60 min). The data represent values
of four independent experiments. *, p < 0.05; **,
p < 0.01 as compared with receptor transfected with
pcDNA3.
-arrestin R169E to promote the
internalization of receptors lacking C-tail CKII sites suggests that
specific receptor phosphorylation sites, required for WT arrestin
binding, were absent from these receptors. Therefore, we examined basal and agonist-stimulated phosphorylation of the
-arrestin-independent receptors, containing no C-tail CKII sites (GnRHR/cf tail and TRHR
3CKII), and compared this to their
-arrestin-dependent counterparts (GnRHR/cf tail +3CKII and TRHR). The HA-tagged GnRHR/cf tail and TRHR have been previously validated and used in
phosphorylation studies (3). The mutagenesis was carried out in the HA
GnRHR/cf tail; thus, the TRHR
3CKII was HA-tagged for this study as
described under "Experimental Procedures." Epitope tagging of this
receptor did not affect receptor function or its
-arrestin-independence (data not shown). COS-1 cells transfected
with HA-tagged versions of these receptors were prelabeled with
[32P]orthophosphate and following exposure to agonist
were immunoprecipitated with an anti-HA antibody. In order to compare
levels of receptor expression, immunoprecipitated proteins were Western
blotted and probed for the HA-epitope. As we have shown in previous
studies, the GnRHR/cf tail undergoes significant
agonist-dependent phosphorylation, which runs as a broad
band between 55 and 80 kDa, representing the mature, glycosylated form
of the receptor (Fig. 9A,
left panel, and Ref. 3). Interestingly,
the GnRHR/cf tail +3CKII did not show an enhanced agonist-promoted
level of phosphorylation of this band (Fig. 9A), despite a
similar level of receptor expression (Western blot, Fig.
9A). Instead, we observed the constitutive phosphorylation
of lower molecular weight forms of the GnRHR/cf tail +3CKII receptor
(see Fig. 9A, left panel,
lanes 5 and 6, arrow),
which were not observed for the GnRHR/cf tail receptor. This result
suggests that the addition of three CKII sites results in additional
constitutive phosphorylation of the GnRHR/cf tail but does not enhance
agonist-induced phosphorylation.
View larger version (107K):
[in a new window]
Fig. 9.
Agonist-induced phosphorylation of receptors
with added or removed CKII consensus sites. Epitope-tagged wild
type and mutated GnRHR/cf tail (A) and TRHRs (B)
were transiently expressed in COS-1 cells, and receptor phosphorylation
(left panels) and expression (right
panels) were determined. Receptor phosphorylation, following
whole cell labeling with [32P]Pi, was
determined in the absence ( ) and presence (+) of 100 nM
GnRH agonist (A) or 100 nM TRH (B),
and shown are representative phosphor images of SDS-polyacrylamide gel
electrophoresis-separated immunoprecipitates. The position of
phosphorylated GnRH and TRH receptors is indicated by brackets, and the
additional phosphorylation of lower molecular weight forms of GnRHR/cf
tail +3CKII is indicated by an arrow. Receptor expression
(right panels) was revealed by probing Western
blots for the HA epitope. Similar results were obtained in three
separate experiments. A, vector controls (lanes
1 and 2), GnRHR/cf tail (lanes
3 and 4), and GnRHR/cf tail +3CKII
(lanes 5 and 6). B, vector
controls (lanes 1 and 2), TRHR
(lanes 3 and 4), and TRHR
3CKII
(lanes 5 and 6).
3CKII
were also examined and are shown in Fig. 9B. The WT and mutant TRHR are strongly phosphorylated upon the addition of agonist (Fig. 9B and Ref. 3) and run as two broad bands in
the range 55-130 kDa that probably represent the mature monomer (band
centered at about 60 kDa) and dimer (band centered at about 120 kDa).
Phosphorylation of the CKII consensus sites in the C-tail of WT TRHR
occurs upon agonist stimulation as removal of these sites resulted in
an approximate 20% reduction of phosphorylated receptor (Fig. 9B,
compare lane 6 with lane
4). The remaining agonist-dependent
phosphorylation of the TRHR
3CKII most likely occurs on other
serines/threonines present in the receptor tail, mediated by additional
kinases involved in TRHR regulation.
-Arrestin-dependent Internalization--
To support our
hypothesis that CKII is involved in the
-arrestin dependence of the
GnRHR/cf tail +3CKII and the WT TRHR, we used the flavanoid compound
apigenin, which is known to be a specific inhibitor of CKII enzymatic
activity (28). COS-1 cells were transfected with receptor in the
presence or absence of
-arrestin. Internalization assays were
carried out on cells pretreated with different kinase inhibitors (PKC
inhibitor (staurosporine) and CKII inhibitor (apigenin)) at
their effective doses (14, 22). Internalization in the absence of
-arrestin of either the GnRHR/cf tail +3CKII sites or the TRHR was
similar to control values following pretreatment with staurosporine or
apigenin (Fig. 10). In the presence of
-arrestin, staurosporine had no effect on the
-arrestin-dependent increase in the internalization of either receptor when compared with control (Fig. 10), while apigenin resulted in a complete inhibition of the
-arrestin-dependent internalization of both receptors.
This selective inhibition supports a role for CKII, and not PKC, in
determining the sensitivity of the WT TRHR and the GnRHR/cf tail +3CKII
to internalize via the
-arrestin-dependent pathway.
View larger version (17K):
[in a new window]
Fig. 10.
Effect of kinase inhibitors on the
-arrestin-dependent internalization of
the GnRH-R/cf tail +3CKII sites and the WT TRHR. COS-1 cells were
transiently transfected with receptor and pcDNA3 (white
bars) or receptor and
-arrestin (black
bars). Cells were pretreated with either Dulbecco's
modified Eagle's medium alone (control); a CKII inhibitor,
apigenin (80 µM, 2 h); or protein kinase C
inhibitor, staurosporine (5 µM, 20 min). Internalization
assays were carried out in duplicate samples following agonist
treatment (60 min). The data represent values of three independent
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin in GPCR regulation has been extensively
studied (29-31); however, the functional receptor domains that determine
-arrestin sensitivity are not fully understood. The objective of the present study was to investigate determinants associated with
-arrestin sensitivity in the C-terminal domain of
GPCRs. By using GnRHR chimeric constructs, we have previously presented
evidence of a causal relationship between
-arrestin sensitivity and
the presence of a functional C-tail (4). Chimeric GnRHRs were generated
by adding a C-tail from another GPCR to the normally
-arrestin-insensitive GnRHR and provided us with a useful tool for
the study of the functionally important receptor C-tail domains. In
addition, the TRHR, a GPCR known to be
-arrestin-sensitive (1, 32,
33) was examined for determinants that confer
-arrestin dependence.
We present the unique finding that CKII consensus sites in the GPCR
C-terminal domain can regulate
-arrestin sensitivity and subsequent
receptor sequestration.
-arrestin-sensitive GPCRs, the TRHR and cfGnRHR,
were used to construct the chimeric GnRHRs. Intriguingly, only the
chimeric GnRHR with the TRHR C-tail exhibited
-arrestin-dependent internalization. This finding
suggests that the presence of a C-tail alone is not the only
requirement and that additional information within this domain is
needed to confer
-arrestin sensitivity. The C-tail has varying
importance in the internalization of different GPCRs. Truncation of the
M2 muscarinic receptor C-tail does not affect its internalization,
since the main site for
-arrestin interaction is within the third
intracellular loop (34, 35), whereas truncation of the TRHR C-tail
abolishes internalization (33). This suggests that the information
necessary for internalization is contained within the TRHR C-tail, and
our finding that the addition of this region to the GnRHR results in
-arrestin dependence supports this notion. Sequence comparisons
between the TRHR C-tail and the cfGnRHR C-tail revealed that both
contained consensus sites for PKC, but there were no consensus sites
for CKII in the cfGnRHR C-tail. This led us to hypothesize that the
presence of C-tail CKII sites can determine
-arrestin dependence.
Therefore, mutant GnRHR/cf tail constructs introducing single, double,
and triple CKII sites into the C-tail were tested for
-arrestin
sensitivity. Introduction of CKII sites into the GnRHR/cf tail chimera
resulted in
-arrestin dependence; however, this was only observed
when three CKII sites were present. The addition of three C-tail CKII sites did not affect levels of ligand binding or the magnitude of
agonist-dependent intracellular signaling. However, for the GnRHR/cf tail +3CKII, a small but significant increase in basal signaling was evident, and the IP dose profile was slightly different with a small increase in the EC50 value compared with the
receptor with no added CKII sites. All three CKII sites were needed for
-arrestin-dependent internalization, since a mutant
containing the third CKII site alone (H354D) was still
-arrestin-independent. Multiple, as opposed to single,
phosphorylation sites are also required for the regulation of other
GPCRs (36-38); also, in a model of arrestin binding, at least three
receptor phosphoacceptor sites were found to be required (39). Our
findings were supported by confocal microscopy of cells expressing
GFP/
-arrestin, in that only cells expressing the chimera with 3CKII
sites displayed a redistribution of GFP/
-arrestin. The time frame of
the
-arrestin translocation for the GnRHR/cf tail +3CKII was similar
to that observed for the GnRHR/TRHR chimera (4). In addition, the
higher levels of internalization for the GnRHR/cf tail +3CKII compared with wild type in HEK 293 cells were similar to levels previously published for the
-arrestin-sensitive GnRHR/TRHR chimera (4).
-arrestin-independent
was surprising, considering that the cfGnRHR is
-arrestin-dependent (4, 40) and C-tail truncations of
this receptor affect internalization promoted by
-arrestin (40). In
addition, the GnRHR/cf tail chimera undergoes
agonist-dependent phosphorylation most likely at the C-tail
serine 363, which is the main site of phosphorylation for the cfGnRHR
but is more important in receptor signaling than internalization (3,
40). The finding that the GnRHR/cf tail chimera is
-arrestin-insensitive suggests that this chimera does not contain
the phosphorylation sites sufficient for
-arrestin-dependent internalization; this is supported
also by the phosphorylation-independent internalization exhibited with
-arrestin R169E. The suggestion that multiple domains of the cfGnRHR
are involved in
-arrestin-dependent internalization (40)
may provide an explanation for this observation. Agonist-dependent phosphorylation was not increased in the
GnRHR/cf tail +3CKII, although additional phosphorylated bands
corresponding to the molecular weight for this receptor in both
untreated and treated samples were evident. A possible explanation is
that the addition of consensus sites for CKII results in constitutively phosphorylated receptor and that the addition of agonist activates the
receptor, which can then promote
-arrestin binding. This fulfills
the two requirements of
-arrestin binding: (i) receptor phosphorylation and (ii) agonist activation. The confocal studies with
-arrestin/GFP and the GnRHR/cf tail +3CKII displayed no translocation prior to agonist stimulation, thus excluding the possibility that constitutive phosphorylation would result in constitutive binding of
-arrestin. In addition, there is the possibility that the mutations that introduced the CKII sites in to the
cf tail also may affect receptor conformation, which, along with
phosphorylation, may enhance accessibility of proteins involved in
receptor endocytosis (40).
-arrestin dependence
of the GnRHR/cf tail chimera was further supported by the analysis of
the role of these sites in a
-arrestin-dependent GPCR,
the TRHR. Sequential mutation of three CKII sites in the TRHR C-tail
did not alter ligand binding or affect the magnitude of
agonist-dependent signaling. However, there was a
significant decrease in basal and agonist-induced signaling at lower
concentrations of TRH. It is unclear why these changes occur for
both the TRHR or GnRHR constructs, but they may reflect changes in
receptor conformation in mutated receptors, which possibly alter
G-protein coupling in the inactive or active state. It seems that the
receptors with C-tail CKII sites (WT TRHR, GnRHR/cf tail +3CKII) have
higher basal signaling than those without C-tail CKII sites (TRHR
3CKII, WT GnRHR/cf tail). This is an interesting observation, which
is under further investigation.
-arrestin; however, this only
occurred when all three sites were removed. Mutation of the third CKII
site alone (S383A) did not alter TRHR internalization compared with WT,
suggesting that at least one CKII site in the TRHR C-tail is sufficient
to promote
-arrestin-dependent internalization. Furthermore, agonist-dependent phosphorylation of the TRHR
3CKII was reduced compared with WT TRHR, suggesting that these sites are indeed phosphorylated, presumably by CKII, and may contribute to
-arrestin binding and subsequent receptor internalization. However,
there was still some degree of receptor phosphorylation of the TRHR
3CKII, probably due to the many other serines/threonines in the TRHR
tail. For other GPCRs (e.g. the PAR1 receptor), all serines/threonines must be mutated to alanine before phosphorylation is
ablated, yet only a portion of these sites influenced receptor function
(i.e. mutation of certain serines/threonines that affected receptor regulation still resulted in full receptor phosphorylation (37)).
-arrestin dependence by the removal of CKII consensus
sites is supported by two previous studies that showed that progressive
truncations of the TRHR C-tail affected receptor internalization in HEK
293 cells (41, 42). It would appear from these studies that receptors
with C-tail truncations that include one or more CKII sites behave like
wild type receptor, and only C-tail truncations with all three CKII
sites removed decrease receptor internalization. We obtained comparable
results in HEK 293 cells where only the internalization of TRHR
3CKII was affected. However, this does raise the question of the significance of having more than one CKII site in the TRHR C-tail, particularly since all three CKII sites are conserved between different mammalian species. It could be argued that the introduction of mutations into
these receptors has resulted in a nonspecific effect on
-arrestin sensitivity and that CKII sites may not be involved in the recruitment of
-arrestin. A mutant form of
-arrestin (R169E) that prevents the protein from discriminating the phosphorylation state of the receptor (27) was used to determine whether receptors could still use
-arrestin following modification of the CKII consensus sites. The
receptors used in this study all showed an increase in internalization
in the presence of
-arrestin (R169E). For WT TRHR and GnRHR/cf tail
+3CKII, there was no difference between wild type and R169E
-arrestin in promoting internalization of both receptors, suggesting
that GRK phosphorylation is not a limiting factor (COS cells express
low levels of GRKs) (26), supporting the involvement of other kinases.
The fact that the two receptor constructs that were
-arrestin-insensitive (WT GnRH/cf tail and TRHR
3CKII) were
capable of undergoing phosphorylation-independent internalization
suggests that the C-tails of these receptors lack phosphorylation sites
required for
-arrestin dependence. In addition, the decrease in
total receptor phosphorylation of the TRHR
3CKII supports this hypothesis.
-arrestin pathway is CKII. This hypothesis is supported by the use
of a specific CKII inhibitor, apigenin (28). Apigenin pretreatment of
cells expressing the WT TRHR and the GnRH/cf tail chimera with three
CKII sites completely inhibited the promotion in internalization by
-arrestin. The PKC inhibitor, staurosporine, had no such effect,
suggesting that PKC is not involved in the
-arrestin sensitivity of
these receptors. The presence of PKC consensus sites in the C-tails of
both
-arrestin-sensitive and -insensitive receptors used in this
study supports this observation.
-arrestin (30, 43, 44).
Although many receptors are phosphorylated by GRKs, this may not
necessarily be involved in receptor internalization (45). In addition,
phosphorylation of some GPCRs is not attributed to GRKs (8, 9), which
raises the question of the regulatory mechanisms involved. It is
possible that CKII or other unidentified kinases may be required
instead of, or in concert with, the GRKs, thus extending the repertoire
of proteins available for the regulation of the GPCR superfamily. While
the functions ascribed to CKII in the literature are fairly extensive,
there have been various reports that provide evidence of a role for
CKII in receptor endocytosis via clathrin-coated pits (21, 46-51). To
date, the only role for CKII in GPCR regulation has been shown for the
frizzled receptor, where CKII phosphorylates the downstream signaling
molecule disheveled (22). To our knowledge, the present study is
the first report of CKII involved in internalization of a GPCR via the
-arrestin-dependent pathway. However, in a study on M3
muscarinic receptor phosphorylation by casein kinase 1
, the authors
report that this receptor was also a substrate for CKII, although in
this receptor it was not agonist-dependent (16). Such
studies support the possibility of additional kinases other than the
GRKs that are involved in regulating GPCR function. There is major
interest in identifying the intracellular components that mediate
specific responses of GPCRs, including proteins that regulate the
-arrestin sensitivity of a receptor, which would provide novel
therapeutic targets.
-arrestin-dependent internalization to the GnRHR, a
C-tail containing consensus phosphorylation sites for CKII is required.
We also propose that CKII sites have an important role in targeting the TRHR to the
-arrestin-dependent pathway. Similar studies
on other GPCRs will be required to determine whether CKII sites have a role in the
-arrestin sensitivity of other members of this receptor family.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Graham Worth for assistance in iodination and Prof. Graeme Martin for comments concerning the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the National Health and Medical Research Council of Australia and the Raine Foundation (to K. A. E).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a Keogh Institute of Medical Research
postgraduate scholarship.
§§ Supported in part by a Block grant from the National Health and Medical Research Council of Australia to the Baker Medical Research Institute.
¶¶ To whom correspondence should be addressed: WAIMR, B Block, Sir Charles Gairdner Hospital, Hospital Ave., Nedlands, Perth, WA 6009, Australia. Tel.: 61 08 9346 1980; Fax: 61 08 9346 1818; E-mail: keidne@waimr.uwa.edu.au.
Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.M009275200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GnRHR, gonadotropin-releasing hormone receptor; GPCR, G-protein-coupled receptor; CKII, casein kinase II; TRH, thyrotropin-releasing hormone; TRHR, TRH receptor; GRK, G-protein coupled receptor kinase; PKC, protein kinase C; C-tail, carboxyl-terminal tail; HEK, human embryonic kidney; cf, catfish; cfGnRHR, catfish GnRHR; HA, hemagglutinin; IP, inositol phosphate; GFP, green fluorescent protein; WT, wild type.
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