From the Istituto Neurologico Mediterraneo
Neuromed, Istituto di Ricovero e Cura a Carattere Scientifico,
86077 Pozzilli, the § Department of Human Physiology and
Pharmacology and ** Department of Experimental Medicine and
Pathology, University of Rome "La Sapienza," 00198 Rome, and
Consorzio Mario Negri Sud, Istituto di Ricerche Farmacologiche
"Mario Negri," 66030 Santa Maria Imbaro, Italy
Received for publication, April 24, 2002, and in revised form, December 18, 2002
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ABSTRACT |
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The metabotropic glutamate 1 (mGlu1) receptor in cerebellar Purkinje cells plays a
key role in motor learning and motor coordination. Here we show that
the G protein-coupled receptor kinases (GRK) 2 and 4, which are
expressed in these cells, regulate the mGlu1 receptor by at
least in part different mechanisms. Using kinase-dead mutants in HEK293
cells, we found that GRK4, but not GRK2, needs the intact kinase
activity to desensitize the mGlu1 receptor, whereas GRK2,
but not GRK4, can interact with and regulate directly the activated
G Metabotropic glutamate
(mGlu)1 receptors, which are
activated by the excitatory amino acid glutamate, are part of an
original family of G protein-coupled receptors (GPCR) called the family 3 GPCRs (1-3). These include all the mGlu receptor subtypes, Ca2+-sensing and GABAB receptors, and some
putative olfactory, pheromone, and taste receptors. Eight subtypes of
mGlu receptors have been identified, which are implicated in different
aspects of physiology and pathology of the central nervous system.
Group I mGlu receptors (mGlu1 and mGlu5), which
stimulate polyphosphoinositide hydrolysis by coupling to
Gq, are localized in the peripheral parts of postsynaptic dendrites and contribute to the regulation of synaptic plasticity. For
example, mGlu1 receptor present in cerebellar Purkinje
cells plays a key role in motor learning and motor coordination.
Similar to many other GPCRs, the signal transduction of the
mGlu1 receptor is strictly regulated by multiple mechanisms
acting at different levels of signal propagation (1). After prolonged
or repeated stimulation, receptors are profoundly desensitized. Protein
kinase C is clearly involved in this process, although a protein kinase C-independent component of mGlu1 receptor desensitization
was also observed (4). The activated We have recently shown that GRK4, one GRK subtype originally identified
in testis and sperm cells, may play a major role in the regulation of
the mGlu1 receptor. In HEK293 cells transfected with GRK4
and in cultured cerebellar Purkinje cells, which naturally express high
levels of GRK4, we demonstrated that this kinase is important for the
desensitization and for rapid internalization of the mGlu1
receptor (8).
The present study shows that the stimulation of the mGlu1
receptor induces the rapid redistribution of Materials--
Polyclonal anti-mGlu1 antibody
was from Upstate Biotechnology; polyclonal anti-RGS4, anti-ERK1, and
anti-G Clones and Mutants--
To generate a fusion protein between GST
and the N-terminal domain of GRK2 (GST-GRK2-Nter) and of GRK4
(GST-GRK4-Nter), we used a PCR-based method as previously described
(12). The GRK4-(K216M,K217M) was prepared as previously described (8).
The following plasmids were generous gifts: GRK2-(K220R) from C. Scorer
(Glaxo Wellcome, Stevenage, UK), G Cell Culture, Transfection, and IP Measurement--
Cerebellar
neurons were prepared from Wistar rats as previously described (8, 13),
with minor modifications to obtain a Purkinje cell-rich culture.
Seven-day-old pups were sacrificed by cervical dislocation and the
cerebella excised and minced with a scalpel. The cerebellar cells were
disgregated with 0.025% trypsin and 0.01% DNase I in Krebs Ringer
plus 0.03% MgSO4, 0.3% BSA for 15 min at 37 °C. The
cells were washed with the same buffer containing 40 µg/ml trypsin
inhibitor and 0.01% DNase I and dissociated by repeated passage
through a fine-tipped pipette. The cell suspension was centrifuged at
400 × g for 2 min, and cells resuspended carefully in
2 ml of the same buffer. After 30-45 min, the Purkinje cells are
enriched from granules by gravity. The upper part of the suspension (granules) was removed very carefully, and the sediment (Purkinje cells) was rinsed with culture medium. Recovered cells were plated at a
density of 20-25 × 104 cells/cm2 onto
poly-L-lysine-coated chamber slides in serum-free defined medium: Eagle's medium supplemented with 1 mg/ml BSA, 10 µg/ml insulin, 0.1 nM L-thyroxin, 0.1 mg/ml
transferrin, 1 µg/ml aprotinin, 30 nM selenium, 100 µg/ml streptomycin, and 100 units/ml penicillin. The cultures were
maintained in a humidified atmosphere of 5% CO2 in air at
37 °C. The cultures, which consisted of ~2-3% Purkinje cells
(assessed by calbindin immunostaining), were used after 15-20 days
in vitro.
HEK293 cells were transfected as described (8). One day after
transfection, the cells were washed in PBS and incubated for 18 h
with Dulbecco's modified Eagle's medium/Glutamax-1 (Invitrogen), then
washed and incubated overnight with minimal essential medium/Glutamax-1 containing 3 µCi/well myo-[3H]inositol
(Amersham Biosciences). On the third day, IP production was measured as
described (8). Briefly, cells were washed twice and incubated for 1-2
h at 37 °C in 1 ml of HEPES-buffered saline (146 mM
NaCl, 4.2 mM KCl, 0.5 mM MgCl2,
0.1% glucose, 20 mM HEPES, pH 7.4), washed again with
HEPES-buffered saline, and pre-incubated for 15 min in the same buffer
containing 10 mM LiCl, 1.8 units/ml glutamic pyruvic
transaminase, 2 mM sodium pyruvate. The stimulus was
carried out for 30 min with 100 µM quisqualate, unless
otherwise indicated. The reaction was stopped by replacing the
incubation medium with 1 ml of ice-cold perchloric acid (5%). Inositol
phosphates were separated by an ion exchange chromatography column of
Dowex AG1-X8 (formiate form) (200-400-mesh, 350-µl bed volume).
Usually 1 × 106 cells were co-transfected with 1 µg
of mGlu1 plasmid along with 5 µg of GRK cDNA, or
empty vector. For mGlu1a 5 µg of the plasmid encoding the
glutamate transporter EAAC1 (8) was included.
Cerebellar Purkinje Cell GRK4 Antisense Oligonucleotide Treatment
and Adenovirus Infection--
For antisense oligonucleotide treatment,
the experiments were performed as previously described (8). Cerebellar
neurons were prepared as described (8) and maintained in culture for 2-3 weeks, at which time two-end phosphorothioate oligonucleotides at
1 µM final concentration were added for 4-5 days. The
sequence of the GRK4 antisense oligonucleotide and of the scrambled
oligonucleotide (used as control) are as described in Ref. 8.
Recombinant adenovirus was prepared according to Ref. 14 with minor
modifications. The Binding of G Proteins to GRK N-terminal and
Immunoprecipitation--
These experiments were performed as described
previously (12). Cytosolic proteins (150 µg) from HEK293 cells
transfected with the G
Immunoprecipitation was done as follows. After treatments, cells were
rapidly washed in ice-cold PBS and solubilized in Triton X-100 lysis
buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl,
1% Triton-X 100, 1 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 mM sodium orthovanadate, 50 mM sodium fluoride, and 10 mM Western Blotting--
After treatments, cells were rapidly
washed in ice-cold PBS and solubilized in Triton X-100 lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton
X-100, 1 mM EDTA, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 mM
sodium orthovanadate, 50 mM sodium fluoride, and 10 mM Immunofluorescence Confocal Analysis--
HEK293 cells
transfected as above and 20-day-old Purkinje cell primary cultures were
fixed with 4% paraformaldehyde in PBS for 15 min at room temperature.
The autofluorescence was quenched by incubation for 30 min in 50 mM NH4Cl, 50 mM glycine in PBS, and
nonspecific interactions were blocked by treatment with blocking solution (0.05% saponin, 0.5% BSA in PBS) for 30 min at room
temperature. Cells were incubated (8) with anti-mGlu1a (0.3 µg/ml, overnight at 4 °C), anti- GRK4-dependent mGlu1 Receptor
Desensitization and Internalization--
GRK2 and GRK4 are the two GRK
subtypes expressed in cerebellar Purkinje cells (8). As these cells
represent one relevant site of the mGlu1 receptor
expression and function, we investigated the role of GRK2 and GRK4 in
the regulation of this receptor. Previous studies have shown that the
agonist-dependent phosphorylation of the mGlu1a
receptor expressed in HEK293 cells is significantly enhanced when GRK2
(9) or GRK4 (8) are co-transfected. To assess the role of
GRK-dependent receptor phosphorylation in the homologous
desensitization of the mGlu1 receptor-stimulated IP production, we used GRK2 and GRK4 kinase-dead mutants in which the
kinase activity was disrupted by site-directed mutagenesis of key amino
acids located in the catalytic domain. Both mutants, which are named
GRK2-(K220R) and GRK4-(K216M,K217M) for GRK2 and GRK4, respectively,
lost their ability to phosphorylate receptor substrates (8, 12). We
determined the agonist-stimulated IP production in HEK293 cells
transfected with the mGlu1 receptor and the effect of
co-expression of different GRK mutants (Fig. 1A). According to our previous
results (8), the co-transfection of either GRK2 or GRK4 resulted in a
35-40% reduction of the agonist-stimulated response. By contrast, the
effect of the two kinase-dead mutants was substantially different; the
GRK4-(K216M,K217M) mutant was ineffective, whereas the GRK2-(K220R)
desensitized the mGlu1 receptor-stimulated signaling to the
same extent as the GRK2 wild type. These results indicated that the
phosphorylation of the mGlu1 receptor was necessary for
GRK4-mediated receptor desensitization, whereas GRK2 utilized, at least
in part, a phosphorylation-independent mechanism for receptor
regulation.
The likely mechanism by which GRK2 could regulate mGlu1
receptor signaling in a phosphorylation-independent manner is provided by the RGS-like domain present in the N terminus of GRK2, because we
and others have previously shown that this is a functionally active
domain able to regulate the GPCR-stimulated Gq signaling by
direct binding and inhibition of the activated G
In our experimental conditions
GRK4 is also primarily involved in mGlu1 receptor
internalization (Fig. 2). In HEK293 cells
transiently expressing the mGlu1 receptor, exposure to
quisqualate for 5 min did not induce a significant level of receptor
internalization and the co-expression of GRK2 resulted in a 2-3-fold
increase of receptor internalization. In cells transfected with GRK2,
the maximal internalization was observed after 20-30 min of agonist
treatment (Fig. 2). The expression of GRK4 drastically enhanced the
internalization of the mGlu1 receptor induced by the
quisqualate, and this effect was rapid with a maximal peak at 5 min of
agonist stimulation and was reversible within 30 min (Fig. 2).
Previous work from our laboratory documented a substantial
co-localization of the mGlu1 receptor and GRK4 both under
basal conditions and after internalization. We investigated whether GRK2 could also be co-localized with the receptor. Agonist-induced receptor internalization was assessed in HEK293 cells co-transfected with the mGlu1 receptor and GRK2, and the reciprocal
co-localization of the receptor and the kinase was determined in cells
untreated or exposed to agonist for 5, 20, and 40 min. By confocal
microscopy analysis, we found that 12 ± 2%, 24 ± 6%,
6 ± 2%, and 9 ± 4% of the mGlu1 receptor and
8 ± 1%, 18 ± 2%, 5 ± 1, and 18 ± 4% of GRK2
staining was reciprocally co-localized respectively after 0, 5, 20, and
40 min of exposure to agonist (n = 20). The limited amount of co-localization found in this time course indicates that the
mGlu1 receptor-GRK2 interaction is less persistent than that of GRK4 with the receptor.
Because GRK4 appeared to be a key regulator of the rapid
agonist-promoted mGlu1 receptor internalization both in
HEK293 cells (Ref. 8 and present results) and in cerebellar Purkinje
cells (8), this kinase was co-transfected with the mGlu1
receptor in subsequent experiments, unless otherwise indicated.
Differential Sorting of mGlu1 Receptor and
We investigated the effect of mGlu1a Receptor-mediated MAPK Activation Is Blunted by
To see whether increasing the overexpression of
In the experiments presented so far, we used HEK293 cells transiently
expressing GRK4. To assess whether this kinase is necessary for
mGlu1 receptor-dependent MAPK activation, we
examined this pathway in cells transfected with the mGlu1
receptor without GRK4 co-transfected. GRK4 is not endogenously
expressed in HEK293 cells. Exposure to quisqualate induced the
activation of MAPK even in the absence of GRK4 (Fig. 6C),
and the time course was similar to that of the earlier experiments
(data not shown). This finding suggests that kinase(s) other than GRK4
could be involved in the mGlu1 receptor-stimulated
Agonist-stimulated mGlu1 Receptor and mGlu1 Receptor-mediated MAPK Activation in Cerebellar
Purkinje Cells--
The activation MAPK by mGlu1 receptor was
documented in Chinese hamster ovary (21) and HEK293 (present study)
cells transfected to overexpress the mGlu1 receptor. We
sought to determine whether this receptor-mediated response also occurs
in cells that physiologically express the mGlu1 receptor.
Using confocal microscopy analysis, we evaluated the activation of MAPK
by quisqualate in cultured cerebellar Purkinje cells (Fig.
8A). Using the
anti-phospho-ERK1/2 antibody, which recognizes the phosphorylated form
of ERK, we found that 5-min treatment with quisqualate induced a robust
increase of the immunoreactivity (Fig. 8A, a and
b). Phospho-ERK1/2 total immunofluorescence intensity
(arbitrary units) was increased from 3586 ± 416 to 56,796 ± 2400 following 5 min of exposure to quisqualate (n = 14). Phosphorylated ERK1/2 was also redistributed and was localized in
the nucleus (Fig. 8A) or in perinuclear regions (data not
shown) depending on the cells examined. The labeling of RGS4, used as
negative control (data not shown), demonstrated that in the same cells
treated with quisqualate the increased immunofluorescence and the
redistribution observed with the anti-phospho-ERK1/2 antibody was
selective. ERK1/2 were not affected by quisqualate treatment in
cultured cerebellar Purkinje cells (Fig. 8A, c
and d). ERK1/2 total immunofluorescence intensity (arbitrary
units) was 41,554 ± 351 and 47,612 ± 215 in control and
treated cells respectively (n = 16).
Involvement of GRK4 and
In cultured cerebellar Purkinje cells, GRK4 was knocked down by the
treatment with an antisense oligonucleotide that selectively decreased
GRK4 by ~ 70%, whereas the treatment with a scrambled oligonucleotide (used as control) was ineffective (Ref. 8 and data not
shown). According to previous findings, the agonist-stimulated mGlu1 receptor internalization was inhibited by 83 ± 7% (n = 12) in cells treated with the antisense
(versus scrambled oligonucleotide-treated cells), whereas in
the same cells the ERK1/2 phosphorylation induced by quisqualate was
similar to that observed in cells treated with the scrambled
oligonucleotide (Fig. 9).
To investigate the involvement of GRK4 mediates the homologous desensitization and the rapid
internalization of the mGlu1 receptor in transfected HEK293
cells and in cultured cerebellar Purkinje cells (8). We have previously shown that, in both cell types, exposure to agonist for 5 min induced
the internalization of the mGlu1 receptor and of GRK4 in
intracellular vesicles where these proteins were substantially co-localized (8). This delineated a rapid GRK4-dependent
mechanism of mGlu1 receptor internalization. The present
study documents that, besides inducing internalization and
co-localization of the receptor and GRK4, the rapid exposure to agonist
promotes the redistribution of GRK4 is a member of the GRK family, which, unlike the other GRKs, is
not ubiquitously expressed but instead is found in only few cell types
(8, 22, 23). This kinase, initially identified in testis and in sperm
cells, is also abundantly expressed in cerebellar Purkinje cells, where
it regulates the mGlu1 receptor (8). In transfected HEK293
cells, the mGlu1 receptor signaling can also be regulated
by GRK2 (8, 9). Because the expression of the mGlu1
receptor is not limited to neurons, it was suggested that GRK2 can play
a role in regulating the mGlu1 receptor in non-neuronal
tissues or, alternatively, in neuronal cell types other than the
cerebellar Purkinje cells (9). Here we show that GRK2 and GRK4 regulate
the mGlu1 receptor signaling by, at least in part,
different mechanisms. GRK4-dependent desensitization was
fully phosphorylation-mediated, whereas GRK2 is also able to regulate
receptor-stimulated IP production by a phosphorylation-independent mechanism, which involves the functional RGS-like domain present within
the GRK2 N terminus. The difference between these two kinases likely
reflects the different ability of their N-terminal domains to interact
with the activated G In HEK293 cells transfected with GRK4, exposure to agonist induces the
rapid (5 min) internalization of the mGlu1 receptor. This
effect resembles the rapid agonist-induced mGlu1 receptor internalization observed in cultured cerebellar Purkinje cells, as the
antisense oligonucleotide-induced knock-down of GRK4 (but not of GRK2)
prevented receptor internalization (8). We therefore focused on this
rapid agonist-stimulated GRK4-dependent mGlu1 receptor desensitization and internalization to assess the role of
Other groups have reported that, in HEK293 cells, the mGlu1
receptor is internalized in an agonist-stimulated manner even when GRK4
was not co-transfected (10, 11). This mechanism seems to be different
from that observed in the presence of GRK4, because the time course is
slower (maximal effect at 30-60 min) and the internalization is
Our data document that in HEK293 cells and in cerebellar Purkinje cells
GRK4 is not necessary for the mGlu1 receptor-stimulated activation of ERK1/2. Experiments in HEK293 cells rather indicate that
GRK2 is involved in this pathway. The mGlu1 receptor
internalization is not sufficient to activate this pathway, and it is
likely that the receptor is not physically associated to the signaling
complex, because the mGlu1 receptor and Based on the available data, we propose the following model for the
mGlu1 receptor signaling and regulation. The activation of
the mGlu1 receptor induces the redistribution of
q. In cells transfected with GRK4 and exposed to
agonist,
-arrestin was first recruited to plasma membranes, where it
was co-localized with the mGlu1 receptor, and then
internalized in vesicles. The receptor was also internalized but in
different vesicles. The expression of
-arrestin V53D dominant
negative mutant, which did not affect the mGlu1 receptor
internalization, reduced by 70-80% the stimulation of
mitogen-activated protein (MAP) kinase activation by the
mGlu1 receptor. The agonist-stimulated differential sorting
of the mGlu1 receptor and
-arrestin as well as the
activation of MAP kinases by mGlu1 agonist was confirmed in
cultured cerebellar Purkinje cells. A major involvement of GRK4 and of
-arrestin in agonist-dependent receptor internalization and MAP kinase activation, respectively, was documented in cerebellar Purkinje cells using an antisense treatment to knock down GRK4 and
expressing
-arrestin V53D dominant negative mutant by an adenovirus
vector. We conclude that GRK2 and GRK4 regulate the mGlu1
receptor by different mechanisms and that
-arrestin is directly
involved in glutamate-stimulated MAP kinase activation by acting
as a signaling molecule.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit of the Gq
(G
q) can in turn be inhibited by RGS (for
regulators of G protein signaling) proteins (5). These RGS proteins work by interacting with G
and by
increasing the intrinsic GTPase activity of G
, acting as
GTPase-activating proteins (6, 7). Recent studies from our and other
laboratories have documented that G protein-coupled receptor kinases
(GRKs) and arrestins are involved in the mechanism of
agonist-stimulated mGlu1 receptor phosphorylation,
desensitization, and internalization. Using transfected HEK293 cells,
it was shown that the mGlu1 receptor is phosphorylated and
desensitized by different GRK subtypes (8, 9) in an
agonist-dependent manner. In these cells agonist treatment
induced the internalization of the mGlu1 receptor, and this
mechanism was
-arrestin- and dynamin-dependent (10, 11).
The mGlu1 receptor is also internalized tonically (i.e. in an agonist-independent manner) by a mechanism that
is
-arrestin- and dynamin-independent and likely involves a
clathrin-mediated endocytic pathway (11). Based on these results and on
studies with different receptor types, it was suggested that multiple endocytic pathways may contribute to the internalization of the same
GPCR (11).
-arrestin, which is first recruited to plasma membranes and then internalized in
intracellular vesicles. Our results support the possibility that
-arrestin acts as a signaling protein that mediates the
mGlu1 receptor-mediated activation of MAP kinases
(MAPK).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q were from Santa Cruz Biotechnology (Santa Cruz,
CA); monoclonal anti-phospho-ERK1/2 was from Cell Signaling Technology;
monoclonal anti-GRK2/3 was from Upstate Biotechnology; polyclonal
anti-GRK4 was from Santa Cruz; monoclonal anti-FLAG M5 was from Eastman
Kodak Co.; Alexa-594 protein labeling kit, Alexa-488 anti-mouse, and
anti-rabbit IgGs were from Molecular Probes (Eugene, OR); monoclonal
anti-
-arrestin antibody (F4C1) was kindly provided by Dr. L. A. Donoso; Cy3-conjugated anti-rabbit IgG was from Sigma.
q from A. Gilman (University of Texas, Dallas, TX), G
q(Q209L) from
N. Dhanasekaran (Temple University, Philadelphia, PA),
-arrestin
V53D from Federico Mayor (Universidad Autonoma de Madrid, Madrid,
Spain), human mGlu1 receptor in pcDNA 3 from M. Corsi
(Glaxo Wellcome, Verona, Italy), the human EAAC1 from J. P. Pin
(CNRS, Montpellier, France) and M. A. Hediger (Harvard Medical
School, Boston, MA), and PAF receptor (PAFr) in pCDM8-FLAG plasmid from
C. Gerard (Harvard Medical School, Boston, MA).
-arrestin V53D cDNA was subcloned into the
multiple cloning site of the shuttle plasmid (pAd-CMV-TRK) by standard
cloning procedures. The purified shuttle plasmid was digested with the
restriction enzyme PmeI to obtain the "rescue fragment."
The fragment was then purified on agarose gel, and 2 µg of purified
rescue fragment was used for homologous recombination. The adenoviral
plasmid pAdEasy-1 (14) was then mixed with the rescue fragment, and the
DNA mixture was transformed into the BJ5183 bacterial strain and
incubated overnight. Colonies were screened by digesting the DNA with
BglII and performing a Southern blot to confirm the presence
of the cDNA insert. The DNA with the proper orientation was
transformed into the DH5a bacterial strain. The recombinant construct,
purified using Qiagen Maxi preparation kit, was digested overnight with
PacI and transfected into HEK293 cells using LipofectAMINE
(Invitrogen). Adenoviral plaques were purified twice by infecting
HEK293 cells in agar. Virus was purified by CsCl gradient
centrifugation, dialyzed, and titrated by plaque assay. The recombinant
adenovirus obtained expresses both green fluorescent protein (GFP) and
-arrestin V53D under independent cytomegalovirus promoters. A virus
expressing only GFP was used as a control. For infection cerebellar
Purkinje cells were incubated with the recombinant adenovirus at a
multiplicity of infection of 50 plaque-forming units/cell for 3 h
at 37 °C in medium without serum. The virus-containing medium was
then removed, and cells were incubated in standard medium plus serum. At 48 h after infection, >95% of the cultured cerebellar
Purkinje cells were infected, as assessed by the expression of GFP. The expression of
-arrestin V53D was confirmed by immunocytochemistry in
cerebellar Purkinje cells or by immunoblot in a U87MG glioblastoma cell line.
q subunit were mixed with 40 µl
of slurry containing GST-GRK-Nter fusion proteins bound to glutathione
agarose beads in a final volume of 400 µl of binding buffer (20 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 100 mM NaCl, 0.1% Lubrol, 10 µM GDP, 3 mM MgCl2), in the presence or absence of 47 mM MgCl2, 30 µM AlCl3, and 20 mM NaF. After 1 h at 4 °C
the beads were washed three times with 1 ml of ice-cold binding buffer
and the resins containing the eventual bound proteins were analyzed by
immunoblotting, using anti-G
q antibody (Santa Cruz
Biotechnology). One fraction of starting material (30-40 µg, ~25%
of the total cytosolic proteins used for binding) was also included in
the gel (indicated as S in Fig. 1).
-glycerophosphate) for 15 min. The lysates
were clarified by centrifugation (10,000 × g for 10 min). Protein concentration of supernatants was determined, and 600 µg of total proteins were incubated with 5 µg of
anti-G
q antibodies for 2h at 4 °C followed by
addition of 50 µl of di-protein A-Sepharose pre-equilibrated in HNTG
buffer (20 mM Hepes, 150 mM NaCl, 0,1% Triton
X-100, 5% glycerol), and an additional 1-h incubation at 4 °C.
Immunoprecipitates were washed four times in HNTG buffer, and the
pellets were boiled in Laemmli buffer for 5 min before electrophoresis.
Immunoprecipitates and starting materials were subjected to 10%
SDS-polyacrylamide gel under reducing condition. After electrophoresis,
proteins were transferred to polyvinylidene difluoride membrane and
immunoblotted using anti-GRK2 (Upstate Biotechnology) and anti-GRK4
(Santa Cruz) antibodies.
-glycerophosphate) for 15 min. The lysates were
clarified by centrifugation (10,000 × g for 10 min),
and 80-100 µg of proteins were separated by SDS-PAGE electrophoresis, blotted onto nitrocellulose, and probed using a
commercial anti-phosphospecific antibody against phosphorylated ERK1/2.
Monoclonal anti-phospho-ERK1/2 antibody was used at 1:2000 dilution.
The membranes were stripped according to instructions from the
manufacturer and reprobed with polyclonal anti-ERK1/2 antibody at
1:5000 dilution (Santa Cruz Biotechnology). Other Western blot analyses
were performed as described (12). The immunoreactive bands were
visualized either by enhanced chemiluminescence using horseradish
peroxidase-linked secondary antibody.
-arrestin (F4C1) (12.5 µg/ml,
1 h, room temperature), anti-phospho-ERK1/2 (4 µg/ml, 1 h,
room temperature), anti-ERK1 (2 µg/ml, 1 h, room temperature),
and anti-RGS4 (2 µg/ml, overnight at 4 °C) antibodies in blocking
solution. The chamber slides were then incubated with blocking solution
containing Alexa-488 anti-rabbit (1:400, Molecular Probes),
Cy3-conjugated anti-rabbit (1:200, Sigma), or Alexa-488
anti-mouse IgG (1:400, Molecular Probes) for 1 h at room
temperature. For PAFr localization, an anti-FLAG M5 monoclonal antibody
directed at MDYKDDDDKEF amino acid sequence at the N-terminal Met-FLAG
fusion protein of PAFr was used; this antibody was conjugated to the
fluorochrome Alexa-594 and diluted at 1 µg/ml in blocking buffer (1 h, room temperature). Each incubation step was carried out in the dark
and followed by careful washes with PBS (six times/3 min each). After
immunostaining the coverslips were mounted on slides with Mowiol 4-88
and analyzed by a Zeiss LSM 510 laser scanning microscope equipped with
an Axiovert 100 M-BP and by the Confocal Imaging System
Ultraview (PerkinElmer Life Sciences). The internalization of the
mGlu1 receptor and of PAF receptors was quantified as
previously described (8). Co-localization was quantified as previously
reported (8) or using the "co-localization and correlation" option
of the Ultraview software.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Regulation of mGlu1a receptor by
GRK2 and GRK4. A, HEK293 cells were transfected with
the mGlu1a receptor and co-transfected with mock
(ctrl), GRK2 or GRK4 wild type (WT), or
kinase-dead mutant (dn) and (100 µM)
quisqualate-stimulated IP formation was measured (means ± S.E. of
5 separate experiments; **, p < 0.01, Tukey's test).
B, binding of GRK2- and GRK4-Nter to activated
G q. Recombinant purified GST-GRK-Nter proteins
conjugated to glutathione-agarose beads were incubated with cytosolic
proteins (150 µg) from G
q-transfected HEK293 cells in
the absence (
) or presence (+) of 30 µM
AlCl3 and 20 mM NaF to activate the
G
q. Incubation (1 h at 4 °C) was stopped by
centrifugation (300,000 × g). After three extensive
washings, G
q bound to the column was detected by
immunoblot using anti-G
q antibody. Starting material
(S) (30-40 µg of cytosolic preparation, about one fourth
of total cytosol used for binding) is also included in the immunoblot.
The experiment shown was repeated three times with similar results.
C, co-immunoprecipitation of G
q and GRKs from
HEK293 cells. HEK293 cells co-expressing G
q
(lanes 1, 2, 5, and
6) or G
q(Q209L) (lanes
3, 4, 7, and 8) and either
GRK2 or GRK4 (as indicated) were untreated (lanes
1, 3, 5, and 7) or exposed
to 100 µM quisqualate (lanes 2,
4, 6, and 8) for 5 min. Cells were
then harvested and lysed, and IP (lanes 1-4) was
performed using an anti-G
q antibody as described under
"Experimental Procedures." Initial cell extract (15%) was included
for comparison (lanes 5-8). The experiment shown
was repeated three times with similar results.
q (12,
15). According to this hypothesis, our results suggest that, unlike GRK2, the GRK4 N terminus, which also contains an RGS homology domain,
should be unable to interact with G
q and to regulate its
signaling cascade at the G protein level. To test this possibility we
prepared a GST-GRK4-Nter fusion protein and we measured the binding of
this domain to G
q, using the GST-GRK2-Nter as a positive control. For binding experiments, the cytosolic proteins from HEK293
cells transfected with G
q were incubated with
agarose-conjugated GST-GRK-Nter fusion proteins. Unbound proteins were
removed by extensive washing, and G
q bound to
GST-GRK-Nter proteins was revealed by immunoblot. According to previous
findings (12), when the incubation was done in the presence of
AlF
q was in the
active state), a substantial fraction of G
q was bound to
GST-GRK2-Nter, whereas in the absence of the AlF
q in the inactive state), the G
q
bound to GST-GRK2-Nter was undetectable (Fig. 1B). When similar experiments were done using GST-GRK4-Nter, the amount of
G
q interacting with this domain was significantly lower
even when G
q was in the active state (Fig.
1B). The amount of the G
q bound to GRK4-Nter
(in the presence of AlF
q in
cells, we investigated the interaction of G
q and GRKs by
co-immunoprecipitation in transfected HEK293 cells. We used both the
wild type G
q or the constitutively active mutant
G
q(Q209L), which can bind in vitro to the
GRK2-Nter even in the absence of AlF
q was immunoprecipitated from cells transfected with
GRK2 or GRK4 plus G
q or G
q(Q209L), and
the presence of GRK subtypes in the immunoprecipitates was assessed by
immunoblot (Fig. 1C). GRK2 was co-immunoprecipitated in an
agonist-dependent manner from cells expressing
G
q, showing that GRK2 and G
q interact in
intact cells and that this binding depends on the active state of
G
q. In cells expressing G
q(Q209L), GRK2
was co-immunoprecipitated even in the absence of agonist, although we
consistently found that the amount of GRK2 co-immunoprecipitated with
G
q(Q209L) was enhanced by quisqualate treatment. This
indicates that the activation of the mGlu1 receptor by
agonist may favor the interaction between GRK2 and G
q in
intact cells, perhaps by modulating the active state of GRK2, which, in
turn, could govern the interaction with G
q. This
hypothesis is consistent with our previous finding showing that the
presence of an agonist-stimulated receptor increased the ability of
GRK2-Nter to inhibit G
q-stimulated IP production (Fig. 4 of Ref. 12). By contrast, GRK4 was never co-immunoprecipitated with
G
q, indicating that this kinase does not interact with
G
q. The levels of expression of GRKs (Fig. 1C), G
q, and G
q(Q209L) (data
not shown) were comparable in different samples.
-arrestin was not
co-immunoprecipitated with GRK2 or with GRK4 in either the presence or
absence of agonist stimulation (not shown).
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Fig. 2.
GRK4-dependent internalization of
mGlu1a receptor. Upper panel,
HEK293 cells transfected with the mGlu1a receptor and
co-transfected with mock (ctrl), GRK2 or GRK4 were untreated
(basal) or treated with quisqualate (100 µM) for 5 min,
and the receptor internalization was assessed by confocal microscopy
analysis. Lower panel, time course of the
mGlu1a receptor internalization in cells transfected with
GRK4 (squares) or GRK2 (triangles) and treated
with quisqualate for the indicated times. Data (means ± S.E.,
n = 15) are the ratio of cytosolic versus
membrane receptor immunofluorescence. Membrane immunofluorescence is
taken as 100%.
-Arrestin
during Agonist-promoted Endocytosis--
We analyzed the intracellular
localization of the transfected mGlu1 receptor and
endogenous
-arrestin in HEK293 cells at various times after agonist
treatment (Fig. 3). In unstimulated cells
-arrestin distribution is largely diffused in the cytosol, whereas
after 2 min agonist stimulation we observed the redistribution of
-arrestin to the plasma membrane, where
-arrestin is
co-localized, at least in part, with the mGlu1 receptor
(Fig. 3f). After 5 min of exposure to quisqualate, both
mGlu1 receptor and
-arrestin are mostly found in
intracellular compartments, but they are localized in distinct
intracellular vesicles (Fig. 3i). After 5 min of quisqualate treatment, 26 ± 5% of the mGlu1 receptor and 22 ± 4% of
-arrestin staining were reciprocally co-localized
(n = 20). This finding indicates that mGlu1
receptor and
-arrestin are not physically bound during
agonist-promoted internalization, suggesting that the mGlu1
receptor is internalized by a
-arrestin-independent mechanism. To
test this possibility, we used the
-arrestin 1 dominant-negative
mutant V53D (
arrV53D), which can block the
-arrestin-dependent internalization of several GPCRs
(16). The agonist-stimulated internalization of the mGlu1a
was not affected by the co-expression of
arrV53D, confirming that
this receptor is internalized by a
-arrestin-independent mechanism
(Fig. 4). After agonist treatment the
amount of mGlu1 receptor intracellular immunofluorescence
(relative to that present on the plasma membranes) was 512 ± 9%
in control cells and it was 430 ± 7% in cells expressing the
arrV53D (n = 50). The PAFr, which is internalized by
a
-arrestin-dependent mechanism (17), was used as
positive control in parallel experiments. As expected we found that,
following agonist treatment, PAFr is internalized and is co-localized
with
-arrestin (not shown) and that the agonist-induced PAFr
internalization was prevented by the co-expression of
arrV53D (Fig.
4). After agonist treatment the amount of PAFr intracellular
immunofluorescence (relative to that present on the plasma membranes)
was 728 ± 4% in control cells and it was 125 ± 6% in
cells expressing the
arrV53D (n = 20).
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Fig. 3.
Agonist-induced redistribution of the
mGlu1a receptor and -arrestin in
HEK293 cells. HEK293 cells transfected with mGlu1a
receptor and treated with vehicle (a-c) or with
100 µM quisqualate for 2 min (d-f)
or for 5 min (g-i) were double-stained with
anti-mGlu1 and anti-
-arrestin antibodies for
immunofluorescence confocal microscopy analysis. The distributions of
the mGlu1a receptor (in red; a,
d, and g) and of
-arrestin (in
green; b, e, and h) are
shown in a single-channel image. The overlay is shown in c,
f, and i, and co-localization is in
yellow (f). Scale bar, 12 µm.
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Fig. 4.
Effect of arrV53D on
mGlu1a and PAF receptor internalization and
-arrestin redistribution. HEK293 cells
transfected with plasmid DNA encoding mGlu1a or PAF
receptor along with the empty vector or the
arrV53D (3 µg/dish)
(+
arr dn) were treated with agonist. The distribution of
mGlu1a and PAF receptor and
-arrestin was determined by
immunofluorescence confocal microscopy. Scale
bar, 7 µm for mGlu1a and PAF receptor and 15 µm for
-arrestin. These experiments are representative of three
similar ones.
arrV53D on the agonist-promoted
redistribution of endogenous
-arrestin. Although the quisqualate treatment for 5 min induced the internalization of
-arrestin (Figs.
3 and 4), in cells transfected with
arrV53D after 5 min of agonist
treatment, the
-arrestin immunofluorescence was predominantly localized to the plasma membranes (Fig. 4). In cells transfected with
arrV53D, after agonist treatment the amount of
-arrestin intracellular immunofluorescence (relative to that present on the
plasma membranes) was 77 ± 14% (n = 29). This
suggests that the
arrV53D could prevent the internalization of
endogenous
-arrestin.
arrV53D--
The experiments reported so far show that, in cells
expressing the mGlu1a receptor, the exposure to agonist
promotes the internalization of the receptor and induces the
redistribution of
-arrestin in distinct intracellular compartments.
We sought to examine whether the agonist-induced
-arrestin
internalization could represent one signaling step for
receptor-stimulated cellular response. We focused on MAPK activation
because it was previously shown that many GPCRs can activate MAPK and
that receptor and
-arrestin internalization is a key step toward
this signaling cascade (18-20). In HEK293 cells expressing the
mGlu1a receptor, exposure to quisqualate stimulated MAPK
activation, as assessed by immunoblot using anti-phospho-ERK antibody,
with a peak observed after 5 min of treatment (Fig. 5). After exposure to quisqualate for 5 min, the level of phospho-ERK1/2 was 349 ± 33% as compared with
untreated cells (n = 9). When
arrV53D dominant
negative mutant was co-expressed, the agonist-stimulated MAPK
activation at 5 min of treatment was reduced by 78 ± 4%
(n = 4). The immunoblot with the F4C1 monoclonal
antibody, which recognizes an epitope common to all the arrestin
subtypes, confirmed the overexpression of the
arrV53D and showed
that
-arrestin 2, which is endogenously expressed in HEK293 cells,
was not affected by the transfection (Fig. 5).
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Fig. 5.
Stimulation of ERK1/2 phosphorylation by
mGlu1a receptor. Cells transfected with the
mGlu1a receptor and co-transfected with empty vector or
with arrV53D dominant negative mutant (+
arr dn) (3 µg/dish), were treated with 100 µM quisqualate for the
indicated times. The experiment shown represents the immunoblot using
anti-phospho-ERK1/2 (P-ERK1/2), anti-ERK1/2 or
F4C1 anti-arrestin (
arr1/2) antibodies
subsequently probed on the same membrane. This experiment is
representative of three similar ones.
arrV53D could result
in a complete blockade of quisqualate-induced MAPK activation, we
performed experiments using 10 µg of
arrV53D plasmid/dish to
transfected HEK293 cells (instead of 3 µg of plasmid/dish, used in
the previous experiments). In cells transfected with 10 µg of
arrV53D plasmid, agonist-stimulated MAPK activation was almost
completely prevented (Fig.
6B). In these cells, after 5 min of quisqualate, the level of phospho-ERK1/2 was 113 ± 14% (n = 4) of that found in untreated cells. By contrast
the higher levels of
arrV53D transfected did not inhibit the
agonist-stimulated mGlu1 receptor internalization (Fig.
6A). After agonist treatment the amount of mGlu1
receptor intracellular immunofluorescence was 425 ± 9% in
control cells and it was 448 ± 15% in cells transfected with 10 µg of
arrV53D (n = 10). By confocal microscopy
analysis, we could demonstrate in single cell that the overexpression
of
arrV53D prevents the agonist-dependent ERK1/2
activation without affecting mGlu1 receptor internalization
(Fig. 6A).
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Fig. 6.
A, overexpression of arrV53D inhibits
ERK1/2 phosphorylation but not receptor internalization. HEK293 cells
transfected with the mGlu1a receptor and GRK4 and
co-transfected with empty vector (a, b,
e, and f) or with
arrV53D (10 µg/dish)
(c, d, g, and h), were
untreated (a, c, e, and g)
or treated with 100 µM quisqualate for 5 min
(b, d, f, and h). The
distributions of the mGlu1a receptor (in red;
a-d) and of phospho-ERK1/2 (in green;
e-h) are shown. Scale bar, 5 µm.
B, immunoblot of phospho-ERK1/2 and total ERK1/2 in HEK293
cells transfected with the mGlu1a receptor and GRK4 and
co-transfected with empty vector or with the
arrV53D (+
arr
dn). C, HEK293 cells transfected with the
mGlu1a receptor and co-transfected with empty vector or
with GRK4 or GRK2 and stimulated with 100 µM quisqualate
for the indicated times, before phospho-ERK1/2 and total ERK1/2
immunoblot. The experiments are representative of three similar
ones.
-arrestin-dependent activation of MAPK. GRK2, which is
endogenously expressed in HEK293 cells, is the obvious candidate,
because the agonist-dependent phosphorylation of the
mGlu1 receptor by GRK2 has been documented (9). We
co-transfected mGlu1 receptor and GRK2 in HEK293 cells, and
we found that the quisqualate-promoted activation of MAPK was increased
(Fig. 6C). In parallel experiments quisqualate treatment (5 min) increased the level of phospho-ERK1/2 expression by 2.57 ± 0.08-fold in HEK293 cells transfected with the mGlu1
receptor alone and by 4.1 ± 0.18-fold in cells with GRK2
co-transfected. Overexpression of GRK4 did not increase
quisqualate-induced ERK1/2 phosphorylation (Fig. 6C).
-Arrestin
Redistribution in Cerebellar Purkinje Cells--
We used cerebellar
Purkinje cells primary culture to assess agonist-dependent
redistribution of the mGlu1 receptor and
-arrestin in
cells that natively express these proteins (Fig.
7). The mGlu1 receptor, which
is expressed at the cell surface in untreated Purkinje cells, is
internalized in intracellular vesicles after 5 min of exposure to
quisqualate.
-Arrestin, which is cytosolic under basal conditions,
is also rapidly redistributed after agonist exposure. In cells exposed
to quisqualate,
-arrestin was found in cytosolic vesicles different
from those where the mGlu1 receptor is internalized.
Quantitative analysis of different experiments confirmed that the
mGlu1 receptor and
-arrestin are not co-localized. Under
basal conditions 13 ± 4% of the mGlu1 receptor and
10 ± 4% of
-arrestin were reciprocally co-localized
(n = 20). After quisqualate treatment for 5 min,
22 ± 5% of the mGlu1 receptor and 26 ± 6% of
-arrestin staining were reciprocally co-localized (n = 20).
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Fig. 7.
Agonist-induced redistribution of the
mGlu1 receptor and -arrestin in
cerebellar Purkinje cells. Cultured rat cerebellar Purkinje cells
treated with vehicle (a-c) or with 100 µM
quisqualate for 5 min (d-f) were double-stained with
anti-mGlu1 receptor and anti-
-arrestin antibodies for
immunofluorescence confocal microscopy analysis. The distribution of
the mGlu1a receptor (in red; a and
d) and of
-arrestin (in green; b
and e) are shown in single-channel images. The overlay is
shown in c and f. Scale
bar, 7 µm.
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Fig. 8.
Agonist-stimulated MAPK activation in
cerebellar Purkinje cells. A, stimulation of ERK1/2
phosphorylation. Cultured rat cerebellar Purkinje cells were stained
with anti-phospho-ERK1/2 (a and b) or with
anti-ERK1/2 (c and d) antibodies for
immunofluorescence confocal microscopy analysis. Serum-starved cells
were treated with vehicle (a and c) or with 100 µM quisqualate for 5 min (b and d).
Scale bar, 10 µm. B, inhibition of
agonist-stimulated ERK1/2 phosphorylation by arrV53D. Cultured
cerebellar Purkinje cells infected with a recombinant adenovirus to
express GFP alone (e and g) or GFP and
arrV53D
(f and h) were treated with 100 µM
quisqualate for 5 min, fixed, and stained with anti-phospho-ERK1/2
antibody for immunofluorescence confocal microscopy analysis. The
expression of GFP (in green; e and f)
and of phospho-ERK1/2 (in red; g and
h) is shown. Scale bar, 20 µm. The
experiments are representative of three similar ones.
-Arrestin in the Agonist-stimulated
mGlu1 Receptor Internalization and MAPK Activation in
Cerebellar Purkinje Cells--
Our data in HEK293 cells indicate that
GRK4 and
-arrestin are major determinants in the mechanism of
agonist-stimulated mGlu1 receptor internalization and MAPK
activation, respectively. We assessed whether these mechanisms are also
important in cells that natively express the mGlu1 receptor
and these regulatory proteins.
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Fig. 9.
GRK4 knock-down inhibits receptor
internalization but not ERK1/2 phosphorylation in cerebellar Purkinje
cells. Cerebellar Purkinje cells treated with the scrambled
oligonucleotide (a, b, e, and
f) or with the GRK4 antisense oligonucleotide (c,
d, g, and h) were untreated
(a, c, e, and g) or treated
with 100 µM quisqualate for 5 min (b,
d, f, and h). The distribution of the
mGlu1a receptor (in red; a-d) and of
phospho-ERK1/2 (in green; e-h) are shown. The
arrows indicate the punctate receptor after agonist
treatment in scrambled oligonucleotide-treated cells. Scale
bar, 7 µm. The experiments are representative of three
similar ones.
-arrestin, we infected the primary
cultured cerebellar Purkinje cells using an adenoviral vector to
express the
arrV53D dominant negative (Fig. 8B). We used
this approach for the following reasons: (i) the attempt to knock down
-arrestin by the antisense treatment was unsuccessful; (ii) the
efficiency of plasmid chemical transfection in primary cultured neurons
is too low (<5%) to transfect cerebellar Purkinje cells, which in
primary cultures represent ~3-4% of the total cell population (8).
Using the adenoviral vector, we obtained a very high infection
efficiency (>85% of cerebellar Purkinje cells) as assessed by GFP,
which is expressed by infected cells. In cells infected with the
adenoviral vector without
arrV53D, exposure to quisqualate induced a
robust ERK1/2 phosphorylation (Fig. 8B, e and
g). This effect was substantially blunted in cells infected
with the adenoviral vector expressing
arrV53D (Fig. 8B,
f and h).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin, which is likely involved
in the mGlu1 receptor-mediated MAPK activation. Upon
agonist stimulation the
-arrestin and the mGluR1 were sorted to
different intracellular vesicles, and the expression of
arrV53D
dominant negative mutant significantly inhibited agonist-promoted MAPK
activation. Our results strongly indicate that
-arrestin is directly
involved in glutamate-stimulated MAPK activation by acting as a
signaling molecule.
q and supports the idea that only
GRK2 and GRK3 (members of the
ARK subfamily) possess a functional RGS-like domain, whereas the members of the GRK4 subfamily (namely GRK4, GRK5, and GRK6) do not interact with the G protein to regulate the signaling (12, 15). While this article was under revision, a paper
from Dhami et al. (24) was published, reporting that in
transfected HEK293 cells the mGlu1 receptor signaling is
regulated by GRK2 by a phosphorylation-independent mechanism. These
data are entirely consistent with the conclusions reached by our study.
-arrestin in receptor trafficking and signaling. In HEK293 cells,
upon agonist exposure
-arrestin is rapidly redistributed to plasma
membranes and then internalized in intracellular vesicles, which are
distinct from those where the receptor is internalized. The
agonist-stimulated differential sorting of the receptor and
-arrestin was also observed in cultured cerebellar Purkinje cells. The ability of an agonist to induce a differential sorting of the
receptor and
-arrestin was already reported for the 5-HT2A receptor,
which is internalized by a
-arrestin-independent mechanism (25).
Consistently we found that the
arrV53D dominant negative mutant did
not inhibit the agonist-stimulated mGlu1 receptor internalization.
-arrestin-dependent. In these studies the
-arrestin
dominant negative mutants used to inhibit the mGlu1 receptor internalization were different from the
arrV53D used in the
present study, and it should be emphasized that different
-arrestin
mutants may interfere differently with the ability of this scaffold
protein to interact with other proteins involved in receptor
trafficking and signaling (18). Interestingly a rapid
(t1/2 = 3.3 min) and extensive tonic internalization
of the mGlu1 receptor has been reported (11), which is not
blocked by
-arrestin dominant negative mutants. This process is
similar to that described in the present study. We suggest that, in the
presence of GRK4, the agonist can increase the rate of the "tonic
recycling" of the mGlu1 receptor.
-Arrestin was first identified as a key regulatory protein important
for the homologous desensitization and internalization of many GPCRs.
Later it was found that
-arrestin could also act as an adaptor
protein, which binds to the GRK-phosphorylated GPCR and, by interacting
with clathrin, promotes receptor redistribution and internalization in
clathrin-coated vesicles. More recently it has been shown that
-arrestin interacts with different proteins, which are either
involved in receptor translocation or in signal transduction events
downstream from the receptor (16, 26, 27). The involvement of
-arrestin in the ubiquitination and degradation of the
2-adrenergic receptor has also been demonstrated (28).
These findings indicate that
-arrestin, besides mediating GPCR
internalization, is involved in several GPCR-mediated signaling cascades. We found that
-arrestin is not directly involved in the
rapid agonist-induced internalization of the mGlu1 receptor, because
-arrestin is not physically associated to the mGlu1
receptor during the receptor internalization and the
arrV53D
dominant negative mutant did not prevent receptor internalization.
However, the exposure to mGlu1 receptor agonist induced a
rapid redistribution of
-arrestin to plasma membranes, where the
receptor and
-arrestin were co-localized. This was followed by
internalization of the receptor and
-arrestin in distinct
intracellular vesicles. We hypothesized that, under these conditions,
-arrestin may act as a signaling protein for intracellular
pathway(s) activated by receptor stimulation. Exposure to the
mGlu1 receptor agonist induces the phosphorylation and
activation of ERK1/2 in transfected cells (Ref. 21 and Fig. 5) and in
cerebellar Purkinje cells (Fig. 8). We therefore used
arrV53D to
assess whether
-arrestin is involved in the mechanism of the
mGlu1 receptor-mediated MAPK activation. A previous study
showed that this mutant was able to inhibit agonist-promoted
sequestration of the
2-AR and 5HT1A receptor
and the activation of MAPK mediated by these receptors (18). The
authors concluded that the
-arrestin-dependent
sequestration of a subset of GPCRs plays a role in the initiation of
mitogenic signals. The present study documents that the expression of
arrV53D did not alter the agonist-stimulated mGlu1
receptor sequestration but did inhibit the agonist-promoted MAPK
activation, indicating that the ability of the
-arrestin dominant
negative mutant to inhibit ERK1/2 phosphorylation is not the result of
the inhibition of receptor sequestration. We suggest that
arrV53D
prevents the interaction of the endogenous
-arrestin with protein(s)
involved in the formation of the signaling complex that mediates the
activation of MAPK. The involvement of
-arrestin in
agonist-dependent MAP kinases activation was confirmed in
cerebellar Purkinje cells using an adenovirus vector to express
arrV53D.
-arrestin are
internalized in different intracellular compartments.
-arrestin, which works as a signaling protein for
receptor-stimulated MAPK activation. In cells that do not express GRK4,
GRK2, which is ubiquitous, is involved in agonist-induced rapid
activation of ERK1/2, G
q-mediated signaling
desensitization, and delayed receptor internalization. GRK2 likely acts
by phosphorylation-independent and/or by
phosphorylation-dependent mechanisms. In cells expressing GRK4 (such as cerebellar Purkinje cells), the
agonist-dependent receptor activation also induces the
rapid internalization of the receptor, which may reinforce the control
of the signaling and could activate other as yet unidentified
signaling pathway(s) requiring receptor internalization.
![]() |
FOOTNOTES |
---|
* This work was supported by Telethon-Italy Grant 1238.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.
¶ Both authors contributed equally to this work.
To whom correspondence should be addressed: INM Neuromed,
IRCCS, località Camerelle, 86077 Pozzilli (IS), Italy.
Tel.: 39-0865-915-241; Fax: 39-0865-927-575; E-mail:
deblasi@neuromed.it.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M203992200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
mGlu, metabotropic
glutamate;
GPCR, G protein-coupled receptor;
G protein, GTP-binding
protein;
GRK, G protein-coupled receptor kinase;
GRK-Nter, the
N-terminal domain of GRK;
IP, inositol phosphate;
GFP, green
fluorescent protein;
G, the
subunit of G protein;
ERK, extracellular signal-regulated kinase;
MAP, mitogen-activated
protein;
MAPK, mitogen-activated protein kinase;
RGS, regulators of G
protein signaling;
GST, glutathione S-transferase;
PBS, phosphate-buffered saline;
BSA, bovine serum albumin;
PAF, platelet-activating factor;
PAFr, PAF receptor.
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REFERENCES |
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