From the Departments of Medicine,
§ Biochemistry and ¶ Cell Biology,
The Howard
Hughes Medical Institute, Duke University Medical Center, Durham,
North Carolina 27710 and
The Geriatrics Research,
Education and Clinical Center, Durham Veterans Affairs Medical Center,
Durham, North Carolina 27705
Received for publication, December 2, 2002
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ABSTRACT |
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By binding to agonist-activated G
protein-coupled receptors (GPCRs), Homologous desensitization of most heptahelical, or G
protein-coupled, receptors
(GPCRs)1 results from the
physical uncoupling of receptor and G protein as a consequence of
arrestin binding. Agonist-occupied GPCRs are rapidly phosphorylated by
specialized G protein-coupled receptor kinases (GRKs). Subsequent high
affinity binding of arrestin to the GRK-phosphorylated receptor results
in steric inhibition of receptor-G protein coupling. In addition, the
two nonvisual arrestins, Data obtained using green fluorescent protein (GFP)-tagged
Recent data from yeast two-hybrid screens and from biochemical
characterization of receptor- Thus, the Materials--
LipofectAMINE was from Invitrogen. FuGENE
6 was from Roche Molecular Diagnostics. Monoclonal M2 anti-FLAG
affinity agarose was from Sigma. Monoclonal anti-hemagglutinin (HA)
affinity agarose was from Covance. Anti-phospho-ERK1/2 antibody was
from Cell Signaling and anti-ERK1/2 antibody was from Upstate
Biotechnology. Goat polyclonal anti-lamin B antibody, rabbit polyclonal
anti-actin, anti-retinoblastoma protein, anti-HA, and anti-FLAG
antibodies were from Santa Cruz Biotechnology. Rhodamine-conjugated
monoclonal anti-HA antiserum was from Molecular Probes. Horseradish
peroxidase-conjugated donkey anti-rabbit antibody was from Amersham
Biosciences. Horseradish peroxidase-conjugated rabbit anti-goat
antibody was from Sigma. Rabbit polyclonal anti- cDNA Constructs--
PCDNA3.1 expression plasmids
encoding GFP-tagged Cell Culture and Transfection--
HEK-293 cells and COS-7 cells
were obtained from the American Type Culture Collection. HEK-293 cells
stably expressing 1.5-2 pmol/mg of the HA-V2R or HA-V2 Confocal Microscopy--
HEK-293 cells were used for confocal
microscopy because of their favorable morphology for comparing the
cytoplasmic and plasma membrane distribution of proteins. Subconfluent
monolayers in 100-mm dishes were transfected with expression plasmids
encoding HA-AT1aR, HA-V2R, HA- Immunoprecipitation and Immunoblotting--
Immunoprecipitation
of FLAG epitope-tagged
For experiments involving the visualization of covalently cross-linked
receptor-
Immunoblotting of FLAG- Quantitation of Nuclear Phospho-ERK1/2--
For the
separation of nuclear and extranuclear pools of endogenous ERK1/2,
COS-7 cells on 100-mm plates were transfected with plasmids encoding
either the HA-V2R or HA-V2 Elk-1 Luciferase Reporter
Assays--
ERK1/2-dependent transcription was measured
using an Elk-1 driven luciferase reporter system as described
previously (12). Briefly, HEK-293 cells stably expressing either HA-V2R
or HA-V2 [3H]Thymidine Incorporation--
HEK-293 cells
stably expressing either V2R or V2 The Stability of the GPCR-
As shown in Fig. 1C, a markedly different pattern was
observed for the AT1aR and V2R. With these receptors, 15 min of agonist exposure led to removal of most of the HA-rhodamine-labeled receptors from the plasma membrane and their accumulation in large endosomal vesicles (left panels, red). An overlapping pattern of
redistribution of GFP-
In overexpression studies, we have previously demonstrated that
To determine whether the stability of the receptor-
As shown in Fig. 2A, 5 min
stimulation of AT1aR, V2R, and
The AT1aR and The GPCR C-terminal Tail Regulates the Activation of
If the ability of a GPCR to activate
Fig. 4C depicts the results of an analogous experiment
performed with the
To address this question, we first analyzed the distribution of
endogenous phospho-ERK1/2 in whole cell lysates and receptor immunoprecipitates following covalent cross-linking with the
membrane-permeable reversible cross-linker, dithiobis(succinimidyl
propionate). COS-7 cells transiently expressing HA-tagged V2 or V2
To further examine the effect of
These data are also consistent with comparisons of the fraction
of The Transcriptional Activity of GPCR-activated ERK1/2 Is
Regulated by the Stability of the Receptor-
Fig. 7A depicts the time
course of vasopressin-stimulated ERK1/2 phosphorylation over 4 h
in the V2R- and V2
Compared with the V2R, activation of V2 The binding of Activation of the ERK cascade by heptahelical receptors is a complex
process that can simultaneously involve multiple mechanisms, and in
which the predominant mechanism varies depending on both the GPCR and
the cellular context in which the receptor is expressed (22, 23). For
example, in S49 lymphoma cells, Given their ability both to dampen receptor-G protein coupling and to
act as scaffolds for ERK activation, Fig. 9 depicts this conceptual role of
-arrestins mediate homologous
receptor desensitization and endocytosis via clathrin-coated pits.
Recent data suggest that
-arrestins also contribute to GPCR
signaling by acting as scaffolds for components of the ERK
mitogen-activated protein kinase cascade. Because of these dual
functions, we hypothesized that the stability of the
receptor-
-arrestin interaction might affect the mechanism and
functional consequences of GPCR-stimulated ERK activation. In
transfected COS-7 cells, we found that angiotensin AT1a and vasopressin
V2 receptors, which form stable receptor-
-arrestin complexes,
activated a
-arrestin-bound pool of ERK2 more efficiently than
1b
and
2 adrenergic receptors, which form transient
receptor-
-arrestin complexes. We next studied chimeric receptors in
which the pattern of
-arrestin binding was reversed by exchanging
the C-terminal tails of the
2 and V2 receptors. The ability of the
V2
2 and
2V2 chimeras to activate
-arrestin-bound ERK2
corresponded to the pattern of
-arrestin binding, suggesting that
the stability of the receptor-
-arrestin complex determined the
mechanism of ERK2 activation. Analysis of covalently cross-linked
detergent lysates and cellular fractionation revealed that wild type V2 receptors generated a larger pool of cytosolic phospho-ERK1/2 and less
nuclear phospho-ERK1/2 than the chimeric V2
2 receptor, consistent
with the cytosolic retention of
-arrestin-bound ERK. In stably
transfected HEK-293 cells, the V2
2 receptor increased ERK1/2-mediated, Elk-1-driven transcription of a luciferase reporter to
a greater extent than the wild type V2 receptor. Furthermore, the
V2
2, but not the V2 receptor, was capable of eliciting a mitogenic
response. These data suggest that the C-terminal tail of a GPCR, by
determining the stability of the receptor-
-arrestin complex,
controls the extent of
-arrestin-bound ERK activation, and
influences both the subcellular localization of activated ERK and the
physiologic consequences of ERK activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin 1 and
-arrestin 2, function as
adapter proteins, binding to clathrin and the
2 adaptin subunit of
the AP-2 complex, and leading to targeting of GPCRs to clathrin-coated
pits where they are internalized (1, 2).
-arrestins and epitope-tagged GPCRs to visualize
-arrestin and receptor trafficking in live cells have demonstrated that most GPCRs
exhibit one of two characteristic patterns of agonist-induced
-arrestin interaction that allow them to be separated into distinct classes (3). One class, represented by the
2 and
1B adrenergic receptors (ARs), and the µ opioid, endothelin A, and dopamine D1A
receptors, binds to
-arrestin 2 with higher affinity than
-arrestin 1. For these receptors, the interaction with
-arrestin is transient.
-Arrestin is recruited to the receptor at the plasma membrane and translocates with it to clathrin-coated pits. Upon internalization of the receptor, the receptor-
-arrestin complex dissociates, such that, as the receptor proceeds into an endosomal pool, the
-arrestin recycles to the cytosol. The second class, represented by the angiotensin AT1a receptor (AT1aR), vasopressin V2
receptor (V2R), and the neurotensin 1, thyrotropin-releasing hormone,
and neurokinin NK-1 receptors, binds
-arrestin 1 and
-arrestin 2 with equal affinity. These receptors form stable complexes with
-arrestin, such that the receptor-
-arrestin complex internalizes
as a unit that is targeted to endosomes. The stability of the
receptor-
-arrestin interaction influences the fate of the
internalized GPCR. The
2AR, which binds
-arrestin transiently, is
rapidly dephosphorylated and recycled to the plasma membrane, whereas
the V2R, which binds
-arrestin stably, recycles slowly. Exchanging
the C-terminal tails of these two receptors not only reverses the
pattern of
-arrestin binding, but also reverses the pattern of
receptor dephosphorylation and recycling (4).
-arrestin complexes have indicated that
-arrestins also have the ability to interact with proteins involved
in signal transduction, suggesting that they may additionally function
in the recruitment of signaling proteins to GPCRs (5-7). One
potentially significant set of interactions is the binding of
-arrestins to components of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinase 3 (JNK3) mitogen-activated protein kinase cascades, which allows them to act as
scaffolds for localized mitogen-activated protein kinase activation (8-12). In KNRK cells, stimulation of the
protease-activated receptor type 2 (PAR2) induces the assembly of
multiprotein complexes that contain the internalized receptor,
-arrestin 1, Raf-1, and activated ERK1/2 (9). The formation of these
complexes, which are sufficiently stable that they can be isolated by
both gel filtration and immunoprecipitation, also affects the cellular distribution of activated ERK. Because
-arrestins are cytosolic proteins, the formation of stable complexes between
-arrestin and
ERK leads to cytosolic retention of ERK activated by PAR2 receptors.
Qualitatively similar results have been obtained for the AT1aR
expressed in HEK-293 and COS-7 cells (11, 12). AT1aR activation causes
the formation of complexes containing the receptor,
-arrestin 2, and
the component kinases of the ERK cascade: cRaf-1, MEK1, and ERK2. Upon
receptor internalization, activated ERK2 appears in the same endosomal
vesicles that contain AT1aR-
-arrestin complexes.
-arrestins appear to play a dual role in GPCR signaling.
They serve both to terminate G protein-dependent signals by
precluding receptor-G protein coupling, and to confer novel signaling
properties upon the receptor by acting as adapters or scaffolds for
signaling proteins. Because of these divergent functions, we
hypothesized that the stability of the receptor-
-arrestin interaction might be a significant factor in determining both the
mechanism of ERK activation employed by a GPCR and the functional consequences of ERK activation within the cell. To test this
hypothesis, we have compared the relative efficiency with which GPCRs
that form transient receptor-
-arrestin complexes (
1bAR and
2AR), and GPCRs that form stable receptor-
-arrestin complexes
(AT1aR and V2R), activate a
-arrestin-bound pool of ERK. We also
compared the signaling properties of chimeric receptors, in which the
C-terminal tail domains have been exchanged so as to alter the
stability of the receptor-
-arrestin interaction (V2
2R and
2V2R). We tested whether these chimeric receptors exhibited altered
activation of
-arrestin-bound ERK, and whether changing the
receptor-
-arrestin interaction altered the cellular distribution of
activated ERK, the ability of ERK to activate transcription of an Elk-1
reporter, and the ability of the receptor to produce a mitogenic
response. We find that the formation of stable receptor-
-arrestin
complexes is associated with enhanced activation of
-arrestin-bound
ERK, cytosolic retention of active ERK, and a reduced transcriptional and mitogenic response to GPCR stimulation. Moreover, this phenotype can be reversed by exchange of the C-terminal tail domain. These data
suggest that the stability of the GPCR-
-arrestin interaction dictates the predominant mechanism of ERK activation and, thereby, the
functional consequences of ERK activation following GPCR stimulation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin was
prepared in the Lefkowitz laboratory.
-arrestin 2, and HA-tagged AT1aR, V2R,
1bAR,
and
2AR, and the chimeric V2
2R and
2V2R were prepared in the
Caron laboratory, as previously described (3, 4). The V2
2R chimera
contains the first 342 amino acids of the V2R (Met-1 to Cys-342) fused
to the last 72 amino acids of the
2AR (Leu-342 to Leu-413), whereas
the
2V2R chimera contains the first 341 amino acids of the
2AR
(Met-1 to Cys-341) fused to the last 29 amino acids of the V2R (Ala-343 to Ser-371). The pcDNA3.1 expression plasmid encoding FLAG
epitope-tagged
-arrestin 2 was created in the Lefkowitz laboratory.
The pEGFP-N1 expression plasmid encoding GFP-ERK2 (9) was provided
by K. A. DeFea and N. Bunnett (University of California, San
Francisco). The pFR-Luc, GAL4-Elk-1, and pRL-tk-luc reporter
plasmids were from Stratagene.
2R were
generated by standard procedures using zeocin selection (0.4 mg/ml).
HEK-293 cells were grown in Eagle's minimal essential medium with
Earle's salt supplemented with 10% fetal bovine serum and 100 µg/ml gentamicin. COS-7 cells were grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum and 100 µg/ml gentamicin. Transient transfection of HEK-293 cells was
performed using FuGENE 6 according to the manufacturers instructions.
Transient transfection of COS-7 cells was performed using LipofectAMINE
as previously described (12). Transfected cells were incubated
overnight in serum-free growth medium supplemented with 10 mM HEPES, pH 7.4, 0.1% bovine serum albumin, and 100 µg/ml gentamicin prior to stimulation.
1bAR, HA-
2AR, HA-V2
2R, or
HA-
2V2R (8 µg/plate) along with GFP-
-arrestin 2 (2 µg/plate).
Twenty-four hours after transfection, cells were passed onto
collagen-coated 35-mm glass bottom dishes and serum-starved overnight.
For visualization of HA epitope-tagged receptors, cell surface
receptors were stained using a 1:500 dilution of rhodamine-conjugated
monoclonal anti-HA IgG in serum starving medium for 1 h at
37 °C. Cells were then washed with serum starving medium, stimulated
as described in figure legends, washed with phosphate-buffered saline,
fixed with 10% paraformaldehyde for 30 min at room temperature, and
again washed prior to examination. Confocal microscopy was performed using a Zeiss LSM510 laser scanning microscope using a Zeiss 63 × 1.4 numerical aperture water immersion lens with dual line switching excitation (488 nm for GFP, 568 nm for rhodamine) and emission (515-540 nm for GFP, 590-610 nm for rhodamine) filter sets.
-arrestin 2 was performed following transient
transfection of COS-7 cells in 100-mm dishes. Cells were transfected
with expression plasmids encoding HA-AT1aR, HA-V2R, HA-
1bAR,
HA-
2AR, HA-V2
2AR, or HA-
2V2AR (2 µg/plate) and GFP-ERK2 (1 µg/plate) with or without plasmid encoding FLAG-
-arrestin 2 (3 µg/plate), as indicated. Stimulation was performed as described in
the figure legends. After stimulation, monolayers were washed with
phosphate-buffered saline, solubilized in 0.8 ml of glycerol lysis
buffer (50 mM Hepes, 50 mM NaCl, 10% (v/v)
glycerol, 0.5% (v/v) Nonidet P-40, 2 mM EDTA, 100 µM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 4 µg/ml leupeptin, 2.5 µg/ml aprotinin), and clarified by centrifugation. For determination of
protein expression and total cellular phospho-ERK1/2, a 50-µl aliquot
of each clarified whole cell lysate was removed and mixed with an equal
volume of 2× Laemmli sample buffer. Approximately 50 µg of protein
from each lysate was resolved by SDS-PAGE and transferred to
polyvinylidene difluoride membrane for immunoblotting. To isolate
-arrestin-bound GFP-ERK2, bovine serum albumin was added to each
lysate to a final concentration of 1%, and immunoprecipitation was
performed using 20 µl of 50% slurry of monoclonal M2 anti-FLAG affinity agarose, with constant agitation overnight at 4 °C. Immune complexes were washed three times with glycerol lysis buffer and boiled
in Laemmli sample buffer. Immunoprecipitated proteins were resolved by
SDS-PAGE and transferred to polyvinylidene difluoride membrane for immunoblotting.
-arrestin-ERK complexes, COS-7 cells in 100-mm dishes were
transfected with expression plasmids encoding HA-V2R or HA-
2V2AR (6 µg/plate). Stimulations were performed at 37 °C in 4.6 ml of
Dulbecco's phosphate-buffered saline. Incubations were terminated by
the addition of 0.4 ml of 25 mM
dithiobis(succinimidylpropionate) in dimethyl sulfoxide. Monolayers
were agitated gently for 30 min at room temperature, washed with
Dulbecco's phosphate-buffered saline containing 50 mM
Tris-HCl, pH 7.4, to neutralize unreacted dithiobis(succinimidyl
propionate), and lysed in 0.5 ml of glycerol lysis buffer. 25-µl
aliquots of clarified cross-linked detergent lysates were mixed with an
equal volume of 2× Laemmli sample buffer with or without
-mercaptoethanol, for electrophoresis under reducing or nonreducing
conditions, respectively. The remainder of each lysate was agitated
overnight at 4 °C with 20 µl of 50% slurry of monoclonal anti-HA
affinity agarose to immunoprecipitate HA-epitope-tagged receptor and
any cross-linked proteins. After washing, immunoprecipitates were
resolved by SDS-PAGE under reducing conditions and subjected to immunoblotting.
-arrestin 2 was performed using rabbit
polyclonal anti-FLAG IgG. Immunoblotting of endogenous
-arrestins was performed using rabbit polyclonal anti-
-arrestin. Endogenous ERK1/2 and phospho-ERK1/2, and GFP-ERK2 and phospho-GFP-ERK2 were detected using polyclonal anti-ERK1/2 and anti-phospho-ERK1/2 IgG.
Horseradish peroxidase-conjugated polyclonal donkey anti-rabbit IgG was
employed as secondary antibody. Immune complexes on polyvinylidene difluoride filters were visualized by enzyme-linked chemiluminescence using the SuperSignal chemiluminescence reagent and immunoblots were
quantified using a Fluor-S MultiImager.
2R (2 µg/plate) and FLAG-
-arrestin 2 (0.5 µg/plate). Serum-starved cells were stimulated with vasopressin
(1 µM) for 5 min. Monolayers were washed twice with
ice-cold phosphate-buffered saline and collected in 2 ml of hypotonic
lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM
NaCl, 3 mM MgCl, 0.3% (v/v) Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml
aprotinin). Cells were incubated on ice for 3 min to allow lysis, and
an aliquot of the lysed cell suspension was removed prior to
centrifugation for measurement of total phospho-ERK1/2. Lysates were
then centrifugation at 500 × g for 5 min to pellet
nuclei. The supernatants, containing a mixture of plasma membrane,
microsomal vesicles, cytoskeleton, and cytosol, represented the
extranuclear fraction. Pellets containing cell nuclei were washed in
lysis buffer without Nonidet P-40 and again pelleted at 500 × g. Both the extranuclear and nuclear fractions were
solubilized in 2× Laemmli sample buffer and phospho-ERK1/2 was
determined by protein immunoblotting. The purity of each fraction was
verified by immunoblotting with antibodies to nuclear lamin B,
retinoblastoma protein, and actin as described previously (12).
2R receptors were plated in 100-mm dishes and transfected
with GAL4-Elk-1 (1 ng/plate), pFR-luc (1 µg/plate), and pRL-tk-luc
(20 ng/plate). The GAL4-Elk-1 plasmid encodes a fusion protein
containing the GAL4 DNA binding domain and the transactivation domain
of Elk-1, pFR-luc encodes the firefly luciferase gene under the control of the GAL4 DNA binding element, and pRL-tk-luc encodes
Renilla luciferase under the control of the thymidine kinase
promoter. One day following transfection, the cells were passed into
6-well dishes and serum-starved overnight. Stimulation with vasopressin (1 µM) was carried out for 6 h. Luciferase
activities were determined using a dual luciferase assay kit (Promega).
Cells were extracted and assayed sequentially for firefly and
Renilla luciferase activities. 5-µl aliquots of cell
lysate were incubated with 50 µl of luciferin reagent and
luminescence was recorded for 5 s. Stop and GloTM
reagent (50 µl) was added and the specific luminescence from Renilla luciferase was recorded for an additional 5 s.
Firefly activities were normalized to Renilla luciferase activity.
2R in 24-well plates were grown to
subconfluence and serum-starved overnight. Subsequently, cells were
incubated with either vasopressin (1 µM) or 2-10% fetal
calf serum for 24 h at 37 °C. During last 4 h of
incubation, [3H]thymidine (0.5 µCi/well) was added.
Cells were washed, DNA was obtained by solubilization in 2 M guanidine in 50% phenol, and incorporated
[3H]thymidine was measured by scintillation counting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Arrestin Complex Determines the
Extent of Activation of a
-Arrestin-bound Pool of ERK--
Oakley
et al. (3) have shown that most GPCRs exhibit one of two
patterns of receptor-
-arrestin interaction. Fig.
1 illustrates each of these patterns,
using anti-HA rhodamine staining and GFP-
-arrestin 2 to track the
cellular localization of an HA epitope-tagged receptor and
-arrestin, respectively. As shown for the HA-
2AR in Fig. 1A, in the absence of agonist, rhodamine-stained receptors
(left panel, red) were confined to the plasma membrane,
whereas GFP-
-arrestin 2 (middle panel, green) was
diffusely cytosolic in distribution. An indistinguishable pattern of
receptor and
-arrestin distribution was observed in unstimulated
cells expressing the
1bAR, AT1aR, and V2R (data not shown). As shown
in Fig. 1B, 15 min of agonist treatment led to the
coalescence of both the
2AR and
1bAR (left panels,
red) and GFP-
-arrestin 2 (middle panels, green) in
small puncta along the plasma membrane. This redistribution reflects the recruitment of
-arrestin to agonist-occupied GPCR and the accumulation of GPCR-
-arrestin complexes in clathrin-coated pits or
nascent endosomes. Internalized receptors, represented by the intracellular accumulation of HA-rhodamine, did not colocalize with
GFP-
-arrestin 2 (right panels), suggesting that the
receptor-
-arrestin complex dissociates at or near the plasma
membrane following receptor internalization.
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Fig. 1.
The pattern of
GFP- -arrestin 2 binding defines two
classes of GPCR. A, distribution of
HA-rhodamine-stained
2AR and GFP-
-arrestin 2 in nonstimulated
HEK-293 cells. HEK-293 cells were transiently transfected with plasmids
encoding HA-
2AR and GFP-
-arrestin 2 as described under
"Experimental Procedures." The HA epitope on the cell surface was
labeled using rhodamine-conjugated anti-HA monoclonal IgG, and
unstimulated cells were fixed in paraformaldehyde and examined by
confocal immunofluorescence microscopy. B, formation of
transient membrane-associated GPCR-
-arrestin complexes. Cells were
transiently transfected with plasmids encoding GFP-
-arrestin 2, and
either HA-
2AR or HA-
1bAR. Cells were prestained with anti-HA
rhodamine, stimulated for 15 min with isoproterenol (1 µM) or phenylephrine (1 µM), respectively,
fixed with paraformaldehyde, and examined by confocal microscopy.
C, formation of stable GPCR-
-arrestin complexes that
colocalize in endosomes. Cells were transiently transfected with
plasmids encoding GFP-
-arrestin 2, and either HA-AT1aR or HA-V2R.
Cells were prestained with anti-HA rhodamine, stimulated for 15 min
with angiotensin II (1 µM) or arginine-vasopressin (1 µM), respectively, fixed with paraformaldehyde, and
examined by confocal microscopy. In each image, the distribution of
rhodamine-labeled HA receptors (red) and GFP-
-arrestin 2 (green) are shown in the single channel images.
Colocalization of HA receptors and GFP-
-arrestin 2 is shown in the
overlay images (yellow).
-arrestin 2 was observed (middle panels,
green; right panels, yellow), indicative of the formation of
stable receptor-
-arrestin complexes that remain associated
throughout receptor endocytosis and sorting.
-arrestins coprecipitate with cRaf-1, MEK1, and ERK2, and that
overexpression of cRaf-1 or stimulation of AT1aRs increases the
phosphorylation of a
-arrestin-bound pool of ERK2 (10, 11),
suggesting that
-arrestins can function as scaffolds for the
component kinases of the ERK1/2 cascade. We have also found that
overexpression of
-arrestin promotes the targeting of endogenous phospho-ERK1/2, along with the AT1aR, to endosomal vesicles and away
from the nucleus, resulting in a smaller nuclear pool of activated
ERK1/2 and a reduction in the Elk-1-driven transcriptional response to
angiotensin II stimulation (12). Similar results have been reported for
the PAR2 (9). However, both the AT1aR and PAR2 form stable
receptor-
-arrestin complexes that remain associated following
internalization of the receptor. Because GPCRs differ in their ability
to form stable complexes with
-arrestins, we hypothesized that they
might also differ in the extent to which they activated
-arrestin-bound ERK.
-arrestin
interaction affects the ability of GPCRs to activate a
-arrestin-bound pool of ERK, COS-7 cells were transfected with HA
epitope-tagged receptor, FLAG epitope-tagged
-arrestin 2, and
GFP-tagged ERK2, and the phosphorylation state of GFP-ERK2 in
FLAG-
-arrestin 2 immunoprecipitates was determined before and after
agonist stimulation. GFP-ERK2 was employed in these studies to
facilitate quantitation of ERK phosphorylation in the transfected cell
pool, because its slower electrophoretic mobility allows it to be
easily resolved from endogenous ERK1/2. GFP-ERK2 has previously been
shown to undergo agonist-stimulated phosphorylation and nuclear
translocation in a manner analogous to endogenous ERK (9).
1bAR expressed in COS-7 cells
produced equivalent levels of whole cell GFP-ERK2 phosphorylation
(top immunoblot). Under these conditions, FLAG-
-arrestin
2 and GFP-ERK2 were constitutively associated, as shown by their
coprecipitation in both the presence and absence of agonist treatment
(second and third immunoblots). The
-arrestin-bound pool of GFP-ERK2 was minimally phosphorylated in the
absence of agonist. In response to stimulation of AT1aR and V2R,
phosphorylation of
-arrestin-bound GFP-ERK2 increased substantially.
In contrast, stimulation of
1bAR had little effect on the
phosphorylation of
-arrestin-bound GFP-ERK2, despite a robust
increase in the level of phospho-GFP-ERK2 in the whole cell lysate
(bottom immunoblot). Fig. 2B, provides a
quantitative assessment of the relative extent of phosphorylation of
-arrestin-bound GFP-ERK2 following stimulation of each of the three
receptors. While no significant difference was observed between the
AT1aR and V2R, the
1bAR was 4-5-fold less efficient at activating
the FLAG-
-arrestin-bound pool of GFP-ERK2.
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Fig. 2.
GPCR-mediated phosphorylation of GFP-ERK2 in
whole cell lysates and
FLAG- -arrestin immunoprecipitates.
A, COS-7 cells were transfected with plasmids encoding
HA-AT1aR, HA-V2R, or HA-
1bAR along with GFP-ERK2 and either empty
vector or FLAG-
-arrestin 2 as indicated. Serum-starved cells were
incubated for 5 min in the presence or absence of angiotensin II
(Ang II, 1 µM), arginine-vasopressin
(Vaso, 1 µM), or phenylephrine
(Phe, 1 µM). Phosphorylation of GFP-ERK2 in
whole cell detergent lysates was determined by immunoblotting using
phospho-ERK1/2-specific IgG as described (upper
immunoblot). Anti-FLAG immunoprecipitates con- taining
FLAG-
-arrestin 2 were probed for FLAG-
-arrestin 2 (second
immunoblot), coprecipitated GFP-ERK2 (third
immunoblot), and phosphorylated GFP-ERK2 (lower
immunoblot). B, bar graph depicting the
amount of phosphorylated GFP-ERK2 present in FLAG-
-arrestin 2 immunoprecipitates following agonist treatment. In each experiment, the
amount phospho-GFP-ERK2 in the FLAG-
-arrestin 2 immunoprecipitate
was normalized to the amount of GFP-ERK2 present. Data are expressed as
a percentage of the
-arrestin 2-associated phospho-ERK signal
observed in angiotensin II-stimulated cells. Data shown represent the
mean ± S.E. from four separate experiments. *, less than AT1aR,
p < 0.05.
1bAR couple primarily to Gq/11 family
heterotrimeric G proteins to stimulate phosphatidylinositol hydrolysis (13), whereas the V2R couples primarily to the Gs-adenylyl
cyclase pathway (4). As shown in Fig. 1, the AT1aR and V2R form stable receptor-
-arrestin complexes, whereas the
1bAR interacts with
-arrestin transiently. Thus, the ability of these receptors to activate
-arrestin-bound ERK2 correlated with their ability to form
stable receptor-
-arrestin complexes, rather than with the activation
of a specific G protein pool.
-Arrestin-bound ERK--
The binding of
-arrestin to an
agonist-occupied GPCR is dependent upon GRK-mediated phosphorylation of
serine and threonine residues located within the C-terminal tail of the
receptor (1, 2). Previous studies have demonstrated that the structure
of the C-terminal tail is also the principal determinant of the
stability of the receptor-
-arrestin interaction (3, 4, 14). As shown
in Fig. 3A, a chimeric GPCR
composed of the V2R, a stable
-arrestin binder, substituted with the
C terminus of the
2AR, a transient
-arrestin binder (V2
2R),
exhibits transient
-arrestin binding like the
2AR. Instead of
accumulating in endosomes along with the receptor, GFP-
-arrestin 2 colocalizes with the V2
2R only at the plasma membrane. As shown in
Fig. 3B, the converse is true of a chimeric
2AR receptor
containing the V2R C terminus (
2V2R), where the characteristic
2AR pattern of receptor-
-arrestin complex formation only at the
plasma membrane is converted into one of colocalized receptor and
-arrestin in endosomes.
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Fig. 3.
Chimeric V2 2
and
2V2 receptors exhibit a reversal of the
pattern of receptor-
-arrestin
interaction. A, comparison of the pattern of
receptor-
-arrestin binding by wild type V2R (V2Rwt) and
the chimeric V2
2R. HEK-293 cells were transiently transfected with
plasmids encoding GFP-
-arrestin 2, and either HA-V2R or HA-V2
2R.
Cells were prestained with anti-HA rhodamine, stimulated for 15 min
with arginine-vasopressin (1 µM), fixed with
paraformaldehyde, and examined by confocal microscopy. Qualitatively
similar redistribution of receptors and
-arrestin 2 was observed in
COS-7 cells transiently expressing HA-V2R and HA-V2
2R and
GFP-
-arrestin 2 (data not shown). B, comparison of the
pattern of receptor-
-arrestin binding by wild type
2AR and the
chimeric
2V2R. HEK-293 cells were transiently transfected with
plasmids encoding GFP-
-arrestin 2, and either
2AR or
2V2R.
Cells were prestained with anti-HA rhodamine, stimulated for 15 min
with isoproterenol (1 µM), fixed with paraformaldehyde,
and examined by confocal microscopy. In each image, the distribution of
rhodamine-labeled HA receptors (red) and GFP-
-arrestin 2 (green) are shown in the single channel images.
Colocalization of HA receptors and GFP-
-arrestin 2 is shown in the
overlay images (yellow).
-arrestin-bound ERK correlates
with the formation of stable receptor-
-arrestin complexes, then one
might expect that exchanging the C terminus of receptors that differ in
their pattern of
-arrestin binding would reverse the pattern of ERK
activation. To test this hypothesis, we compared the ability of the V2R
and
2AR to activate
-arrestin-bound GFP-ERK2, with that of the
chimeric V2
2R and
2V2R. As shown in Fig.
4A, 5 min stimulation of COS-7
cells expressing V2R and V2
2R produced equivalent activation of
GFP-ERK2 measured in the whole cell lysate. However, when the
phosphorylation state of
-arrestin-bound GFP-ERK2 was compared, the
V2R and V2
2R differed significantly. As shown quantitatively in Fig.
4B, the V2R was about three times more effective than the
V2
2R at mediating the activation of
-arrestin-bound GFP-ERK.
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Fig. 4.
Effect of exchanging the C terminus of the
V2R and 2AR on GPCR-mediated phosphorylation
of GFP-ERK2 in whole cell lysates and
FLAG-
-arrestin immunoprecipitates.
A, COS-7 cells were transfected with plasmids encoding
either HA-V2R or HA-V2
2R along with GFP-ERK2 and either empty vector
or FLAG-
-arrestin 2 as indicated. Serum-starved cells were incubated
for 5 min in the presence or absence of arginine-vasopressin
(Vaso, 1 µM). Phosphorylation of GFP-ERK2 in
whole cell detergent lysates was determined as described (upper
immunoblot). Anti-FLAG immunoprecipitates containing
FLAG-
-arrestin 2 were probed for FLAG-
-arrestin 2 (second
immunoblot), coprecipitated GFP-ERK2 (third
immunoblot), and phosphorylated GFP-ERK2 (lower
immunoblot). B, bar graph depicting the
amount of phosphorylated GFP-ERK2 present in FLAG-
-arrestin 2 immunoprecipitates following stimulation of V2R and V2
2R.
C, cells were transfected with plasmids encoding either
HA-
2AR or HA-
2V2R along with GFP-ERK2 and either empty vector or
FLAG-
-arrestin 2 as indicated. Serum-starved cells were incubated
for 5 min in the presence or absence of isoproterenol (Iso,
1 µM). Phosphorylation of GFP-ERK2 in whole cell
detergent lysates was determined as described (upper
immunoblot). Anti-FLAG immunoprecipitates containing
FLAG-
-arrestin 2 were probed for FLAG-
-arrestin 2 (second
immunoblot), coprecipitated GFP-ERK2 (third
immunoblot), and phosphorylated GFP-ERK2 (lower
immunoblot). D, bar graph depicting the
amount of phosphorylated GFP-ERK2 present in FLAG-
-arrestin 2 immunoprecipitates following stimulation of
2AR or
2V2R. For the
experiments shown in panels B and D, the amount
phospho-GFP-ERK2 in the FLAG-
-arrestin 2 immunoprecipitate was
normalized to the amount of GFP-ERK2 present. The response of the
chimeric receptors is expressed as a percentage of the
-arrestin
2-associated phospho-ERK signal observed in cells expressing the
corresponding wild type receptor. Data shown represent the mean ± S.E. from four separate experiments. *, greater or less than wild type,
p < 0.05.
2AR and
2V2R. Unlike the V2R and V2
2R, the
wild type
2AR was a relatively poor activator of ERK2 in COS-7
cells. Substitution of the V2R tail, which causes the
2V2R chimera
to bind
-arrestin 2 tightly, produced a receptor capable of
eliciting a substantially greater increase in GFP-ERK2 phosphorylation, even when ERK phosphorylation was measured in the whole cell lysate. Associated with this increase in overall GFP-ERK2 phosphorylation, we
observed a 2-fold increase in isoproterenol-stimulated phospho-ERK2 bound to
-arrestin for the
2V2R. This result, shown
quantitatively in Fig. 4D, suggests that the increase in
whole cell ERK phosphorylation seen with the V2
2R reflects increased
utilization of a
-arrestin scaffold by the chimeric receptor.
-Arrestin Binding Affects the Cellular Distribution of
Endogenous Phospho-ERK1/2 following Stimulation of the Wild Type V2R
and Chimeric V2
2R--
One reported consequence of the formation of
-arrestin-ERK complexes is the cytosolic retention of
-arrestin-bound ERK (9, 11, 12). Whereas the preceding data suggest
that some fraction of the ERK activated in response to GPCR stimulation
is associated with
-arrestin, and that the stability of the
GPCR-
-arrestin complex affects the efficiency with which
-arrestin-bound ERK is activated, they do not address whether the
-arrestin-bound ERK pool represents a large enough fraction of the
total cellular pool of GPCR-activated ERK to affect its overall
cellular distribution.
2
receptors were stimulated for varying times prior to cross-linking and
detergent lysis. As shown in Fig.
5A, when these lysates were
resolved by SDS-PAGE under nonreducing conditions, which preserve the
covalent cross-links, agonist stimulation resulted in the appearance of a heterogenous population of high apparent molecular weight bands that
immunoblotted for HA-tagged receptor,
-arrestin, ERK1/2, and
phospho-ERK1/2. When the same samples were resolved under reducing
conditions, which break the cross-links, these high molecular weight
complexes were absent. As shown in Fig. 5B, when receptor immunoprecipitates from cross-linked lysates were resolved under reducing conditions, we observed agonist-dependent
coprecipitation of endogenous
-arrestin and phospho-ERK with the
epitope-tagged receptor. Thus, the migration of receptor,
-arrestin,
and ERK as high molecular weight species in cross-linked whole cell
detergent lysates reflected, as least in part, the agonist-induced
formation of receptor-
-arrestin-ERK complexes. To directly compare
the effect of
-arrestin binding on the distribution of V2R-activated endogenous ERK1/2, we measured the fraction of the total phospho-ERK1/2 in cross-linked whole cell lysates that was present in complexes with
apparent molecular weight >100,000 following stimulation of V2R
and V2
2R. As shown in Fig. 5C (upper immunoblot and
bar graph), ~75% of the phospho-ERK1/2 generated by the V2R was
present in these complexes, compared with ~35% of that generated by
the V2
2R. Conversely, about 20% of the phospho-ERK1/2 migrated at 42-44 kDa when the V2R was stimulated, compared with 55% for the V2
2R. In these experiments, the total amount of phospho-ERK1/2 generated by the two receptors was indistinguishable, as demonstrated when the cross-linked lysates were electrophoresed under reducing conditions (lower immunoblot). As the V2R and V2
2R differ
only in the stability of
-arrestin binding (3), these data suggest that stable
-arrestin binding is associated with the enhanced formation of high molecular weight complexes containing the receptor,
-arrestin, and ERK, and that these complexes contain a significant fraction of the phospho-ERK generated by V2R.
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Fig. 5.
Analysis of the distribution of
endogenous -arrestin, ERK1/2, and
phospho-ERK1/2 in covalently cross-linked cell lysates following
stimulation of wild type V2R and chimeric
V2
2R. COS-7 cells expressing either
HA-V2R or HA-V2
2R were stimulated with vasopressin (1 µM) for the indicated times prior to covalent
cross-linking with dithiobis(succinimidyl propionate) and preparation
of detergent lysates. A, cross-linked detergent lysates from
stimulated V2R-expressing cells were resolved by SDS-PAGE under
reducing (
-ME, mercaptoethanol) or nonreducing
conditions. Identical immunoblots were probed for HA-epitope
(left panel),
-arrestin (second panel), ERK1/2
(third panel), and phospho-ERK1/2 (right panel)
as described. The representative immunoblots shown are from one of six
separate experiments. B, HA-V2R was immunoprecipitated from
cross-linked detergent lysates and resolved by SDS-PAGE under reducing
conditions. Identical immunoblots were probed for HA epitope
(left panel), and coprecipitated endogenous
-arrestin
(center panel) and phospho-ERK1/2 (right panel).
The representative immunoblots shown are from one of three separate
experiments. C, cross-linked detergent lysates from
stimulated V2R- and V2
2R-expressing cells were resolved by SDS-PAGE
under reducing (lower immunoblot) or nonreducing
(upper immunoblot) conditions. The percent of the total
phospho-ERK1/2 signal present in each stimulated lane that migrated
with an apparent molecular weight of 42,000-44,000 or >100,000 was
quantified by scanning densitometry. The bar graph compares
the distribution of phospho-ERK1/2 between pools of different apparent
molecular weight, following V2R and V2
2R stimulation. Data shown
represent the mean ± S.E. from three to six separate
experiments.
-arrestin binding on the cellular
distribution of active ERK1/2, we compared the fraction of total
cellular phospho-ERK1/2 that translocated to the nucleus following
stimulation of the V2R and V2
2R. In these experiments, COS-7 cells
transiently expressing HA-tagged V2 or V2
2 receptors were stimulated
for 5 min prior to isolation of cell nuclei by differential
centrifugation. As shown in Fig.
6A, we found that with the V2R
less than 10% of the total cellular phospho-ERK1/2 was present within
the nucleus 5 min after stimulation, compared with about 25% with the
V2
2 chimera. Conversely, as shown in Fig. 6B, the amount
of phospho-ERK1/2 present in the extranuclear fraction, representing
plasma membrane, microsomes, cytoskeleton, and cytosol, was
proportionally greater for the V2R than the V2
2R. At this time
point, the total amount of phospho-ERK1/2 in the V2 and V2
2
receptor-expressing cells was indistinguishable. Thus, the data
obtained by measuring the fraction of active ERK1/2 in high molecular
weight complexes (Fig. 5) and those obtained by measuring the nuclear
fraction of phospho-ERK1/2 are complementary, and suggest that
stimulation of the wild type V2R generates more phospho-ERK1/2 in large
multiprotein complexes and less phospho-ERK1/2 in the nucleus than
stimulation of the V2
2 receptor.
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Fig. 6.
Effect of exchanging the C terminus of the
V2R and 2AR on nuclear translocation of
activated endogenous ERK1/2 following agonist stimulation. COS-7
cells were transfected with plasmids for either HA-V2R or HA-V2
2R.
Serum-starved cells were stimulated with vasopressin (1 µM) for 5 min, cell nuclei were isolated as described,
and the phospho-ERK1/2 content of the nuclear and extranuclear
fractions was determined by immunoblotting. A,
phospho-ERK1/2 content of the whole cell lysate collected prior to
fractionation (upper immunoblot) and the nuclear fraction
(lower immunoblot) following stimulation of V2R and V2
2R.
B, phospho-ERK1/2 content of whole cell lysate collected
prior to fractionation (upper immunoblot) and the
extranuclear fraction (lower immunoblot) following
stimulation of V2R and V2
2R. In the bar graphs, data are
expressed as the percentage of the total cellular phospho-ERK1/2 pool
present in the nuclear or extranuclear fraction. Data shown represent
the mean ± S.E. of four independent experiments. *, greater or
less than V2R, p < 0.05.
-arrestin-associated phospho-ERK1/2 formed in response to
stimulation of the wild type PAR2 receptor and a mutant receptor lacking GRK phosphorylation sites that does not bind
-arrestin (9).
Using a gel filtration approach, these authors found that greater than
80% of the phospho-ERK formed in response to wild type receptor
activation coeluted with the receptor, Raf-1, and
-arrestin, whereas
less than 2% of the phospho-ERK coeluted with the mutant receptor.
These authors also found that the wild type receptor activated a
predominantly non-nuclear pool of ERK, whereas the mutant predominantly
activated nuclear phospho-ERK. The presence of a nuclear export signal
in
-arrestin 2, which was recently shown to account for the nuclear
exclusion of
-arrestin 2-bound JNK3 in cells overexpressing
-arrestin 2 (15), may account for the effect of
-arrestin binding
on ERK distribution.
-Arrestin
Interaction--
The preceding data demonstrate that the ability of a
GPCR to bind stably to
-arrestin correlates with increased
activation of
-arrestin-bound of ERK1/2, and decreased nuclear
translocation of phospho-ERK1/2. To determine whether the functional
consequences of ERK activation are affected by the stability of the
receptor-
-arrestin interaction at endogenous levels of
-arrestin
and ERK1/2 expression, we employed stably transfected HEK-293 cell
lines expressing the V2R and V2
2R. These cell lines exhibit
comparable levels of cAMP production in response to vasopressin
stimulation, but differ in the stability of the receptor-
-arrestin
interaction and the pattern of receptor internalization,
dephosphorylation, and recycling.
2R-expressing HEK-293 cells. At short time points,
up to ~10 min, the responses were indistinguishable. Although the
overall level of ERK1/2 phosphorylation declined dramatically after 30 min for both receptors, the level of persistent ERK1/2 phosphorylation
was modestly, but significantly, greater for the V2
2R than the V2R.
Previous work has shown replacement of the V2R tail with that of the
2AR increases the rate of receptor dephosphorylation and recycling
after endocytosis (3). Thus, this persistent signal may represent the
effect of
-arrestin binding on the rate of receptor recycling and
the steady state level of undesensitized receptors on the plasma
membrane in the continuous presence of agonist. Fig. 7B
compares the ability of V2R and V2
2R to induce transcription of an
Elk1-driven luciferase reporter. This response is dependent on
activation of endogenous ERK1/2, because it is completely eliminated by
incubation with the MEK inhibitor, PD98059 (data not shown). As shown,
the chimeric receptor produced a transcriptional response that was
~8-fold greater than the wild type receptor. The response to
stimulation of endogenous epidermal growth factor (EGF) receptors was
comparable between the two cell lines.
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Fig. 7.
Effect of exchanging the C terminus of the
V2R and 2AR on the time course of
vasopressin-induced phosphorylation of endogenous ERK1/2 and induction
of an Elk1-luciferase reporter. A, HEK-293 cells stably
expressing either V2R (upper immunoblot) or V2
2R
(lower immunoblot) were stimulated for the indicated times
with vasopressin (1 µM) and whole cell phospho-ERK1/2 was
determined by immunoblotting. The graph represents the
mean ± S.E. of five separate experiments. B, HEK-293
cells stably expressing either V2R or V2
2R were transfected with
plasmids for pFR-luciferase, GAL-4-Elk-1, and TK-Renilla as
described. Luciferase activity was determined after 4 h incubation
of serum-starved cells in the presence or absence (NS) of
vasopressin (1 µM) or EGF (10 ng/ml). Luciferase activity
is expressed as the -fold increase relative to unstimulated V2R cells
and represents mean ± S.E. from three separate experiments. *,
greater than V2R, p < 0.05.
2R resulted in greater
nuclear translocation of activated ERK1/2 (Fig. 6), more persistent ERK1/2 activation, and enhanced ERK1/2-dependent
transcription (Fig. 7). Because nuclear translocation of ERK1/2 is
required for growth factor-stimulated mitogenesis (16), we compared the ability of the V2R and V2
2R to stimulate DNA synthesis in the two
cell lines. As shown in Fig. 8, the V2R,
despite its ability to mediate robust activation of ERK1/2, failed to
elicit a detectable increase in [3H]thymidine
incorporation into DNA. In contrast, the V2
2R was weakly, but
significantly, mitogenic, eliciting a response comparable with
refeeding with 2% fetal calf serum. Because these two receptors differ
only in their C termini, these data suggest that the stability of the
receptor-
-arrestin interaction influences the mitogenic potential of
the V2R. It is likely that this effect is because of both binding of
ERK1/2 to
-arrestin leading to its retention in the cytosol, and to
-arrestin-dependent removal of receptors from the cell
surface leading to a diminished steady state level of ERK
activation.
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Fig. 8.
Effect of exchanging the C terminus of the
V2R and 2AR on vasopressin-stimulated DNA
synthesis. Serum-starved HEK-293 cells stably expressing either
V2R or V2
2R were incubated for 24 h in the presence or absence
(NS) of vasopressin (1 µM) or fetal bovine
serum (FBS, 2% v/v) prior to the determination of
[3H]thymidine incorporation into DNA. Results are
expressed as the -fold increase relative to unstimulated cells.
Experiments were performed in duplicate and the data shown represent
mean ± S.E. from three independent experiments. For reference,
refeeding with medium containing 10% fetal calf serum resulted in a
10-12-fold increase in [3H]thymidine incorporation. *,
greater than NS, p < 0.05.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestins to agonist-occupied GPCRs serves two
broad functions. By uncoupling the receptor from heterotrimeric G
proteins and targeting it for endocytosis,
-arrestin binding is
central to the processes of GPCR desensitization and sequestration that
lead to rapid termination of G protein-dependent signals. At the same time,
-arrestins function as adapter proteins, bringing various signaling molecules into complex with the receptor and initiating additional
-arrestin-dependent signaling events.
The finding that
-arrestins can interact directly with potential enzymatic effectors such as Src family tyrosine kinases (8, 17-19) and
ubiquitin ligases (20), as well as components of the ERK1/2 (8, 9, 11,
12) and JNK3 (10, 15, 21) mitogen-activated protein kinase modules,
suggests that
-arrestins may serve in a variety of signaling roles.
2AR-mediated ERK1/2 activation
occurs via a Gs-, adenylyl cyclase-, and
PKA-dependent pathway that causes activation of the small
GTPase, Rap1 (24, 25), whereas in fibroblasts, cardiomyocytes, and
pancreatic acinar cells ERK1/2 activation is achieved largely through a
Ras-dependent mechanism involving activation of pertussis
toxin-sensitive G proteins and "transactivation" of EGF receptors
(26-29). Other receptors, notably the AT1aR, and the PAR2 and
neurokinin NK-1 receptors, appear to utilize
-arrestins as scaffolds
to a significant extent (8, 9, 11, 12), although AT1aRs are also
clearly capable of mediating ERK activation through cross-talk with EGF or platelet-derived growth factor receptors (30-32). We have
previously shown that overexpression of
-arrestins in COS-7 cells
enhances AT1aR-mediated ERK1/2 activation, but leads to cytosolic
retention of ERK and a diminished transcriptional response (11,
12).
-arrestins are uniquely
positioned to influence the balance between these different mechanisms.
Furthermore, because the mechanism used to activate ERK influences its
spatial distribution and transcriptional activity, the binding of
-arrestin and receptor might dictate the cellular response to ERK
activation. In a number of systems, the proliferative response to GPCR
stimulation involves cross-talk between GPCRs and EGF receptors (31,
33). In contrast,
-arrestin-dependent ERK activation
does not appear to lead to proliferative signaling. Wild type PAR2
receptors, which mediate
-arrestin-dependent activation of a predominantly cytosolic pool of ERK1/2 in KNRK cells, do not
stimulate [3H]thymidine incorporation or cell replication
(9). As we have shown, altering the stability of the V2R
receptor-
-arrestin interaction by replacing the C-terminal tail of
the V2R with that of the
2AR was sufficient to confer mitogenic
potential on a receptor that, in this system, did not detectably
stimulate [3H]thymidine incorporation (Fig. 8).
-arrestins in regulating the mechanism and functional consequences
of GPCR-stimulated ERK activation. Activation of ERK1/2 via classical G
protein-dependent mechanisms, potentially including
transactivation of receptor tyrosine kinases, leads to nuclear
translocation of ERK1/2, activation of ERK1/2-dependent
transcription and a mitogenic response. In contrast, activation of
ERK1/2 in a
-arrestin-bound pool leads to localized ERK1/2 activity,
with less nuclear phospho-ERK1/2 and little or no mitogenic potential.
Significantly,
-arrestin binding to the receptor not only confers
the ability to activate
-arrestin-bound ERK1/2, it also attenuates
signaling via G protein-dependent pathways and determines
the rate of receptor recycling. As we have shown, altering the
stability of the receptor-
-arrestin interaction affects not only the
efficiency of GPCR-mediated activation of a
-arrestin-bound ERK
pool, but also the balance between the nuclear and cytosolic
phospho-ERK1/2 and the steady-state level of ERK1/2 activity during
prolonged stimulation. This shift undoubtedly reflects the role of
-arrestins in receptor desensitization, sequestration, and recycling
as much as it does the enhanced utilization of
-arrestin scaffolds
by receptors that bind stably to
-arrestin.
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Fig. 9.
Proposed model of the effect of
-arrestin binding on ERK1/2 activation
and function. The binding of agonist to a GPCR initially leads to
ERK activation via G protein-dependent pathways. The
binding of
-arrestin simultaneously inhibits G
protein-dependent ERK1/2 activation, by inducing homologous
receptor desensitization and sequestration, and initiates the
activation of a
-arrestin-bound pool of phospho-ERK1/2. For GPCRs
that form stable receptor-
-arrestin complexes, activation of the
-arrestin-dependent pathway is more pronounced, leading
to the formation of a functionally distinct pool of
phospho-ERK1/2.
The extent to which "G protein-dependent" and
"-arrestin-dependent" ERK activation are genuinely
independent processes is unclear. In murine fibroblasts derived from
-arrestin 1/
-arrestin 2 homozygous null embryos, lysophosphatidic
acid-stimulated ERK1/2 activation is sensitive to inhibitors of the EGF
receptor tyrosine kinase, suggesting that GPCR-stimulated EGF receptor
transactivation can occur in a
-arrestin null
background.2 On the other
hand,
-arrestin recruitment requires GRK-mediated receptor
phosphorylation, and receptor phosphorylation by GRK2 or GRK3 requires
G
subunit-mediated membrane translocation of the kinase. Thus,
one might expect that
-arrestin-dependent ERK activation
would not be G protein-independent. Rather, it would represent a shift
in the mechanism of ERK activation coincident with homologous
desensitization that leads to the localized activation of a
functionally distinct pool of ERK1/2. Interestingly, however, Seta
et al. (34) have recently reported that in Chinese hamster ovary cells, a mutant AT1aR that is markedly impaired in G protein coupling is still fully competent to induce ERK1/2 activation, but that
ERK activated by the mutant receptor is retained in the cytosol and is
transcriptionally inactive (34). While these authors do not implicate
-arrestins in the putatively "G protein-independent" activation
of ERK, their data do suggest the existence of one or more mechanisms
of ERK activation that do not require G protein activation, and that,
like the
-arrestin-dependent activation of ERK, leads to
localized ERK activation outside the of the cell nucleus.
Our data demonstrate the significant impact of the
receptor--arrestin interaction on the mechanism and functional
consequences of ERK activation. The activation of ERK bound to
-arrestin, which is favored in the setting of a stable
receptor-
-arrestin interaction, limits nuclear translocation of ERK
and attenuates Elk-1-driven transcription. However, any additional
functions of
-arrestin-bound ERK1/2 remain to be discovered. ERK1/2
are known to phosphorylate multiple plasma membrane, cytoplasmic, and
cytoskeletal substrates (16), including several proteins involved in
heptahelical receptor signaling, such as
-arrestin 1 (35), GRK2 (36,
37), and the G
-interacting protein GAIP (38). Thus, one role
of
-arrestin-ERK complexes could be to target ERK1/2 to substrates
involved in the regulation of GPCR signaling or intracellular
trafficking. Alternatively,
-arrestin-bound ERK1/2 may phosphorylate
other cytosolic kinases involved in transcriptional regulation, such as
p90 RSK (39), which in turn relay signals to the nucleus. In such a
model, transcriptional events mediated directly by the nuclear pool of
ERK1/2 would be attenuated, whereas alternate pathways of
ERK-dependent transcription would persist, resulting in an
altered pattern of GPCR-stimulated transcription.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Donna Addison and Julie Turnbough for excellent secretarial assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DK55524 (to L. M. L.), HL16037 (to R. J. L.), and NS19576 (to M. G. C.).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.
** Investigators with the Howard Hughes Medical Institute.
§§ To whom correspondence should be addressed: N3019 GRECC, Durham Veterans Affairs Medical Center, 508 Fulton St., Durham, NC 27705. Tel.: 919-286-0411 (ext. 7196); Fax: 919-416-5823; E-mail: luttrell@receptor-biol.duke.edu.
Published, JBC Papers in Press, December 6, 2002, DOI 10.1074/jbc.M212231200
2 T. Kohout and R. J. Lefkowitz, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GPCR, G protein-coupled receptor; AR, adrenergic receptor; AT1aR, angiotensin II type 1a receptor; ERK1/2, extracellular signal-regulated kinases 1 and 2; G protein, heterotrimeric guanine nucleotide-binding protein; GFP, green fluorescent protein; GRK, G protein-coupled receptor kinase; HA, influenza virus hemagglutinin; JNK3, c-Jun N-terminal kinase 3; PAR2, protease-activated receptor type 2; V2R, vasopressin receptor type 2; EGF, epidermal growth factor.
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REFERENCES |
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