From the Howard Hughes Medical Institute
Laboratories, Departments of Cell Biology and Medicine, Duke University
Medical Center, Durham, North Carolina 27710 and the § John
P. Robarts Research Institute, Departments of Physiology and
Pharmacology and Toxicology, University of Western Ontario, P.O. Box
5015, 100 Perth Drive, London, Ontario N6A 5K8, Canada
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ABSTRACT |
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Signal transduction via G protein-coupled receptors
(GPCRs)1 is intimately
associated with a wide variety of biological processes including
neurotransmission, chemoattraction, cardiac function, olfaction, and
vision. Recent studies suggest that In the present study, we used a green fluorescent protein conjugate of
Materials--
Human embryonic kidney (HEK 293) cells were
provided by the American Type Culture Collection (ATCC). Tissue culture
media and fetal bovine serum were obtained from Life Technologies, Inc. Isoproterenol and dopamine were purchased from Research Biochemicals International. Endothelin and neurotensin were from Peninsula Laboratories, and angiotensin II was from Sigma. Rabbit anti-HA polyclonal antibody and mouse anti-HA 12CA5 monoclonal antibody were
obtained from Babco and Boehringer Mannheim, respectively. Fluorescein
isothiocyanate-conjugated goat anti-mouse secondary antibody was
purchased from Sigma, and rhodamine-conjugated goat anti-rabbit Fabs
was obtained commercially from Organon Teknika. [125I]Cyanopindolol was purchased from NEN Life
Science Products.
DNA Construction--
All recombinant DNA procedures were
carried out following standard protocols. Cell Culture and Transfection--
HEK 293 cells were grown in
Eagle's minimal essential medium with Earle's salt (MEM) supplemented
with 10% (v/v) heat-inactivated fetal bovine serum and gentamicin (100 µg/ml). The cells were seeded at a density of 2.5 × 106 cells/100-mm dish and transiently transfected with the
cDNAs described in the figure legends by a modified calcium
phosphate method (22). Following transfection (~18 h), the cells were incubated with fresh medium and allowed to recover 7-9 h before being
reseeded into 35-mm glass-bottomed culture dishes (MatTek) or into
six-well dishes (Falcon) containing or not containing 22-mm square
glass coverslips coated with collagen (Sigma). The use of HEK 293 cells
expressing Receptor Expression--
Confocal Microscopy--
Confocal microscopy was performed on a
Zeiss LSM-410 laser scanning microscope using either Zeiss 40 × 1.3 or Zeiss 63 × 1.4 numerical aperture oil immersion lenses.
For characterizing the pharmacology of Immunofluorescent Labeling--
For performing colocalization
studies of Data Analysis--
The changes in As described previously (18), in the absence of receptor
activation -Arrestins are multifunctional
proteins identified on the basis of their ability to bind and uncouple
G protein-coupled receptors (GPCR) from heterotrimeric G proteins. In
addition,
-arrestins play a central role in mediating GPCR
endocytosis, a key regulatory step in receptor resensitization. In this
study, we visualize the intracellular trafficking of
-arrestin2 in
response to activation of several distinct GPCRs including the
2-adrenergic receptor (
2AR),
angiotensin II type 1A receptor (AT1AR), dopamine D1A receptor (D1AR), endothelin type A receptor
(ETAR), and neurotensin receptor (NTR). Our results reveal
that in response to
2AR activation,
-arrestin2
translocation to the plasma membrane shares the same pharmacological
profile as described for receptor activation and sequestration,
consistent with a role for
-arrestin as the agonist-driven switch
initiating receptor endocytosis. Whereas redistributed
-arrestins
are confined to the periphery of cells and do not traffic along with
activated
2AR, D1AR, and ETAR in
endocytic vesicles, activation of AT1AR and NTR triggers a
clear time-dependent redistribution of
-arrestins to
intracellular vesicular compartments where they colocalize with
internalized receptors. Activation of a chimeric AT1AR with
the
2AR carboxyl-terminal tail results in a
-arrestin
membrane localization pattern similar to that observed in response to
2AR activation. In contrast, the corresponding chimeric
2AR with the AT1AR carboxyl-terminal tail
gains the ability to translocate
-arrestin to intracellular
vesicles. These results demonstrate that the cellular trafficking of
-arrestin proteins is differentially regulated by the activation of
distinct GPCRs. Furthermore, they suggest that the carboxyl-tail of the receptors might be involved in determining the stability of
receptor/
arrestin complexes and cellular distribution of
-arrestins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Arrestin proteins play an important role in regulating the
responsiveness of GPCRs by contributing to mechanisms involved in both
GPCR desensitization and resensitization (1-5).
-Arrestins regulate
GPCR desensitization by binding and uncoupling the receptors from
heterotrimeric G proteins once they have been phosphorylated by G
protein-coupled receptor kinases (GRKs) (1, 3). In addition, they are
also required for the sequestration (endocytosis) of a growing number
of GPCRs, including the CCR-5, follitropin receptor,
lutropin/choriogonadotropin receptor, m2 muscarinic acetylcholine
receptor, mu opioid receptor, substance P receptor, and the
2-adrenergic receptor (
2AR) (4, 6-11). At least in the case of the
2AR, the
agonist-dependent sequestration of the receptor to an
endosomal compartment not only promotes receptor dephosphorylation
but is essential for the re-establishment of normal receptor
responsiveness (5, 12-15).
-arrestins participate in GPCR
sequestration by directing receptors to clathrin-coated vesicles (4,
16, 17).
-Arrestins have been shown to undergo redistribution in
response to receptor activation both in live cells and following the
fixation of cells, and to co-localize with clathrin (7, 11, 18, 19).
However, while the phenomenon of
-arrestin cellular trafficking is
potentially important for understanding mechanisms underlying GPCR
internalization and resensitization, the detailed pharmacology of the
receptor-mediated
-arrestin redistribution has never been
characterized. As a consequence, it is not clear whether the
pharmacological profile of
-arrestin translocation recapitulates the
pharmacology described for GPCR endocytosis. Moreover, the cellular
fate of
-arrestin proteins following association with various GPCRs
also remains unknown, as well as where and when
-arrestins
dissociate from each receptor. In the case of rhodopsin, it was
demonstrated that the interaction of visual arrestin with rhodopsin
prevented the dephosphorylation and resensitization of the receptor
(20, 21). Therefore, it is likely that the dissociation of the
-arrestin/receptor complex contributes to the regulation of
responsiveness for other GPCRs.
-arrestin2 (
arr2GFP) (18) to examine the cellular trafficking of
-arrestin upon stimulation of several distinct GPCRs.
Our data demonstrate that the pharmacology of
-arrestin2 translocation in living cells could account for the agonist dependence of
2AR sequestration. Moreover,
-arrestin2 was
observed to redistribute to distinct subcellular locations in response
to activation of different GPCRs. This differential redistribution of
-arrestins likely involves the function of the carboxyl-terminal
region of the receptors.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Arrestin1 with GFP
conjugated to its COOH terminus (
arr1GFP) was
constructed in a manner similar to
arr2GFP (18) by
replacing the terminal stop codon of
-arrestin1 with a
SalI restriction site and inserting the modified cDNA in
frame into the polylinker of p(S65T)GFP-N3
(CLONTECH) (18).
pcDNA3-AT1A R-
2AR-CT and
pcDNA1-Amp-
2AR-AT1AR-CT were constructed
by polymerase chain reaction. The chimeric AT1AR with the
2AR carboxyl-terminal tail
(AT1AR-
2AR-CT) contains the first 302 amino
acids (Met1-Tyr302) of the AT1AR
fused to the last 87 amino acids
(Cys327-Leu413) of the
2AR. The
chimeric
2AR with the AT1AR
carboxyl-terminal tail (
2AR-AT1AR-CT)
includes the first 348 amino acids
(Met1-Lys348) of the
2AR fused
to the last 38 amino acids (Ala324-Glu359) of
the AT1AR. The sequences of the DNA constructs were
confirmed by DNA sequencing.
2AR-GFP was described previously (23).
2AR expression was
measured by saturating [125I]cyanopindolol binding done
at 30 °C for 60 min (24). The expression of other receptors was
measured by flow cytometry and normalized according to the
2AR expression measured at the same time by both flow cytometry and saturating binding (16). The receptor expression levels
was between 2000 and 4000 fmol/mg of whole cell protein for the
experiments assessing
-arrestin translocation with confocal microscope (see below) and between 1000 and 2000 fmol/mg of whole cell
protein for all other experiments.
-arrestin translocation in
living cells, HEK 293 cells expressing
2AR and low
levels of
arr2GFP were plated on 35-mm glass-bottomed
culture dishes and kept warm at 30 °C in serum-free MEM on a heated
microscope stage.
arr2GFP fluorescent signals were
collected sequentially using the Zeiss LSM software time scan function
in the photon counting mode using single line excitation (488 nm).
Drugs were applied to the cells either prior to or during the scanning
of
arr2GFP labeled cells. For studying
-arrestin
trafficking in response to activation of other receptors, HEK 293 cells
expressing the receptor of interest and low levels of
arr2GFP were seeded on 22-mm square glass coverslips,
stimulated with saturating concentrations of drugs at 30 °C for
1 h or as indicated, and then fixed with 3.7% paraformaldehyde in
phosphate-buffered saline.
arr2GFP fluorescent signals
were collected using single line excitation (488 nm). Colocalization
studies of
arr2GFP and rhodamine-labeled receptor
fluorescence were performed using dual excitation (488, 568 nm) and
emission (515-540 nm, GFP; 590-610 nm, rhodamine) filter sets.
Specificity of labeling and absence of signal cross-over were
established by examination of single-labeled samples.
arr2GFP and rhodamine-labeled
2AR in live cells, HEK 293 cells expressing HA
epitope-tagged
2AR and
arr2GFP grown on
35-mm glass-bottomed culture dishes were incubated in serum-free MEM
containing anti-HA polyclonal antibody at 37 °C for 30 min. Cells
were washed three times with ice-cold MEM and incubated for 30 min on
ice in the presence of rhodamine-conjugated goat anti-rabbit Fabs.
Cells were washed an additional three times with serum-free MEM at
30 °C and imaged by confocal microscopy as described above. For
studying colocalization of
arr2GFP and
AT1AR, HEK 293 cells expressing HA epitope-tagged AT1AR were grown on 22-mm square glass coverslips and
incubated in serum-free MEM containing rhodamine-conjugated anti-HA
12CA5 monoclonal antibody on ice for 45 min. Cells were then washed, stimulated with a saturating concentration (500 nM) of
angiotensin II for 1 h, washed again, fixed with 3.7%
paraformaldehyde in phosphate-buffered saline, and imaged by confocal
microscopy as described above.
arr2GFP
distribution to the plasma membrane were analyzed with IP Labs
software. The magnitude of
arr2GFP fluorescence at the
plasma membrane were determined by integration of the fluorescence
signal along the cell perimeter. The relative magnitude of
arr2GFP distribution along a linear slice of the cell
was quantitated by the line scan function provided with the Zeiss LSM
410 image analysis software.
arr2GFP translocation time
course and dose-response curves were analyzed using GraphPad Prism. All
data points represent the mean ± S.D.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
arr2GFP fluorescence was evenly distributed
throughout the cytoplasm and exhibited no apparent enhanced plasma
membrane localization (Fig. 1,
A and C; control). However, upon
agonist-activation of the
2AR, a
time-dependent rapid redistribution of
arr2GFP to the plasma membrane occurred (Fig. 1,
A and B). The time course of
-arrestin
translocation determined here, t1/2 = 2.3 min
(Fig. 1B), followed
2AR phosphorylation
(t1/2 = 15-40 s) (24, 25) and preceded
2AR internalization (t1/2 = 10 min) (4). At first
arr2GFP appeared diffusely at the
plasma membrane, but with time a punctate pattern became apparent.
Moreover, the redistribution of
arr2GFP from the cytosol
to the plasma membrane was agonist dose-dependent (Fig. 1,
C and D). The half-maximal effective
concentration (EC50) of agonist was calculated to be 6 nM (Fig. 1D), a value comparable to that
reported for
2AR sequestration in HEK 293 cells (11 nM) (24). No significant
arr2GFP
translocation in response to agonist exposure was observed in cells
lacking overexpressed
2AR (data not shown). To further
test the agonist specificity of
arr2GFP translocation,
cells were treated 5 min with 1 µM isoproterenol to
induce
arr2GFP translocation and then exposed to a
saturating concentration of the antagonist propranolol in the presence
of the agonist (Fig. 1E). Following the treatment of cells
with the antagonist, the distribution of
arr2GFP
fluorescence reversed, with
arr2GFP redistributing over
time from the plasma membrane back to the cytoplasm. However, the
redistribution of
arr2GFP back into the cytoplasm was
not immediate, but proceeded over a time course of 5-10 min consistent
with previous reports describing agonist-dependent and
independent steps of receptor internalization (26). Since the
pharmacology of
arr2GFP translocation accurately
reflected the agonist dependence of
2AR sequestration, these results suggest that
-arrestin translocation and receptor binding serve as the agonist-dependent switch triggering
endocytosis of the
2AR.
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Fig. 1.
Pharmacology and agonist dependence of
arr2GFP translocation in response
to
2AR activation in HEK 293 cells. Visualization (A) and quantitation
(B) of the time course for
arr2GFP membrane
translocation in HEK 293 cells expressing
2AR and
arr2GFP in response to stimulation with 25 µM isoproterenol for 0-15 min. Shown are representative
confocal microscopic images of
arr2GFP fluorescence
obtained prior to (control) and 90 s, 3 min, and 10 min following
the addition of agonist to the medium. Visualization (C) and
quantitation (D) of the agonist dose-dependent
membrane translocation of
arr2GFP in HEK 293 cells in
response to 5-min exposures to increasing concentration of
isoproterenol 10
10 to 10
5 M.
Shown are representative confocal microscopic images of
arr2GFP fluorescence in HEK 293 cells obtained prior to
(control) and following the addition of 10
10
M, 10
8 M, and 10
6
M isoproterenol. (E) Effect of treating cells
with the antagonist propranolol on the localization of
arr2GFP redistributed to the plasma membrane in response
to receptor activation. Shown are representative confocal microscopic
images of
arr2GFP fluorescence in HEK 293 cells prior to
(control) treatment for 5 min with 1 µM isoproterenol
(agonist), following which antagonist (300 µM
propranolol) was added to the agonist containing medium to compete for
receptor binding sites containing medium and time scanned for an
additional 10 min (agonist + antagonist). All cells were transfected
with 1 µg of pGFP-N3/
arr2 and 5 µg of 12CA5 epitope-tagged
2AR in pcDNA1-Amp, and experiments were performed
independently on 4-8 different cells. Experiments were performed on a
heated microscope stage set at 30 °C. Data points represent the
mean ± S.D. of 8 (B) and 5 (D) different
cells from separate transfections. Increased membrane localized
fluorescence was quantitated using IPLab spectrum image analysis
software (Signal Analytics Corp.). The inset bars
represent 10 µm.
While arr2GFP was observed to translocate to the plasma
membrane and cluster at coated pits (18),
arr2GFP
labeling of intracellular endocytic vesicles following
2AR activation was never observed. The overall
distribution pattern of
arr2GFP appeared different from
that of a GFP-conjugated
2AR (23) (Fig.
2, A and B). In
response to agonist stimulation, the
2AR-GFP
redistributed from a diffuse plasma membrane localization to a
membrane-associated vesiculated pattern, followed by the appearance of
2AR-GFP in endocytic vesicles randomly distributed
throughout the cytosol of the cell (Fig. 2B). Therefore, we
examined the agonist-induced intracellular trafficking of both the
2AR and
arr2GFP in the same living cells.
To do this,
2ARs engineered with an amino-terminal HA
epitope tag were expressed in HEK 293 cells with
arr2GFP
and labeled with 12CA5 monoclonal antibodies, which were themselves labeled with rhodamine-conjugated anti- Fabs.
2ARs
labeled in this manner were still able to respond normally to agonist
activation (Fig. 2C). In the absence of agonist,
2AR immunofluorescence (red) was localized
solely to the plasma membrane, whereas
arr2GFP fluorescence (green) was limited to the cytoplasm (Fig. 2C).
In response to agonist activation of
2ARs,
arr2GFP translocated to the receptors at the plasma
membrane. This was followed by the redistribution of both the receptors
and
-arrestin to clathrin-coated pits, as denoted by the appearance
of yellow hot spots (Fig. 2C). However, while
yellow vesicles could be observed close to the membrane
surface, no colocalization of
arr2GFP with
2AR-bearing vesicles was ever observed in the cytoplasm
of the cell (Fig. 2C). A similar agonist-mediated
redistribution of
arr1GFP to plasma membrane-localized
2AR, but not
2AR localized in endocytic vesicles, was also observed (data not shown). These results demonstrate that
2AR/
-arrestin complex dissociates at or close to
the plasma membrane, and therefore
-arrestins are excluded from
endocytic vesicles shortly following their formation.
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In a previous study, we have demonstrated that although the
AT1AR can utilize a distinct endocytic mechanism,
overexpression of exogenous -arrestins mobilizes the receptor for
internalization via clathrin-mediated endocytosis similar to that
utilized by the
2AR (16). To further characterize the
interaction of
-arrestin with the AT1AR,
receptor-mediated
arr2GFP trafficking was examined in
HEK 293 cells co-expressing the AT1AR and
arr2GFP. The cells were stimulated with angiotensin II
for various periods of time at 30 °C and observed under confocal
microscope. Similar to that observed with the
2AR,
arr2GFP was evenly distributed throughout the cytoplasm
in the absence of agonist, but underwent a rapid translocation to the
plasma membrane in response to AT1AR activation (Fig.
3A). However, activation of
the AT1AR for a longer period of time (>4 min) resulted in
a clear redistribution of
arr2GFP to intracellular
endocytic vesicles.
arr1GFP was also observed to undergo
a similar redistribution to AT1AR-containing endocytic vesicles.2 With time, the
arr2GFP-containing vesicular structures grew in size and
were mobilized to cluster at the perinuclear region of the cells (Fig.
3A). In contrast, under parallel conditions,
arr2GFP remained confined to the plasma membrane even
when the
2AR was activated by isoproterenol for 1 h
(Fig. 3B). To further confirm that the redistribution of
-arrestins is receptor-driven, we examined the localization of
agonist-activated AT1AR and
arr2GFP in the
same HEK 293 cells. To do this, AT1ARs engineered with an
amino-terminal HA epitope tag were expressed in HEK 293 cells with
arr2GFP and labeled with rhodamine-conjugated anti-HA
12CA5 monoclonal antibodies. When the cells were activated by
angiotensin II, an agonist-dependent colocalization of
AT1AR red immunofluorescence and
arr2GFP
green fluorescence was observed and persisted for up to 1 h, as
reflected by the predominant intracellular yellow vesicular
structures located at the perinuclear region (Fig.
4). These data demonstrate a functional
interaction between the AT1AR and
-arrestin proteins. In
addition, our results also indicate that, unlike the
2AR/
-arrestin complex, the
AT1AR/
-arrestin complex remains intact and is mobilized
to the interior of the cell driven by receptor internalization.
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Since the activation of 2AR and AT1AR
promoted
-arrestin trafficking to distinct subcellular locations, we
examined the redistribution of
arr2GFP in response to
activation of several other GPCRs to address the generality of the two
different
-arrestin translocation patterns. To do this,
arr2GFP was co-expressed in HEK 293 cells with different
GPCRs including dopamine D1A receptor (D1AR), endothelin
type A receptor (ETAR) and neurotensin receptor (NTR). As
shown in Fig. 5, in response to
activation of the D1AR and the ETAR,
arr2GFP underwent a rapid membrane translocation and
remained in a punctate pattern at the periphery of the cells for as
long as 1 h, similar to that observed following
2AR
stimulation. In contrast, in HEK 293 cells overexpressing the NTR,
neurotensin stimulation resulted in the redistribution of
arr2GFP fluorescence to intracellular vesicular
structures with a pattern reminiscent of that observed following
AT1AR activation. Therefore, different GPCRs either
separate from
-arrestins at the level of plasma membrane or
internalize with
-arrestin in intracellular vesicles. This property
appears independent of both the types of G protein-coupling and
agonists.
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Previous studies of GPCR internalization have suggested that the
carboxyl-terminal region is important for receptor interaction with
-arrestins (4). Therefore, to examine whether the carboxyl-terminal tail of the receptors contributes to the differential trafficking of
-arrestin, we engineered chimeric mutants of the AT1AR
and
2AR with their carboxyl-terminal region exchanged
for that of the other. Like the wild-type AT1AR and
2AR, the chimeric AT1AR and
2AR mutants (namely
AT1AR-
2AR-CT and
2AR-AT1AR-CT) underwent rapid
internalization in response to agonist stimulation (Fig. 6B). However, when
co-expressed with
arr2GFP, angiotensin II activation of
the AT1AR-
2AR-CT did not result in
-arrestin trafficking to intracellular vesicles, but rather resulted
in a
-arrestin membrane localization pattern similar to that
observed in response to
2AR activation (Fig.
6A). In fact,
arr2GFP fluorescence was retained at the plasma membrane for up to 1 h and was never
localized to intracellular vesicular structures in response to
activation of the chimeric AT1AR mutant. In contrast, the
corresponding
2AR chimeric mutant with AT1AR
carboxyl-tail displayed "AT1AR-like" phenotype and
acquired the ability to mediate
-arrestin translocation to
intracellular vesicles (Fig. 6A). In addition, a
2AR-
arr2GFP fusion protein with
arr2GFP attached to the carboxyl terminus of the
2AR was also localized to large intracellular vesicular structures similar to the vesicular population containing
arr2GFP following AT1AR stimulation (Fig.
6C). These results demonstrate that the carboxyl-terminal
region of the receptors is involved in determining the mode of
interaction of the receptors with
-arrestin proteins, and
subsequently the agonist-dependent redistribution of
-arrestins.
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DISCUSSION |
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In the present work, we use green fluorescent protein-conjugated
-arrestin2 to study the cellular trafficking of
-arrestin proteins in response to several different GPCRs in both living and
fixed cells. By doing so, we show that the pharmacology of
-arrestin
translocation underlies the inherent agonist dependence of
2AR endocytosis. More interestingly, our results
demonstrate that the cellular fate of
-arrestin2 (or
-arrestin1)
is differentially regulated following activation of distinct GPCRs. A
popular assumption in the field has been that
-arrestins are
associated with internalized receptors in intracellular vesicles
(reviewed in Refs. 21, 27, and 28). While this appears to be true for
some receptors such as the AT1AR and NTR, we found that in
the case of the
2AR, D1AR, and
ETAR,
-arrestins do not traffic with the receptors to
endosomes and appear to dissociate from receptor-bearing vesicles
shortly following their formation. In addition, swapping of the
carboxyl-terminal regions between the AT1AR and
2AR switches the phenotype of both receptors in terms of
their ability to mobilize plasma membrane-associated
-arrestin to
cytosolic vesicular structures. This indicates that the
carboxyl-terminal region of the receptors is important in determining
receptor/
-arrestin association and receptor-mediated
-arrestin
cellular distribution to the plasma membrane and/or endosomes.
Considerable effort has been expended to uncover receptor endocytic
motifs underlying the agonist-dependent endocytosis of GPCRs. The expectation was that discrete amino acid motifs on the
GPCRs, similar in function to those utilized by single transmembrane spanning receptors, would be identified (21, 29, 30). However, the
matching pharmacology for -arrestin translocation and
2AR sequestration described here strongly indicates that
-arrestin binding replaces the exposure of discrete amino acid
motifs as the agonist-driven switch regulating receptor endocytosis. In contrast, the agonist dependence of the endocytosis of receptor tyrosine kinases is thought to involve the exposure of
tyrosine-containing motifs on the receptors (30-34).
It is likely that the effect of agonist binding to the receptor is to
induce an intramolecular rearrangement of multiple intracellular GPCR
domains. This results in a generalized receptor conformation that is
necessary to promote GRK phosphorylation and -arrestin binding.
Interestingly, this conformational requirement may account for the
failure of some opioid agonists to induce the sequestration of the mu
opioid receptor and the sequestration defect described for the
2AR-Y326A mutant (11, 24, 35). Furthermore, experiments using
2AR/
3AR chimeric receptors showed
that normal sequestration of the resulting chimeras required the
swapping of several intracellular domains, including the first and
second intracellular loops and the carboxyl tail between the two
receptor proteins (36). Thus, it would appear that the endocytic
switching function of
-arrestins is related to their role in
receptor desensitization, i.e. the binding to and uncoupling
of the receptor from its G protein. Once receptors are bound to
-arrestin, they then gain the ability to traffic to clathrin-coated
pits. Indeed, photobleaching experiments of
2AR-GFP in
HEK 293 cells suggest that desensitized receptors (i.e.
complexed with
-arrestins) are free moving in the plasma membrane
and that their movement to coated pits is not rate-limiting for
2AR endocytosis (23). This suggests that the interaction of
-arrestin with the
2AR represents the initiating
event for receptor endocytosis.
Whereas there is growing evidence supporting -arrestins as a general
endocytic intermediate for many GPCRs, it is somewhat surprising that
-arrestins do not traffic to the same cellular compartments upon
activation of distinct receptors. The internalization of
-arrestin
with some GPCRs but not with others suggests that the properties of
-arrestin/receptor interactions differ for different GPCRs. For
example, in the case of the
2-adrenergic receptor,
-arrestins were demonstrated to bind to the third intracellular loop, whereas
-arrestin interactions with the
2AR
appear to involve multiple receptor domains including the receptor
carboxyl terminus (36, 37). In addition, peptide inhibition studies suggest that the third and, to a lesser extent, the first intracellular loops of rhodopsin may play an important role in arrestin binding to
light-activated forms of rhodopsin (38). In the present study, these
differences are highlighted by the ability of
-arrestin2 to
internalize with the AT1AR and NTR, but not the
2AR, D1AR, and ETAR. In the case
of the AT1AR, it appears that the carboxyl-terminal tail
contributes directly to
-arrestin interactions. While it is
plausible that these differences are the consequence of the high
affinity and slow off-rate of peptidic ligands that might trap
receptors in a conformation favoring stable
-arrestin binding, the
observation that the ETAR do not internalize with
-arrestin bound does not support this apparently simple explanation.
Rather, the present experiments with an
AT1AR-
2AR carboxyl-terminal tail chimera as
well as a
2AR-AT1AR carboxyl-terminal tail
chimera suggest that these differences appear to be regulated by
differences in either the tertiary structure or
-arrestin-interacting sequences in the carboxyl-terminal domains of
these receptors. Presumably, the carboxyl-terminal domain in
conjunction with other intracellular receptor domains determines the
relative stability of receptor/
-arrestin complexes.
In a previous study, we have reported that, although the
AT1AR is capable of utilizing a dynamin- and
-arrestin-independent endocytic pathway, co-expression of
-arrestins significantly increases the level of
dynamin-dependent AT1AR internalization (16).
This suggests that the AT1AR has the ability to directly interact with
-arrestins. In this study, using the GFP-conjugated
-arrestin2, we were able to visualize an
agonist-dependent co-trafficking of the receptor with
-arrestins to endocytic vesicles. This represents the first direct
demonstration of AT1AR association with
-arrestins. As
GRKs were shown to phosphorylate and desensitize the AT1AR (39), it is probable that, similar to their role in
2AR
function,
-arrestins also play an important role in
AT1AR regulation by binding to the GRK-phosphorylated form
of the receptor. In addition to the AT1AR and
2AR, we also visualized the trafficking of several other
receptors with
-arrestins, including the D1R,
ETAR, and NTR. The activation of the NTR resulted in an
"AT1AR-like"
-arrestin distribution pattern, whereas
the activation of the other two receptors triggered a membrane
localization of
-arrestins similar to that observed for the
2AR. These observations indicate that, while
GPCR/arrestin interactions represent a general GPCR regulatory mechanism, the stability of receptor/
-arrestin complexes differs from receptor to receptor.
The binding of arrestin proteins to GRK-phosphorylated GPCRs serves to
desensitize various GPCRs, following which, the agonist-promoted receptor internalization is proposed to contribute to receptor dephosphorylation and resensitization (21, 40). A critical step leading
to effective GPCR dephosphorylation and resensitization is the
dissociation of the GPCR/arrestin complex, since dephosphorylation of
rhodopsin was demonstrated to be blocked when the receptor is
arrestin-bound (20). Our results indicate that -arrestins dissociate
from the
2AR shortly following the redistribution of
-arrestins to coated pits. This early dissociation of
GPCR/
-arrestin complexes is presumably appropriate to allow the
2AR to associate with receptor phosphatase and
dephosphorylate in early endosomes (15). On the other hand, for GPCRs
that internalize with
-arrestins bound, it might be expected that
the kinetics of dephosphorylation of these receptors would be slower.
While
-arrestin binding to receptors may be a general feature of
GPCR regulation, our results suggest that the nature of this
association or the stability of receptor/
-arrestin complex differs
depending upon the receptor studied. Therefore, studies on the
dissociation of receptor/
-arrestin complex should be valuable for
understanding the mechanisms by which receptor desensitization and
resensitization are achieved. The development of mutant or chimeric
receptors with altered ability to interact with
-arrestins should
greatly facilitate this goal.
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ACKNOWLEDGEMENT |
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We thank Sobha Budduluri for expert technical assistance.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant NS 19576, an unrestricted Neuroscience Award from Bristol-Myers Squibb, and an unrestricted grant from Zeneca Pharmaceutical Co. (to M. G. C.), Heart and Stroke Foundation of Ontario Grant NA-3349 (to S. S. G. F.), and National Institutes of Health Grant HL 03422 (to L. S. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of a Merck Frosst Canada postdoctoral fellowship.
Recipient of a fellowship award from the Heart and Stroke
Foundation of Canada.
** Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, Box 3287, Duke University Medical Center, Durham NC 27710. Tel.: 919-684-5433; Fax: 919-681-8641.
Recipient of a McDonald scholarship from the Heart and Stroke
Foundation of Canada.
2 R. Oakley, personal communication.
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ABBREVIATIONS |
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The abbreviations used are:
GPCR, G
protein-coupled receptor;
AT1AR, angiotensin II type 1A
receptor;
AT1AR-2AR-CT, chimeric
AT1AR mutant with the
2AR carboxyl-terminal
tail;
2AR,
2-adrenergic receptor;
2AR-AT1AR-CT, chimeric
2AR
mutant with the AT1AR carboxyl-terminal tail;
arr2GFP, green fluorescent protein conjugate of
-arrestin2;
arr1GFP, green fluorescent protein
conjugate of
-arrestin1;
D1AR, dopamine D1A receptor;
ETAR, endothelin type A receptor;
GFP, green fluorescent
protein;
GRK, G protein-coupled receptor kinase;
HA, hemagglutinin;
MEM, minimal essential medium;
NTR, neurotensin receptor.
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REFERENCES |
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