From the Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232
Received for publication, September 17, 2002, and in revised form, January 6, 2003
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ABSTRACT |
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The vast majority of G protein-coupled
receptors are desensitized by a uniform two-step mechanism:
phosphorylation of an active receptor followed by arrestin binding. The
arrestin·receptor complex is then internalized. Internalized receptor
can be recycled back to the plasma membrane (resensitization) or
targeted to lysosomes for degradation (down-regulation). The
intracellular compartment where this choice is made and the molecular
mechanisms involved are largely unknown. Here we used two arrestin2
mutants that bind with high affinity to phosphorylated and
unphosphorylated agonist-activated G protein-coupled receptors
(GPCRs)1 are the largest
known group of sensor proteins. There are over 1,000 members of this
family that respond to a wide variety of stimuli: light, odorants,
hormones, neurotransmitters, peptides, extracellular calcium, etc. (1). Activated GPCRs catalyze GDP/GTP exchange on heterotrimeric G proteins,
whereupon the GTP-liganded G protein Agonist bound to the internalized arrestin·receptor complex likely
dissociates due to low internal pH in the endosomes (1). The loss of
active receptor conformation promotes arrestin dissociation, whereupon
the receptor can be dephopshorylated and recycled to the plasma
membrane. The release of receptor-bound arrestin, kinetically limited
by the stability of the arrestin·receptor complex, is the first step
in this process. Both non-visual arrestins preferentially bind to the
phosphorylated agonist-activated form of their cognate GPCRs (10-13).
However, their binding to the phosphorylated inactive form is also
relatively high (11-13), suggesting that arrestin dissociation from
internalized phosphoreceptor may be rate-limiting. Recently we have
constructed several structurally diverse mutants of both non-visual
arrestins that bind to activated receptors in a
phosphorylation-independent fashion (10, 11, 13). In contrast to wild
type (WT) arrestin2 binding to phosphoreceptor, the binding of the
arrestin2(R169E) and arrestin2(3A) phosphorylation-independent mutants
to unphosphorylated receptor is strictly
activation-dependent. We expected that in cells these
mutants would bind primarily to the unphosphorylated receptor and that
upon internalization the mutant arrestin·receptor complex would
dissociate substantially faster than the complex of WT arrestin2 with
phosphoreceptor. Here we used phosphorylation-independent mutants to
examine how this change in the properties of the arrestin·receptor
complex affects receptor trafficking in cells.
Note that in this paper we use the systematic names of arrestin
proteins. The synonyms of arrestin2 are Direct arrestin-binding assay was performed with purified
reconstituted In vitro receptor phosphorylation assay was performed by
incubating 450 fmol of purified reconstituted Cell Culture and Transient Transfection--
HEK-tsA201 cells, a
clone of human embryonic kidney (HEK) 293 cells stably expressing the
simian virus 40 large T antigen, were used. The cells were grown in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal calf serum at 37 °C in a 5% CO2 environment. At
60-70% confluence in T75 flasks, the cells were co-transfected with 5 µg of pcDNA3- Receptor Internalization and Down-regulation Assays--
Cells
were harvested by trypsin/EDTA 48 h post-transfection. The cells
were then divided and treated in suspension in PBS supplemented with 1 mM ascorbic acid (for 1 and 2 h incubation) with or
without 10 µM ( Western Blotting--
The expression of arrestins and GRK2 in
each experiment was measured by quantitative Western blotting (with
corresponding purified proteins as standards), as described (11). Mouse
monoclonal anti-arrestin F4C1 (15) and anti-GRK 2/3 (Upstate
Biotechnology) were used as the primary antibodies, and horseradish
peroxidase-conjugated goat anti-mouse (Roche Molecular Biochemicals)
was used as the secondary antibody. The bands were visualized with
Super Signal ECL reagent (Pierce) and quantified, as described (11,
16).
Confocal Microscopy--
This was performed on an Olympus
Axiovert dual laser fluorescent confocal microscope. The cells were
grown, treated, and subsequently fixed (3.7% paraformaldehyde; 20 min
on ice) and permeabilized (0.1-0.05% Triton X-100; 3 min) on Lab-Tek
chambered cover glass (Nunc). Arrestins were visualized with F4C1 (15)
followed by rhodamine-conjugated (red) goat anti-mouse antibody
(Molecular Probes); HA- Quantitative Image Analysis and Statistics--
To make confocal
microscopy data as reliable and quantitative as the data obtained by
direct measurements of arrestin and ligand binding, for each time point
in every experiment we collected 25-70 images, quantified relevant
signals, and statistically analyzed the results. Images collected on
the confocal microscope were analyzed using C-Imaging systems software
Simple 32 (Compix Inc, Cranberry Township, PA).
To analyze intracellular antibody deposition in the presence of WT
arrestin2 or R169E mutant (Fig. 4), two-color confocal images of green
Internalization and Recycling Assays--
Assays using
reversible surface receptor biotinylation (Fig. 5) were performed
essentially as described (17, 18). Briefly, HEK-tsA201 cells were
transfected with a plasmid encoding
For each experiment, three wells were not biotinylated (negative
control), three were left unreduced after biotinylation (total biotinylated receptor, control for the efficiency of biotinylation), and three wells were reduced immediately after biotinylation (control for the efficiency of biotin removal). The amount of biotinylated In Vivo Receptor Phosphorylation--
This was performed
essentially as described (47). HEK 293 cells were transfected with
HA- The Binding of Phosphorylation-independent Arrestin2 (R169E) to
Phosphorylation-independent Arrestin2 Mutant Reduces the Proportion
of Internalized Receptor and Prevents Agonist-induced
To quantify cell surface and total
Interestingly, the effect of the R169E mutant on receptor
down-regulation increases with its expression level in the range of
2.7-21.2 fmol/µg of total protein (i.e. 3-25-fold molar
excess over endogenous WT arrestin2 in HEK 293 cells, as determined by quantitative Western blot) (F(1,27) = 36.4;
p = 0.0001). The effect reaches its maximum at
the expression level of the mutant higher than 10 fmol/µg of total
protein (>10-fold molar excess over endogenous). In contrast,
the level of WT arrestin2 overexpression in the same range does not
affect Both WT Arrestin2 and the R169E Mutant Support Rapid Endocytosis of
To quantify anti-HA antibody accumulation inside the cell, we analyzed
the internalization of the red antibody at different time points upon
agonist exposure (0, 10, 30, and 60 min). To this end, 48-71 cells per
group, expressing
To ascertain that this is the case using a different approach, we
employed reversible surface biotinylation (17, 18). In this paradigm,
surface proteins including
To compare the rates of recycling directly, after the same 2 h
incubation with isoproterenol to achieve receptor internalization, we
removed the agonist and NH4Cl and allowed the cells to
recycle internalized receptor for 10 min in the presence of 10 µM antagonist alprenolol prior to the removal of surface
biotin. Note that only the percentage of the receptor remaining
inside the cell can be measured (Fig. 5). Thus, the difference between
the amount of internalized (biotinylated) receptor after agonist
incubation with and without subsequent antagonist incubation represents
recycled receptor. Therefore, we calculated the percent of the recycled receptor as the ratio of this difference to the total amount of the
receptor internalized. We found that the same proportion of internalized receptor (61-63%) is recycled in 10 min in control cells
and in cells overexpressing WT arrestin2, even though the amount of
internalized receptor was significantly higher in the latter case (Fig.
5). A somewhat smaller proportion of the large amounts of receptor
internalized in the presence of NH4Cl is recycled in 10 min. Again, it is the same (39-41%) in control cells and cells
overexpressing WT arrestin2. In sharp contrast, over 90% of the
receptor internalized in complex with arrestin2(R169E) mutant was
recycled, even in cells that were forced to internalize 75% of the
receptor by endosome alkalinization (Fig. 5). These results clearly
demonstrate that the rate of
To summarize, three independent lines of evidence suggest that
arrestin2(R169E) promotes Overexpression of GRK2 Rescues The Effects of a Structurally Distinct Phosphorylation-independent
Arrestin2(3A) Mutant Mimic the Effects of R169E--
To ascertain that
phosphorylation-independent binding of R169E to the receptor rather
than some unanticipated peculiarity of this particular mutant is indeed
the cause of the observed change in receptor trafficking, we tested
another structurally distinct arrestin2 mutant with a similar phenotype
(13) (Fig. 7). For these experiments, we
chose arrestin2(I386A,V387A, F388A), referred to below as
arrestin2(3A). In contrast to R169E, the 3A mutation does not affect
the polar core and destabilizes a different intramolecular interaction
holding arrestin in its basal (inactive) conformation: the
three-element interaction between N-terminal Overexpression of Both Phosphorylation-independent Arrestin2
Mutants Suppresses Agonist-induced Increase in
Collectively, these data strongly suggest that the ability of the R169E
and 3A mutants to bind agonist-activated unphosphorylated Receptor sorting in endocytic pathways is one of the fundamental
issues in cell biology. This problem has two distinct aspects. First,
the cell can simultaneously direct different receptors to different
recycling and/or degradative pathways (28-32). This appears to be
accomplished via selective interaction of some receptors with certain
components of the sorting machinery (reviewed in Ref. 1). Second, cells
apparently change the trafficking and ultimate fate of the same
receptor under different conditions. Here we are attempting to address
this latter aspect using extensively characterized A wealth of experimental evidence indicates that agonist-activated
GRK-phosphorylated Short-term agonist exposure usually does not lead to any appreciable
receptor down-regulation, whereas persistent GPCR stimulation results
in a progressive loss of the receptor due to its degradation in the
lysosomes (1, 14, and references therein). An increase in the time of
Certain GPCRs tend to recycle rapidly, whereas others stay internalized
for a long time (30, 32, 37). The presence of clusters of serines and
threonines phosphorylated by GRKs in the C termini of several receptors
has been shown to determine how long arrestin stays in the complex with
the receptor and how rapidly the receptor resensitizes (32, 34).
Different receptors also demonstrate varying preferences for the two
non-visual arrestins (12, 37-39), which, in their turn, differentially
regulate trafficking even of the same receptor (35, 37-39). Certain
mutations in GPCR C termini or an exchange of these elements between
GPCRs often switch their arrestin preferences and/or trafficking
patterns (30, 32, 38-40). All these experimental approaches have been extensively used to delineate the mechanisms of receptor trafficking and substantially improved our understanding of differential sorting of
different GPCRs in the same cell (14, 32, 37-40). However, evidence
that the two non-visual arrestins as well as the C-terminal regions of
the receptors themselves interact with numerous other partners
(reviewed in Refs. 1 and 31) is accumulating rapidly. These additional
interactions are likely to be affected by the above mentioned
experimental manipulations, which makes these approaches unsuitable for
the elucidation of mechanisms that change the fate of the same receptor
in the same cell under different conditions.
Phosphorylation-independent mutants of non-visual arrestins provide a
unique tool for selective manipulation of the arrestin-receptor interface without introducing any collateral changes in the process. According to the widely accepted model of GPCR de- and re-sensitization (1, 31), internalized receptor exists in three main forms: arrestin·phosphoreceptor complex, free phosphoreceptor, and partially and fully dephosphorylated receptor (Fig.
9). Because high-affinity arrestin
binding requires receptor multi-phosphorylation (2, 24, 41-43), free
phosphoreceptor is heterogeneous as the result of the variation in its
original phosphorylation level and its progressive dephosphorylation.
Experimental evidence suggests that only fully dephosphorylated
receptor is recycled (25, 33), which makes perfect sense biologically.
There are two distinct types of conceivable mechanisms directing
internalized receptor to the lysosomes. Free phosphoreceptor and/or
receptor·arrestin complex may be specifically recognized and
transported there. Arrestin-dependent
ubiquitination of 2-adrenergic receptor to
manipulate the receptor-arrestin interface. We found that mutants
support rapid internalization of
2-adrenergic receptor similar to
wild type arrestin2. At the same time, phosphorylation-independent arrestin2 mutants facilitate receptor recycling and sharply reduce the
rate of receptor loss, effectively protecting
2-adrenergic receptor
from down-regulation even after very long (up to 24 h) agonist
exposure. Phosphorylation-independent arrestin2 mutants dramatically
reduce receptor phosphorylation in response to an agonist both in
vitro and in cells. Interestingly, co-expression of high levels
of
-adrenergic receptor kinase restores receptor down-regulation in
the presence of mutants to the levels observed with wild type
arrestin2. Our data suggest that unphosphorylated receptor
internalized in complex with mutant arrestins recycles faster than
phosphoreceptor and is less likely to get degraded. Thus, targeted
manipulation of the characteristics of an arrestin protein that binds
to a G protein-coupled receptors can dramatically change receptor
trafficking and its ultimate fate in a cell.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit and the free
-dimer modulate the activity of various effectors, including adenylyl cyclase, phospholipase C, cGMP phosphodiesterase, ion channels, etc. (1). The same active receptor conformation that interacts with G proteins is phosphorylated by GPCR-kinases (GRKs) (1).
Arrestins then bind to the active phosphorylated state of the receptor
(2). Arrestin binding prevents further G protein interaction
(apparently, by simple steric exclusion (3)), often targeting receptors
to the coated pits due to the high affinity of non-visual arrestins for
various components of the internalization machinery: clathrin (4),
clathrin adaptor AP2 (5), and N-ethylmaleimide-sensitive fusion protein
(NSF) (6). Arrestins also couple GPCRs to alternative, G
protein-independent signaling pathways (1), such as activation of Src
(7), c-Jun NH2-terminal kinase 3 (8), cRaf-1 (9), and
extracellular signal-regulated kinase (7, 9).
-arrestin and
-arrestin1; arrestin3 is also called
-arrestin2.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptor (
2AR) and in
vitro-translated radiolabeled arrestins, as described (12).
Briefly, the receptor was phosphorylated with GRK2 in the presence of
50 µM of isoproterenol and washed to remove the agonist.
In vitro-translated tritiated arrestins (50 fmol) were mixed
in 50 mM Tris-HCl, pH 7.5, 0.5 mM
MgCl2, 50 mM potassium acetate with
phosphorylated or unphosphorylated
2AR (100 fmol/assay) in the
presence of 50 µM
-agonist isoproterenol or 50 µM
-antagonist alprenolol in a final volume of 50 µl. The samples were then incubated for 35 min at 30 °C,
immediately cooled on ice, and loaded onto 2-ml Sepharose 2B columns
equilibrated with 10 mM Tris-HCl, pH 7.5, and 100 mM NaCl. The bound arrestin eluted with the
receptor-containing membranes in the void volume (between 0.5 and 1.1 ml). Nonspecific binding determined in the presence of 0.3 µg
liposomes (<10% of the total binding and <1% of the arrestin
present in the assay) was subtracted.
2AR with 400 fmol of
purified GRK2 in the presence of 50 µM of isoproterenol
in 10 µl of 20 mM Tris-HCl, pH 7.5, 2 mM
EDTA, 5 mM MgCl2, 50 µg/ml bovine serum
albumin, and 10 µM cold ATP + [
-32P]ATP
(final specific activity 10-15 dpm/fmol) with or without the indicated
concentrations of Escherichia coli-expressed purified WT
arrestin2 or R169E mutant (10, 11) for 10 min at 30 °C. After both
arrestin and GRK2 were added to the receptor, the reactions were
started by the addition of ATP and stopped by the addition of 5 µl of
SDS sample buffer. The samples were subjected to SDS-PAGE, the gels
were dried, exposed,
2AR bands were cut out, and the incorporated
radioactivity was quantified in a liquid scintillation counter.
2AR-GFP (14) and 6-10 µg of
pcDNA3-arrestin2 or its R169E or 3A mutants. In the experiments
shown in Figs. 6 and 7, the cells were also co-transfected with 10 µg
of pcDNA3-GRK2 (14). For the experiments shown in Fig. 4, the cells
were transfected with 5 µg of the indicated arrestin2 construct along
with 2 µg of pcDNA3-
2AR-GFP plus 2 µg of hemagglutinin
(HA)-
2AR (7) in 60-mm tissue culture dishes. LipofectAMINE
(Invitrogen) was used according to the manufacturer's instructions for transfection.
)isoproterenol (Sigma). At the end of the incubation, the cells were washed three times with ice-cold PBS.
For 24-h incubation the cells were treated in six well plates and
harvested after the wash. Total and cell surface receptor was measured
with 0.2 nM [125I]pindolol (1 h; 22 °C)
and 15 nM [3H]CGP-12177 (PerkinElmer Life
Sciences) (3 h; 14 °C), respectively, as described (14). Nonspecific
binding was determined in the presence of 10 µM
(
)alprenolol (Sigma) and subtracted. Internalized receptor was
calculated as the difference between total and cell-surface receptor.
Cells expressing 1-2.5 pmol
2AR per 1 mg of total protein were used.
2AR was labeled with monoclonal anti-HA
rhodamine-conjugated antibody (Boehriger Mannheim), the signal was
amplified with rhodamine-conjugated goat anti-mouse antibody;
2AR-GFP (green) was detected by its intrinsic fluorescence.
2AR-GFP and red HA-
2AR labeled with anti-HA rhodamine-conjugated
antibody were collected and saved as two-color and red single-color
images. The membranes of the cells were carefully outlined using
two-color images (green
2AR-GFP and red HA-
2AR) to improve the
visibility of the membrane, particularly after longer
incubations. Using red single-color images converted to grayscale, the
area of red fluorescence corresponding to the receptor was thresholded
based on the grayscale, and the total amount of red fluorescence
(anti-HA rhodamine-conjugated antibody) and intracellular red
fluorescence were measured for each cell. To measure only intracellular
receptor, we excluded the receptor associated with the cell membrane by
disqualifying any fluorescent object that touched the membrane outline.
The results were expressed for each cell as a percentage of the total
cell-associated red fluorescence localized inside the cell. The data
were analyzed by two-way analysis of variance with protein (WT
versus R169E) and incubation time as main factors. To
determine the dependence of intracellular antibody accumulation on
incubation time, separate analysis of variance for each arrestin
protein with incubation time as the main factor was used. Post hoc
comparison and contrasts to compare individual time points were used
where appropriate. For all statistical analyses, p < 0.05 was considered significant.
2AR-GFP with and without
plasmids encoding WT arrestin2 and R169E mutant. 24 h
post-transfection the cells were re-plated in
poly-D-lysine-covered 24-well plates and serum-starved
overnight. The cells were washed three times with ice-cold PBS
supplemented with 1 mM CaCl2 and 0.5 mM MgCl2 (PCM), biotinylated on ice with 6-10
mg/ml of Sulfo-NHS-SS-Biotin (Pierce) in PCM for 40-50 min. The cells
were washed 3 times with PCM with 1 mg/ml bovine serum albumin and
incubated for 2 h at 37 °C in DMEM supplemented with 1 mM ascorbic acid in the presence or absence of 10 µM isoproterenol with or without 20 mM
NH4Cl (to inhibit recycling) (25). At the end of the
incubation, plates were cooled on ice and washed three times with
ice-cold PCM. To quantify internalized receptor, surface biotin was
removed by two 15-min cycles of reduction with ice-cold 150 mM glutathione, pH 8.75, in 150 mM NaCl,
followed by neutralization for 20 min with 50 mM
iodoacetamide in PCM. To determine the rate of receptor recycling, the
plates after agonist incubation and wash were further incubated in DMEM
with 10 µM antagonist alprenolol for 10 and 20 min at
37 °C and then reduced and neutralized. The cells in each well were
lysed at room temperature in 0.1 ml of extraction buffer (EB) (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 mM lysine, 2 mM
benzamidine, 1 mM phenylmethylsulfonyl fluoride) supplemented with 1% SDS. The lysates were transferred to an Eppendorf tube, diluted with 0.5 ml of EB with 1% Triton X-100, and sonicated for 10 s on ice. Cell debris were pelleted by centrifugation for 20 min at 4 °C. The upper 0.5 ml were transferred to an Eppendorf tube containing 15 µl of UltraLink immobilized avidin gel (Pierce) and incubated overnight with gentle rolling at 4 °C (the remainder was used to quantify arrestin expression by Western). To remove unbound
protein, the gel was washed twice with EB with 1% Triton X-100 and
twice with EB. The gel was then resuspended in 30 µl of SDS sample
buffer. The samples were subjected to SDS-PAGE and quantitative Western
blotting using primary anti-GFP Living Colors A.v. peptide antibody
(Clontech) (1:1,000) and secondary horseradish peroxidase-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories) (1:12,500). The bands were visualized with Super Signal
ECL reagent (Pierce) and quantified, as described (11, 16), on Fluor-S
MultiImager (Bio-Rad) using Quantity One 4.2.0 software.
2AR-GFP in these samples was quantified as above. The total
biotinylated receptor in each experiment was used as a reference point
(100%) for the quantification of internalized receptor. Only the data from the experiments where the amount of biotinylated
2AR-GFP was
high and the removal of the biotin was complete were used. In all
experiments three wells per experimental condition were used.
2AR to achieve receptor expression of 3-5 pmol/mg protein and
either control pcDNA3 vector or constructs expressing WT
arrestin2, arrestin2(R169E), or arrestin2(3A) mutants (>25 fmol/µg
of total protein, i.e.
25-fold endogenous arrestin2). Transfected cells were re-plated into 12-well plates, serum-starved overnight, washed 3× with 2 ml of phosphate-free DMEM (PF-DMEM), incubated in PF-DMEM for 7 min, and washed with the same medium 3×
with 2 ml per well. For receptor and arrestin quantification by
[125]IPIN binding and Western, the cells were scraped into 50 mM Tris-Hcl, pH 8.0, 100 mM NaCl, 5 mM EGTA, 2 mM benzamidine, 1 mM
phenylmethylsulfonyl fluoride, and homogenized by pipetting. For
[32P] labeling, the cells were incubated for 2 h in
1 ml of PF-DMEM supplemented with 0.25 mM ascorbic acid,
170 µCi of [32P]phosphoric acid (PerkinElmer Life
Sciences) with or without 2.5 µM isoproterenol, as
well as with 2.5 µM isoproterenol plus 20 mM
NH4Cl (to inhibit receptor recycling) at 37 °C.
Following incubation, the cells were chilled on ice, washed with
ice-cold PBS, and solubilized in 0.5 ml per well of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Igepal
CA-630, 0.5% sodium deoxycholate, 0.1 mM
Na3VO4, 10 mM
Na4P2O7, 10 mM NaF, 5 mM EGTA, 3 mM benzamidine, 1 mM
phenylmethylsulfonyl fluoride). Lysates were centrifuged for 20 min at
100,000 rpm (rotor TLA100, Beckman TL-100 tabletop ultracentrifuge). To
reduce background, supernatants were pre-cleared by incubation with 10 µl of protein G-agarose (Boehringer-Mannheim) for 3 h at
4 °C. After the removal of precipitated material, the supernatants
were transferred to a fresh tube and incubated for 1 h with 1 µg
of 3F10 high-affinity rat monoclonal anti-HA antibody (Boehringer-Mannheim) at 4 °C. After the addition of 10 µl of protein G-agarose beads, incubation was continued overnight (14-15 h)
at 4 °C. Beads were washed twice for 20 min at 4 °C with 1 ml of
lysis buffer, twice with high salt buffer (lysis buffer containing
0.1% Igepal, 0.05% sodium deoxycholate, and 500 mM NaCl),
and twice with the same buffer without NaCl. Immune complexes were then
resuspended in 2× SDS sample buffer and separated on 8% SDS-PAGE gel.
After autoradiography, 32P incorporation into receptor band
was quantified on Fluor-S MultiImager (Bio-Rad) using Quantity One
4.2.0 software. The identity of the radiolabeled band with HA-
2AR
was further confirmed by Western blotting of immunoprecipitated
material with HA-Tag mouse monoclonal antibody (Cell Signaling).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic Receptor Is Strictly Activation-dependent
and Inhibits Receptor Phosphorylation by GRK2--
A major
conformational rearrangement of the arrestin molecule is necessary for
high-affinity receptor binding (2, 19-22). The basal (inactive)
conformation of arrestin is stabilized in part by the polar core, an
unusual network of buried solvent-excluded charged residues in the
fulcrum of the two-domain arrestin molecule (20-24). Receptor-attached
phosphates intrude upon the polar core and upset its charge balance
(20-24). Charge reversal mutations of the main phosphate sensor in the
polar core, Arg-175 in visual arrestin (16, 20, 23), Arg-169 in
arrestin2 (10, 11), and Arg-170 in arrestin3 (13), sufficiently
destabilize the polar core to allow the mutant to bind with high
affinity to any activated form of the receptor, phosphorylated or not
(10, 11, 13, 16, 20-24). Although the binding of WT arrestin2 to
phosphorylated
2AR (P-
2AR) is enhanced by receptor activation,
its binding to inactive P-
2AR (in the presence of an antagonist) is
quite substantial (Fig. 1A).
The phosphorylation-independent R169E mutant demonstrates similar high
binding to active and inactive P-
2AR. In contrast, its binding to
unphosphorylated
2AR is strictly activation-dependent
(Fig. 1B). By virtue of high-affinity binding to
unphosphorylated receptor, arrestin2(R169E) can be expected to compete
with receptor kinases. Indeed, in sharp contrast to WT arrestin2,
purified R169E mutant effectively reduces
2AR phosphorylation by
GRK2 in vitro (Fig. 1C). In these experiments we
used 40 nM GRK2 because physiological concentrations of
GRK2 in the human brain are in the range of 16-60
nM.2 Because both
proteins compete for the same agonist-activated receptor, a substantial
molar excess of R169E mutant (2.5-25-fold) is necessary for
significant inhibition of receptor phosphorylation (Fig.
1C). These functional characteristics of arrestin2(R169E) suggest that upon its overexpression in cells the phosphorylation of
2AR by endogenous GRK2 will be attenuated and that unphosphorylated
2AR will form complexes with the mutant arrestin2, which are likely
to dissociate upon receptor deactivation due to agonist loss in
endosomes faster than the complexes of WT arrestin2 with phosphorylated
2AR.
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Fig. 1.
Comparative functional characteristics of WT
arrestin2 and R169E phosphorylation-independent mutant. WT
arrestin2 (A) binds with high selectivity to phosphorylated
activated (by 50 µM agonist isoproterenol)
(P- 2*) and inactive (in the presence of 50 µM antagonist alprenolol) (P-
2)
2AR, whereas arrestin2(R169E) (B) also binds to activated
unphosphorylated (
2*) receptor. As a result, E. coli-expressed purified (10, 11) R169E mutant, but not purified WT
arrestin2, effectively competes with GRK2 for agonist-activated
2AR, thereby inhibiting
2AR phosphorylation by GRK2
(C). Means ± S.D. of 3 experiments performed in
duplicate as described under "Experimental Procedures" are
presented.
2AR
Down-regulation in HEK 293 Cells--
To test how the change in
arrestin2 functional characteristics due to the R169E mutation affects
the trafficking of
2AR, we co-transfected HEK 293 cells with
2AR-GFP fusion (which is a fully functional
2AR (14)) with WT
arrestin2 and R169E mutant. Both arrestins are rapidly mobilized to the
plasma membrane upon agonist stimulation and can be subsequently
detected co-localized with the receptor in intracellular vesicles near
the membrane (Fig. 2), suggesting that
the R169E mutant supports receptor internalization. However, at later
time points (e.g. 60 min in Fig. 2, A and
B) we found a substantially lower proportion of
2AR-GFP
in the endocytic vesicles in the presence of the R169E mutant than with
WT arrestin2. In cells expressing the R169E mutant, most of the
receptor is present either on the membrane or in small endocytic
vesicles close to it, and most of the R169E mutant itself appears in
the cytoplasm (Fig. 2B). In all cells examined, a virtually
contiguous green outline of the plasma membrane is visible after 1-2 h
of agonist incubation. In contrast, in cells expressing WT arrestin2 most of
2AR-GFP and detectable amounts of arrestin2 gather in large
intracellular vesicles, and a substantially smaller proportion of
2AR-GFP is found at or near the plasma membrane, so that the green
outline is no longer contiguous and often is not visible at all.
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Fig. 2.
Both WT arrestin2 and R169E mutant are
rapidly mobilized by activation of 2AR.
HEK 293 cells expressing
2AR-GFP and either WT arrestin2
(A) or R169E mutant (B) were challenged with 10 µM isoproterenol for the indicated periods and fixed. The
receptor was visualized by its own fluorescence (green), and
arrestins were visualized by immunocytochemistry with F4C1
anti-arrestin primary (15) and rhodamine-labeled secondary antibodies
(red). Arrestin-receptor co-localization appears as the
yellow color. The images of typical cells shown were
processed using Adobe Photoshop software.
2AR in cells expressing either
form of arrestin2, we measured the binding of the hydrophylic ligand
[3H]CGP-12177 and the hydrophobic membrane-penetrating
ligand [125I]iodopindolol, respectively (4, 14). Under
continuous stimulation with a saturating concentration of the
2AR
agonist isoproterenol in the presence of WT arrestin2, an increasing
proportion of the receptor is found inside the cells (i.e.
is labeled with [125I]iodopindolol but not with
[3H]CGP-12177). In contrast, only about 10% of the
receptor is internalized at any time point in the presence of the R169E
mutant (Fig. 3A). In our
experiments the abundance of internalized receptor correlates with the
rate of receptor down-regulation, in agreement with a previous report
(14). In the presence of the R169E mutant there is very little loss of
2AR even after 24 h of incubation with the agonist (Fig.
3B). In contrast, almost half of the receptor is lost with
WT arrestin2 after 24 h (Fig. 3B).
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Fig. 3.
Expression of arrestin2(R169E)
mutant reduces the proportion of internalized
2AR and suppresses receptor down-regulation.
HEK 293 cells expressing
2AR-GFP and either WT arrestin2 or R169E
mutant were challenged with 10 µM isoproterenol for the
indicated periods at 37 °C and washed 3 times with ice-cold PBS.
Control cells were incubated without isoproterenol and washed in
parallel. Their cell surface and total
2AR was measured with
hydrophilic ligand [3H]CGP-12177 and with
[125I]pindolol, respectively. A shows
internalized receptor (the difference between the total and cell
surface receptor) expressed as a percentage of the total receptor.
B shows the difference between the amount of total receptor
measured in isoproterenol-treated and control cells at each time point
expressed as a percentage of the total receptor in control cells. Note
that by definition A shows the percentage of functional
internalized receptor that does not bind hydrophilic ligand
[3H]CGP-12177 but still binds
[125I]pindolol at the time of measurement. In contrast,
B shows the percentage of the receptor that is no longer
functional, i.e. the receptor that has lost the ability to
bind [125I]pindolol from time 0 to the time of
measurement. Means ± S.D. of 5-7 experiments in which arrestin
expression levels exceeded 10 fmol/µg protein are presented. *,
p < 0.05; ***, p < 0.001, as compared
with WT.
2AR down-regulation (F(1,30) = 1.053;
p = 0.313). These data support the notion that receptor
trafficking in the presence of WT arrestin2 and its
phosphorylation-independent mutant is fundamentally different and that
a substantial molar excess of the mutant is necessary to out-compete
endogenous arrestin2 (and likely endogenous receptor kinase,
cf. Fig. 1C). In a separate series of
experiments with WT arrestin2 and R169E mutant overexpression we found
that receptor down-regulation at all time points correlates with its
transport to lysosomes (visualized using LysoTracker Red (Molecular
Probes)) (not shown), in accord with previous reports (14).
2AR in HEK 293 Cells--
The very low proportion of internalized
2AR at all time points along with minimum receptor transport to
lysosomes and down-regulation in the presence of arrestin2(R169E) could
be explained by an impaired ability of the mutant to support receptor
internalization. Alternatively, rapid
2AR endocytosis (as suggested
by the association of the mutant with
2AR-GFP on the membrane and in
the endocytic vesicles shown in Fig. 2) coupled with an accelerated
recycling back to the plasma membrane may account for these
observations. To ascertain which is the case, we took advantage of the
sensitivity of the antibody-antigen interaction to pH. We reasoned that
a substantial proportion of an antibody bound to the extracellular
surface of
2AR will dissociate during the time the receptor spends
in the endosomes, thus leaving evidence of its presence there even
after the receptor itself moves out of this compartment. To this end, we co-expressed
2AR-GFP (to follow receptor localization) and HA-tagged
2AR with WT arrestin2 and R169E mutant. Cells were then
pre-labeled with rhodamine-conjugated anti-HA antibody. The free
antibody was washed away, and the cells were challenged with isoproterenol to induce receptor internalization, which was stopped at
different time points. The cells were fixed, and the anti-HA antibody
was visualized either directly by its own fluorescence or after signal
amplification with a rhodamine-conjugated secondary antibody. As shown
in Fig. 4, most of the antibody
(red) stays on the plasma membrane in unchallenged cells,
whereas upon isoproterenol stimulation it accumulates in endocytic
vesicles in cells expressing WT arrestin2 and R169E mutant. The amount
of anti-HA antibody detected inside the cell in the absence of an
agonist does not appreciably change with time (not shown).
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Fig. 4.
R169E mutant supports rapid
2AR internalization, as evidenced by the transport
of anti-receptor antibody into endocytic vesicles. Cells
co-expressing
2AR-GFP (green) and HA-
2AR along with WT
arrestin2 (left) or R169E mutant (right) were
pre-labeled with rhodamine-conjugated anti-HA antibody (red)
then challenged with 10 µM isoproterenol. Images of
typical cells (A) and quantification of antibody
internalization (B) are shown. Statistical analysis of
48-71 cells per experimental group is presented in the text. *,
p < 0.01, as compared with WT.
2AR-GFP and HA-
2AR at comparable levels (as
judged by green and red fluorescence, respectively), were selected in
random view fields. The internalized red as the percentage of total
cell-associated red was quantified and analyzed. Exposure to an agonist
induced rapid internalization of the red antibody in the presence of
both WT arrestin2 and R169E mutant (F(3,449) = 70.0, p = 0.0001) After 10 min incubation, about 50% of the
total red antibody was found inside the cells (Fig. 4B). At
this early time point, the R169E mutant was even more efficient than WT
arrestin2 in mediating internalization of the red antibody. Over the
60-min incubation period, the percentage of the red antibody
internalized in cells expressing the R169E mutant was somewhat lower
than in the cells expressing WT arrestin2. In cells expressing the
mutant arrestin, the percentage of internalized antibody reached a
maximum after 10 min of stimulation and did not change much afterward;
whereas, in cells expressing WT arrestin2, the percentage of
internalized antibody increased with time up to 60-70% at the latest
time point tested. Conceivably, the antibody brought in early on in the
presence of R169E is subsequently transported to lysosomes and
destroyed. We did not further investigate the reason for this
difference because it is quite small and cannot account for the almost
total lack of receptor down-regulation seen in the presence of the
R169E mutant. These data indicate that the receptor is effectively
internalized with both WT arrestin2 and R169E mutant. Interestingly, in
cells expressing phosphorylation-independent arrestin2(R169E), most of
the GFP-labeled receptor (green) is seen on the plasma
membrane even after 1 h-long agonist stimulation, whereas in cells
expressing WT arrestin2, a substantial proportion of the green receptor
co-localizes with red anti-HA antibody in endocytic vesicles. These
data support the idea that the rates of
2AR internalization with
both forms of arrestin2 are similar. Thus, the major difference lies in
the rapid recycling of the receptor internalized in complex with the
R169E mutant.
2AR-GFP are labeled with a biotin
derivative containing an S-S bond between the attachment point and the
biotin moiety. Receptor internalization is then induced by agonist
incubation for 2 h. Reduction with glutathione (which cannot
penetrate the membrane) at this point removes biotin from surface
receptor, so that only internalized
2AR-GFP remains biotinylated
(Fig. 5). When the agonist is washed away
and the cells are further incubated with a saturating concentration of the antagonist alprenolol for 10 min prior to reduction, the biotin moiety is also removed from
2AR-GFP that was recycled back to the
plasma membrane, so that a smaller fraction of the receptor (inversely
proportional to the rate of recycling) remains biotinylated (Fig. 5).
The cells are then lysed and biotinylated
2AR-GFP is bound to
immobilized avidin beads, washed, and subsequently eluted with SDS
sample buffer. Bound biotinylated
2AR-GFP is then visualized and
quantified by Western using anti-GFP antibody (Fig. 5). In control
cells 35 ± 4% of
2AR-GFP is internalized after 2 h of incubation with 10 µM isoproterenol. Overexpression of WT
arrestin2 increases the fraction of internalized receptor from 35 ± 4% to 60 ± 7%, (p < 0.01, as compared with
control), whereas overexpression of R169E mutant significantly
decreases it (to 8 ± 5%; p < 0.01, as compared with
control and WT arrestin2-expressing cells), in good agreement with
radioligand binding data (compare Figs. 3A and 5).
Interestingly, when endosome acidification and receptor recycling was
prevented by 20 mM NH4Cl (25), essentially the same fraction of
2AR-GFP (67-75%) was internalized in all cases (Fig. 5), corroborating the idea that WT and mutant forms of arrestin2 equally support receptor internalization and suggesting again that the
low proportion of
2AR-GFP found inside the cell at any given moment
in the presence of R169E is due to its accelerated recycling.
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Fig. 5.
Accelerated recycling of
2AR-GFP internalized in the presence of
arrestin2(R169E) mutant. Surface receptor was reversibly
biotinylated in HEK 293 cells overexpressing
2AR-GFP (1-2 pmol/mg
protein) alone (C) or co-expressing receptor with 10-14
fmol/µg protein of WT arrestin2 (WT) or arrestin2(R169E)
mutant (R169E), as described under "Experimental
Procedures." The receptor was allowed to internalize for 2 h at
37 °C in the presence of 10 µM
-agonist
isoproterenol (ISO, 2 h) or 10 µM
isoproterenol with 20 mM NH4Cl (ISO,
NH4Cl, 2 h) to inhibit
recycling. In parallel plates, after the internalization with or
without NH4Cl, the agonist was removed and the cells were
allowed to recycle internalized
2AR-GFP for 10 min at 37 °C in
the presence of 10 µM
-antagonist alprenolol
(ISO, 2 h, ALP, 10 min, and
ISO, NH4Cl, 2 h, ALP, 10 min, respectively). Cells
incubated in the absence of ligands served as controls
(Control). Surface biotin was then removed, the cells were
lysed, and biotinylated proteins were isolated on immobilized avidin
resin, eluted with SDS sample buffer, subjected to SDS-PAGE,
transferred to polyvinylidene difluoride membrane, and visualized using
anti-GFP antibody (top panel). The bands were quantified,
and the amount of biotinylated
2AR-GFP was expressed as a percentage
of the total biotinylated
2AR-GFP. Means ± S.D. of 4 independent experiments each performed in triplicate are shown in the
lower three panels. *, p < 0.01, as
compared with WT; a, p < 0.01, as compared
with C; b, p < 0.05, as compared
with C.
2AR-GFP recycling depends on the nature
of the arrestin it is internalized with and that
phosphorylation-independent arrestin2 mutant ensures unusually rapid
recycling of the receptor.
2AR internalization as effectively as wild
type arrestin2. We observed rapid mobilization of R169E mutant to the
receptor in response to agonist (Fig. 2), rapid accumulation of
anti-receptor antibody in endosomes in the presence of R169E (Fig. 4),
as well as high receptor internalization in the presence of R169E
mutant when recycling is blocked by NH4Cl (Fig. 5). These
data are consistent with recent in vitro findings that
arrestin2(R169E) interacts with clathrin and AP2 with even higher
affinity than WT arrestin2 (26). Thus, very fast recycling of
2AR-GFP internalized in complex with R169E mutant (Fig. 5) is the
main reason for the low levels of internalized
2AR in R169E-expressing cells at all time points during agonist incubation (Fig. 3).
2AR Down-regulation in the
Presence of the Phosphorylation-independent
-arrestin
Mutant--
The main mechanistic difference between WT arrestin2 and
the R169E mutant is that the latter binds to unphosphorylated
2AR. We hypothesized that the observed difference in receptor
down-regulation is the result of the formation of an arrestin complex
with unphosphorylated receptor, which is highly sensitive to receptor
deactivation (compare the binding of R169E mutant to inactive
2AR
and agonist-activated
2AR* on Fig. 1B). If that is true,
then enhanced receptor phosphorylation should abolish the difference
(compare the binding of both WT arrestin2 and R169E mutant to inactive
P-
2AR and agonist-activated P-
2AR* on Fig. 1). To test this
hypothesis, we overexpressed GRK2 along with WT and mutant arrestin2,
challenged the cells with isoproterenol, and measured total
2AR
using [125I]iodopindolol. In these experiments, GRK2
expression was 8-24 fmol/µg of total protein (as determined by
quantitative Western blot), i.e. the concentration of the
kinase in these cells was equal to or higher than that of arrestin to
ensure that the mutant cannot effectively compete with the kinase for
2AR* (cf. Fig. 1). As before (Fig. 3B),
without GRK2 we observed a significant time-dependent
receptor loss with WT arrestin2 and very little down-regulation with
R169E (Fig. 6). However, in the presence of overexpressed GRK2 there is a substantial
2AR loss with both forms of arrestin2 (Fig. 6). GRK2 overexpression facilitates
down-regulation in both cases (F(1,27) = 47.7;
p = 0.0001). The effect is most dramatic after 24 h of incubation with agonist, particularly in cells expressing the
R169E mutant, where receptor loss increases from about 14% without
GRK2 to 47% in the presence of GRK2 (F(1,16) = 19.1, p = 0.0005). In fact, there is no significant
difference in the degree of receptor loss in the presence of the two
forms of arrestin2 after 24 h stimulation when GRK2 is
simultaneously overexpressed (F(1,16) = 2.3, p = 0.15), suggesting that
2AR internalized in
complex with arrestin2(R169E) can be successfully targeted to lysosomes
and degraded. These results rule out any unanticipated differences in
the ability of the two forms of arrestin2 to interact with the
internalization machinery that could have affected receptor
trafficking.
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Fig. 6.
Simultaneous overexpression of GRK2
rescues 2AR down-regulation in the presence of
the R169E mutant. HEK 293 cells expressing
2AR-GFP and either
WT arrestin2 or R169E mutant (10-12 fmol/µg protein) with or without
simultaneous overexpression of GRK2 (8-24 fmol/µg protein) were
challenged with 10 µM isoproterenol for the indicated
periods and washed. Total
2AR was measured with
[125I]pindolol. The difference in receptor binding
between control and agonist-treated cells is presented as the receptor
lost due to agonist treatment. Means ± S.D. of 4 experiments in
which arrestin and GRK2 expression levels exceeded 10 and 8 fmol/µg
protein, respectively, are presented. *, p < 0.01, as
compared with WT.
-strand I,
-helix I,
and
-strand XX of the C-tail (13, 21, 22, 24, 27). Similar to its
visual arrestin analog (13, 27) and the R169E mutant (Fig. 1),
arrestin2(3A) demonstrates the ability to bind to unphosphorylated
receptor in a strictly activation-dependent fashion (Fig.
7A). The expression of arrestin2(3A) in HEK 293 cells at the
same level as R169E in the experiments described above results in a
sharp decrease in
2AR down-regulation upon prolonged agonist
exposure, as compared with WT arrestin2 (Fig. 7B)
(F(1,8) = 25.33; p = 0.001).
Simultaneous expression of GRK2 with this mutant (at levels equal to or
higher than that of arrestin) fully rescued receptor down-regulation (Fig. 7B) (F(1,16) = 22.35;
p = 0.0002). Thus, similar selectivity profiles of the
two structurally diverse phosphorylation-independent mutants (compare
Fig. 1B and Fig. 7A) translate into virtually identical effects of their expression on receptor trafficking in cells
(compare Fig. 6 and Fig. 7B).
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Fig. 7.
The structurally distinct
phosphorylation-independent arrestin2(3A) mutant affects
2AR down-regulation similarly to R169E. HEK
293 cells expressing
2AR-GFP and either WT arrestin2 or 3A mutant
(10-14 fmol/µg protein) with or without simultaneous overexpression
of GRK2 (8-24 fmol/µg protein) were challenged with 10 µM isoproterenol for the indicated periods and washed.
Total
2AR was measured with [125I]pindolol. The
difference in receptor binding between control and agonist-treated
cells is presented as the receptor lost due to agonist treatment.
Means ± S.D. of 3 experiments in which arrestin and GRK2
expression levels exceeded 10 and 8 fmol/µg protein, respectively,
are presented. *, p < 0.01, as compared with WT.
2AR Phosphorylation
in Vivo--
To ascertain that cells expressing high levels of
arrestin2(R169E) and arrestin2(3A) mutants actually internalize
unphosphorylated
2AR, we compared isoproterenol-induced in
vivo phosphorylation of HA-
2AR in cells with no exogenous
arrestin and in those expressing > 20 fmol/µg protein of WT or
phosphorylation-independent forms of arrestin2 (Fig.
8). In these experiments we used a lower
concentration of the agonist isoproterenol to minimize the contribution
of receptor phosphorylation by protein kinase A (1). The cells
were treated with isoproterenol for two hours to maximize
internalization (cf. Figs. 3 and 5). In parallel, the cells
were also treated with agonist in the presence of 20 mM
NH4Cl to make sure that the great majority of the receptor
is internalized (cf. Fig. 5), so that the overall HA-
2AR
phosphorylation level measured by this method primarily reflects that
of the internalized receptor. We found that isoproterenol treatment
significantly increases 32P incorporation into HA-
2AR
without arrestin2 overexpression (i.e. with endogenous WT
arrestin) and upon overexpression of WT arrestin2 (Fig. 8, B
and C). The increase in receptor phosphorylation remains
essentially the same when receptor recycling is inhibited by 20 mM NH4Cl under continuous stimulation. In sharp
contrast, virtually no isoproterenol-induced increase in HA-
2AR
phosphorylation was observed in cells expressing either of the
phosphorylation-independent forms (Fig. 8). In the presence of R169E or
3A mutants the level of receptor phosphorylation did not increase even
in the presence of isoproterenol and 20 mM
NH4Cl (Fig. 8), conditions that result in the
internalization of over 70% of
2AR (Fig. 5). We found no
statistically significant differences in isoproterenol-induced HA-
2AR phosphorylation between cells expressing receptor only and
receptor + WT arrestin2, suggesting that HA-
2AR phosphorylation depends on the functional properties of the arrestin present, but not
on its expression level per se. No difference in
receptor phosphorylation was found between cells expressing
arrestin2(R169E) and arrestin2(3A), indicating that their common
ability to bind to an active receptor in a phosphorylation-independent
fashion is responsible for the suppression of agonist-induced receptor phosphorylation in living cells. These data indicate that in the presence of high levels of phosphorylation-independent mutant forms of
arrestin2,
2AR is predominantly internalized in the unphosphorylated
state, which apparently results in its accelerated recycling (Fig. 5)
and minimal down-regulation (Figs. 3, 6, and 7).
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Fig. 8.
Phosphorylation-independent arrestin mutants
prevent agonist-induced increase in HA- 2AR
phosphorylation in vivo. HEK 293 cells
overexpressing HA-
2AR (3-5 pmol/mg protein) alone (
) or
co-expressing receptor with >25 fmol/µg protein of WT arrestin2
(WT) or arrestin2(R169E) (R169E) or arrestin2(3A)
(3A) mutants were loaded with [32P]phosphate,
as described under "Experimental Procedures." The cells were
incubated for 2 h without agonist stimulation (
) or challenged
with 2.5 µM isoproterenol (ISO) alone or in
the presence of 20 mM NH4Cl (ISO,
NH4Cl) to prevent receptor recycling.
Cells were lysed, and solubilized HA-
2AR was immunoprecipitated with
rat monoclonal anti-HA antibody 3F10. A, the identity of
immunoprecipitated HA-
2AR band and yield uniformity was confirmed by
Western blotting of 1/10 of the sample using mouse monoclonal HA-Tag
antibody. B, immunoprecipitated sample was resolved on 8%
SDS-PAGE. The gel was dried and subjected to autoradiography to
visualize [32P]HA-
2AR bands. C,
radioactivity in the receptor band was quantified on Fluor-S
MultiImager and expressed as the percentage of receptor-associated
radioactivity in unstimulated cells. Typical blot and autoradiogram are
presented in A and B, respectively. Means ± S.D. of three experiments are presented in C. *,
p < 0.01 as compared with cells expressing only
HA-
2AR and p < 0.05 as compared with cells
expressing HA-
2AR + WT arrestin2; **, p < 0.01 as
compared with cells expressing only HA-
2AR and p < 0.01 as compared with cells expressing HA-
2AR + WT arrestin2.
2AR is
responsible for the dramatically different fate of the internalized
receptor observed in our experiments. The binding of these mutants to
2AR is strictly dependent on receptor activation (compare the
binding of both mutants to inactive
2AR and agonist-activated
2AR* on Figs. 1B and 7A). We believe that the
consequent higher sensitivity of R169E·
2AR and 3A·
2AR
complexes to receptor deactivation (ligand dissociation in endosomes),
as compared with the WT arrestin2·P-
2AR complex (Figs. 1 and 7),
is likely to play an important role in the change in receptor flow.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2AR and
phosphorylation-independent arrestin2 mutants R169E and 3A that
effectively desensitize this receptor (11, 13) and fundamentally change
the properties of the arrestin·receptor complex (Figs. 1 and 7).
2AR is internalized via the coated pits in
complex with arrestin (1, 4, 5, 7, 14, 29, 33, 34) (Fig.
2A). This is a relatively stable complex with high agonist
affinity (10, 14), in which bound arrestin appears to shield
receptor-attached phosphates (35). Low internal pH in the endosomes
likely induces agonist dissociation. Ensuing receptor return into the
inactive state facilitates arrestin release, whereupon
receptor-attached phosphates become accessible to protein phosphatases.
Inhibitors of protein phosphatases prevent effective receptor recycling
(25, 33), suggesting that only dephosphorylated receptor is transported
back to the plasma membrane. This is a dynamic process, as the receptor
undergoes repetitive endocytosis and recycling in the continuous
presence of an agonist (33, 34).
2AR intracellular "tenure" was observed after 18 h of
agonist treatment (36). However, it is not clear whether any additional
sorting mechanisms are turned on by prolonged stimulation, or the
apparent receptor diversion from the recycling to the degradative pathway is just a cumulative effect of the same cycling mechanisms operating for a long time.
2AR that enhances receptor degradation (44) is a
recently described example of a mechanism of this type. Alternatively,
there may be a steady-state "nonspecific" transport of proteins
from endosomes to lysosomes, the net result of which (on the background
of selective recycling of dephosphorylated receptor) is
"preferential" trafficking of phosphoreceptor and/or arrestin·receptor complex to the lysosomes by virtue of pure
kinetics. In our view, the simplicity of the latter mechanism and its
obvious applicability to the majority of GPCRs makes it rather
attractive (Fig. 9). Obviously, receptor- and/or functional
state-specific and purely kinetic mechanisms are by no means mutually
exclusive. Most likely, an intricate interplay of several mechanisms of
different types functioning side by side underlies the remarkable
variety of GPCR trafficking patterns observed experimentally in various cells.
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Fig. 9.
A model of receptor cycling in the presence
of WT and phosphorylation-independent arrestin. A,
multiple receptor phosphorylation is required for high-affinity binding
of WT arrestin2. The arrestin·phosphoreceptor complex is then
internalized. Low internal pH in the endosomes facilitates agonist
dissociation. Consequent return of the receptor into its inactive state
induces arrestin dissociation. The emerging multi-phosphorylated
receptor goes through sequential dephosphorylation steps before the
fully dephosphorylated, recycling-competent form is produced.
B, constitutively active mutants (R169E and 3A) bind to
unphosphorylated active receptor. The dissociation of the
arrestin·receptor complex in endosomes immediately yields
recycling-competent receptor, thereby reducing the probability of its
transport to lysosomes and degradation.
The introduction of a phosphorylation-independent arrestin mutant into
cells can be expected to change the rates of several steps in the
sequence of events. First, the majority of 2AR in complex with
mutant arrestin will be unphosphorylated for two reasons: 1) its
binding does not depend on receptor phosphorylation (Figs.
1B and 7A), and 2) it competes with GRK2 (Fig.
1C). Accordingly, we found that overexpression of
phosphorylation-independent forms of arrestin2 (either R169E or
3A) precludes agonist-induced increase in
2AR phosphorylation in
living cells even under conditions where the majority of the receptor
is internalized (Fig. 8). Second, the life span of the
receptor·arrestin complex is likely to decrease, because the loss of
active receptor conformation dramatically reduces the affinity of the
mutant for unphosphorylated
2AR (Figs. 1B and
7A), whereas deactivation of phosphorylated
2AR has only a moderate effect on WT arrestin2 binding (Fig. 1A). Third,
upon arrestin dissociation most of the receptor will emerge in
unphosphorylated (i.e. recycling-competent) form. Thus, in
the pool of internalized
2AR the proportion of receptor in complex
with arrestin and that of free phosphoreceptor will be sharply reduced,
whereas the proportion of unphosphorylated receptor ready to be
transported to the plasma membrane will increase dramatically (Fig. 9).
The relatively short half-life of arrestin complex with
unphosphorylated receptor is also likely to result in reduced receptor
ubiquitination (44). In short, such a shift in the distribution of
functional forms of the internalized receptor can only shorten the
tenure of the receptor in the endocytic compartment, facilitate
its recycling, and make its degradation less probable (Figs. 3, 6, and
7), regardless of the mechanism of receptor targeting to lysosomes that
the cell actually employs.
Conversely, overexpression of GRK2 enhances receptor phosphorylation, thereby stabilizing the complex of either arrestin with receptor (Figs. 1, A and B, and 7A) and slowing down subsequent receptor dephosphorylation. Both effects prolong receptor tenure in endosomes and reduce the proportion of recycling-competent fully dephosphorylated receptor, thus increasing time-dependent receptor down-regulation in the presence of both WT and mutant arrestin2, as observed (Figs. 6 and 7).
Thus, phosphorylation-independent arrestin2 mutants bind to and induce
the internalization of unphosphorylated 2AR (Figs. 1, 7, and 8),
thereby shortening and simplifying the chain of events necessary to
bring internalized receptor into the recycling-competent state (Fig.
9). This greatly reduces intracellular receptor transit time, as
evidenced by the combination of its effective internalization (Figs. 4
and 5) and low proportion of internalized receptor at any given moment
(Figs. 3A and 5). Indeed, unphosphorylated receptor internalized in complex with arrestin2(R169E) recycles substantially faster than phosphoreceptor internalized with WT arrestin2 (Figs. 5 and
8). An interesting net result of this accelerated cycling is minimum
receptor loss even after very long agonist exposure (Figs.
3B, 6, and 7). It has been suggested that excessive receptor phosphorylation, internalization, and down-regulation significantly contribute to the development of congestive heart failure (45) and
nephrogenic diabetes insipidus (46). The ability of
phosphorylation-independent mutants of non-visual arrestins to
"protect" receptors from phosphorylation, prolonged
internalization, and down-regulation in conjunction with their
demonstrated effectiveness in promoting short-term desensitization (11,
13) makes these arrestins promising tools for therapeutic intervention
in pathological processes of this kind.
Different elements in non-visual arrestins are involved in their
binding to GPCRs and numerous non-receptor partners. As a result, these
interactions can be independently manipulated by appropriate mutations
(1, 2, 4-13). We believe that "custom-designed" mutant arrestins
with unusual functional characteristics represent a novel class of
tools for studies of various facets of GPCR signaling and trafficking,
as well as for gene therapy of various disorders associated with
hereditary errors in these processes.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. J. L. Benovic for purified
GRK2, pcDNA3-2AR-GFP, and pcDNA3-GRK2 constructs, J. J.
Onorato for purified
2AR, L. A. Donoso for F4C1 anti-arrestin
antibody, and M. G. Caron for HA-
2AR construct. We thank Dr. N. L.
Schramm for expert advice on surface biotinylation, T. A.
Vishnivetskaya for technical assistance, and Y. V. Gurevich for the
artwork in Fig. 9.
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FOOTNOTES |
---|
* This work was supported in part by NIH Grants EY11500 and GM63097 (to V. V. G.) and MH62651 (to E. V. G.).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.
To whom correspondence should be addressed: Dept. of Pharmacology,
Vanderbilt University School of Medicine, RRB, Rm. 454, Nashville, TN 37232. Tel.: 615-322-7070; E-mail:
Vsevolod.Gurevich@mcmail.vanderbilt.edu.
Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M209532200
2 E. V. Gurevich, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
GPCR, G
protein-coupled receptors;
GRK, GPCR-kinase;
WT, wild type;
2AR,
2-adrenergic receptor;
HEK, human embryonic kidney;
DMEM, Dulbecco's modified Eagle's medium;
GFP, green fluorescence protein;
HA, hemagglutinin;
PBS, phosphate-buffered saline;
PCM, PBS
supplemented with 1 mM CaCl2 and 0.5 mM MgCl2;
P, phosphorylated.
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