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INTRODUCTION |
G protein-coupled receptors
(GPCRs)1are heptahelical
receptors that couple to heterotrimeric G proteins. Upon agonist
stimulation GPCRs initiate second messenger signaling cascades through
activated G protein subunits. Agonist stimulation also leads to rapid
receptor phosphorylation by serine/threonine kinases known as G
protein-coupled receptor kinases. The receptor, thus
phosphorylated, has increased affinity for cytosolic proteins called
-arrestins.
-Arrestin binding leads to the uncoupling of the
receptor from its cognate G proteins, causing dampening or
desensitization of GPCR signaling via the downstream second messenger
molecules (1). Recently, novel adaptor and scaffold functions of
-arrestins have been discovered. Thus, while terminating G protein
signals,
-arrestin binding can initiate new signal waves from GPCRs
(2). For example,
-arrestins serve as adaptors that bring
nonreceptor tyrosine kinases such as Src to form signaling complexes
with the internalizing receptor (3).
-Arrestins function as
GPCR-regulated scaffolds for mitogen-activated protein kinase modules
such as ASK-MKK4-JNK3 and RAF-MEK-ERK1/2 (4, 5). In addition,
-arrestins interact with proteins of the endocytic machinery such as
clathrin,
-adaptin subunit 2 of the AP2 complex, and Arf-6 and thus
promote internalization of receptors via clathrin-coated vesicles
(6-8).
Ubiquitination, a post-translational attachment of ubiquitin residues
to the lysines of substrate proteins, has been implicated in the
internalization of yeast pheromone receptors (9, 10), the endocytosis
of several mammalian cell surface receptors (11-13), and sorting to
the multivesicular bodies in yeast (14). The prototypic mammalian GPCR,
2AR is ubiquitinated in an agonist- and
-arrestin-dependent manner (15). Interestingly, receptor ubiquitination is not crucial for its internalization but is essential for proper trafficking to lysosomes for degradation. On the other hand,
2AR internalization requires the agonist-promoted
ubiquitin modification of the adaptor protein
-arrestin2 catalyzed
by a RING domain containing E3 ubiquitin ligase Mdm2 (15).
It is now evident that G protein-coupled receptor kinase
phosphorylation and subsequent
-arrestin binding promote
internalization of numerous GPCRs. Furthermore, recently developed
fluorescent-tagged
-arrestins have aided in visualizing the
translocation of cytosolic
-arrestins to activated receptors at the
cell surface using confocal microscopy (16). By employing such methods,
two distinct patterns of
-arrestin trafficking within the cell have
been delineated resulting in the classification of GPCRs as follows:
class A (e.g.
2AR,
1b-adrenergic receptor, µ opioid receptor, endothelin
1A, and dopamine D1A receptors), where
-arrestin interacts with the receptor at the cell surface but does not endocytose into vesicles, thus showing a transient interaction with the receptor; and class B
(e.g. vasopressin receptor V2, angiotensin AT1a, neurotensin 1, thyrotropin-releasing hormone, and neurokinin NK-1 receptors), in
which
-arrestins and receptor traffic together from the cell membrane to endocytic vesicles (17). These two classes of receptor also
differ with regard to their affinity for different
-arrestin isoforms. Class A receptors preferentially bind
-arrestin2, whereas class B receptors bind to
-arrestin1 and
-arrestin2 with equal affinity.
We now report that the distinct intracellular trafficking patterns of
-arrestin with the two classes of receptors are a function of the
differing pattern of
-arrestin ubiquitination and deubiquitination, which they induce. The pattern of
-arrestin ubiquitination is transient with
2AR (class A) and is sustained with V2R
(class B), which parallels the intracellular trafficking of
-arrestin2 with respect to these two receptors. Moreover a
-arrestin-ubiquitin fusion protein changes the trafficking pattern
such that a transient
-arrestin binder such as
2AR
now shows stable
-arrestin interaction because
-arrestin-Ub and
2AR traffic together into cells and colocalize in
endocytic vesicles. Our data strongly suggest that
-arrestin
ubiquitination stabilizes the receptor-
-arrestin complex and that
the intracellular trafficking of
-arrestin is determined by its
ubiquitination status.
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EXPERIMENTAL PROCEDURES |
Materials--
LipofectAMINE was from Invitrogen. FuGENE 6 was
from Roche Diagnostics. M2 anti-FLAG affinity agarose beads,
monodansylcadaverine, isoproterenol, arginine vasopressin
peptide, anti-FLAG M1 and M2 antibodies, and fluorescein isothiocyanate
anti-mouse secondary IgG were from Sigma. pEYFPC1, anti-GFP monoclonal
antibody (which recognizes YFP fusion proteins as well) were from
Clontech. Ubiquitin antibody UbP4D1 was from Santa
Cruz Biotechnology. Monoclonal antibody 12CA5 to HA epitope was from
Roche Molecular Biochemicals. Easy tagTM express protein labeling mix,
35S- and 125I-labeled (
)iodocyanopindolol
were from PerkinElmer Life Sciences. Horseradish peroxidase-conjugated
secondary antibodies were from Amersham Biosciences. A1CT is a rabbit
polyclonal antibody to
-arrestin generated in the Lefkowitz Laboratory.
The expression plasmids for YFP-
arrestin2-wild type,
HA-
2ARV2CT, and HA-V2R
2CT (Refs. 16-18)
were kindly provided by Dr. Marc Caron at Duke University.
The plasmids pcDNA3-
-arrestin2-Ub and pEYFPC1-
-arrestin2-Ub
were constructed as follows. A 1250-bp DNA fragment encoding
-arrestin2 was amplified to contain
5'-KpnI-HindIII and 3'-SalI ends; a
240-bp DNA fragment encoding ubiquitin was amplified to contain
5'-SalI and 3'-XbaI ends. The two fragments were
ligated together with pcDNA3 with HindIII and
XbaI ends to obtain
-arrestin2-Ub expression plasmid. A
1500-bp DNA fragment encoding
-arrestin2-Ub was subcloned into the
KpnI and ApaI sites of pEYFPC1 vector to obtain
the expression plasmid for YFP-
-arrestin2-Ub. All constructs were
confirmed by DNA sequencing.
Cell Culture and Transfection--
COS-7 and HEK-293 cells were
obtained from American Type Culture Collection. COS-7 cells were
maintained in Dulbecco's modified Eagle's medium (Invitrogen)
supplemented with 10% fetal bovine serum and 1% penicillin
streptomycin and transiently transfected with LipofectAMINE reagent.
HEK cells were maintained in minimal essential medium supplemented with
fetal bovine serum and transiently transfected with FuGENE 6 reagent.
Immunoprecipitation and Immunoblotting--
To detect
ubiquitinated
-arrestin2,
-arrestin2 with a C-terminal FLAG
epitope was transiently transfected into COS-7 cells or HEK-293 cells.
Cells were serum starved for at least 2 h and then stimulated or
not for the stipulated times with the appropriate agonists. Cells were
solubilized in a lysis buffer containing 50 mM HEPES (pH
7.5), 0.5% Nonidet P-40, 250 mM NaCl, 2 mM
EDTA, 10% (v/v) glycerol, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl
fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 1 µg/ml pepstatin
A, 100 µM benzaminidine, and 10 mM NEM.
Soluble extracts were mixed with FLAG M2 affinity beads and rotated at
4 °C overnight. Nonspecific binding was eliminated by repeated
washes with lysis buffer, and bound protein was eluted with sample
buffer containing SDS. The proteins were separated on a gradient gel
(4-20%, Invitrogen) and transferred to nitrocellulose membrane for
Western blotting. Chemiluminescent detection was performed using
SuperSignal® West Pico reagent (Pierce).
Metabolic Labeling--
COS-7 cells were transiently transfected
with YFP-
-arrestin2 or YFP-
-arrestin2-Ub. Prior to labeling with
isotope, the cells were washed twice with methionine/cysteine-free
medium and incubated in this medium for 10 min. Cells were incubated
with radiolabel solution (Dulbecco's modified Eagle's medium, 10 mM HEPES (pH 7.5), 2% dialyzed fetal bovine serum, 300 µCi/ml 35S) for 20 min at 37 °C, after which the cells
were washed three times with regular medium and incubated in medium for
0, 2, 4, 6, 8, 10, and 12 h at 37 °C. At the end of incubation
at each time point, the cells were lysed in glycerol lysis buffer
(above), and immunoprecipitations of both constructs were done with
either GFP beads (n = 2) or using A1CT antibody
(n = 1).
Receptor Internalization--
FLAG or HA epitope-tagged
receptors expressed in HEK-293 cells in 12-well dishes were treated
with or without agonist for 30 min in serum-free medium at 37 °C.
Cell surface receptors were labeled with M1 FLAG or 12CA5 monoclonal
antibody and fluorescein isothiocyanate-conjugated goat antibody to
mouse IgG as secondary antibody. Receptor internalization was
quantified as loss of cell surface receptors as measured by flow
cytometry (flow cytometry facility, Duke University).
Receptor Degradation--
Degradation assays were done with
[125I](
)iodocyanopindolol ([125I]CYP)
radioligand binding as reported before (15) on whole cells gently
resuspended in Dulbecco's modified Eagle's medium buffered with 10 mM HEPES (pH 7.5). Binding was performed in triplicate with
400 pM [125I]CYP in the presence or absence
of the hydrophobic antagonist propranolol (10 µM, to
define nonspecific binding). Binding was terminated by rapid dilution
and filtration on Whatman GFC glass fiber filters. For degradation
assays incubation was at 30 °C for 30 min, and the receptor number
(total specific [125I]CYP binding sites) was determined
after 24 h of isoproterenol treatment and expressed as percent of
receptor number assessed in nonstimulated cells.
Confocal Microscopy--
HEK-293 cells have a favorable
morphology for examining sections of cytoplasm and nucleus
simultaneously and hence were used in these experiments. HEK-293 cells
on 10-cm dishes were transiently transfected with
HA-
2AR, HA-V2R, HA-
2ARV2CT, or
HA-V2R
2CT, along with
-arrestin2-GFP,
YFP-
-arrestin2, or YFP-
-arrestin2-Ub. 24 h post-transfection
cells were plated on collagen-coated 35-mm glass bottom plates. Cells
were starved for at least 2 h in serum-free medium prior to
stimulation. After stimulation cells were fixed with 4%
paraformaldehyde. For visualizing HA-tagged receptors, fixed cells were
permeabilized with 0.01% Triton® X-100 in phosphate-buffered saline containing 2% bovine serum albumin for 20 min and incubated at
room temperature with 12CA5 antibody at 1:500 dilution. The secondary
antibody, anti-mouse IgG conjugated to Texas Red, followed this.
Antibody incubations were done for 1 h followed by repeated washes
using phosphate-buffered saline. Cells expressing low and equivalent
levels of the fluorescent proteins were selected carefully to examine
YFP-
-arrestins. Confocal images were obtained on Zeiss LSM510 laser
scanning microscope using dual excitation (488, 568 nm) and emission
(515-540 nm GFP, YFP; 590-610 nm, Texas Red) filter sets. Live images
were acquired using a heated (37 °C) microscope stage and collected
sequentially using single line excitation (488 nm).
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RESULTS |
-Arrestin Ubiquitination and Deubiquitination Correlate with the
Stability of the GPCR-
-Arrestin Complex during Agonist-promoted
Internalization--
Stimulation of
2AR in COS-7 cells
with the agonist isoproterenol can lead to robust ubiquitination of
-arrestin2 at about 1 min after agonist treatment (Fig,
1A, left panel, and
Ref. 15). However, this ubiquitination signal diminishes within 15 min. In contrast, stimulation of either V2 vasopressin receptors or angiotensin AT1a receptor leads to a stable ubiquitination pattern (Fig. 1B, left panel, and data not shown). With
these receptors
-arrestin2 is ubiquitinated robustly within 1 min,
but the signal does not diminish at 15 min. Interestingly
-arrestin2
is mostly polyubiquitinated (attachment of the first ubiquitin to the
substrate lysine followed by formation of a polyubiquitin chain on the
first ubiquitin) or possibly multiubiquitinated (ubiquitination of
substrate at several lysines). Similar agonist-stimulated
ubiquitination patterns were also observed for the
-arrestin1
isoform with the aforementioned receptors (not shown). Identical
ubiquitination time courses for
-arrestin with the above receptors
were seen in yet another cell system, namely, HEK-293 cells (Fig. 1,
A and B, right panels). In addition,
we found a higher level of basal ubiquitination in some experiments
when the receptors were overexpressed.

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Fig. 1.
-Arrestin ubiquitination
and deubiquitination parallel its association with and dissociation
from GPCR. Endogenous or overexpressed 2AR
(A) or overexpressed V2R (B) in COS-7 cells
(left panels) and HEK-293 cells (right panels)
were stimulated with the agonist 10 µM isoproterenol
(ISO) or 1 µM AVP for the indicated times, and
overexpressed -arrestin2-FLAG ( -arr2 Flag) was
immunoprecipitated (IP) with anti-FLAG beads. The
immunoprecipitate was probed (IB) for ubiquitinated forms
using Ub antibody (Santa Cruz). Horseradish peroxidase-linked
anti-mouse IgG (Amersham Biosciences) was used as the secondary
antibody. Unmodified -arrestin-FLAG has a mobility of ~51 kDa. The
blots are representative of three independent experiments.
C, isoproterenol stimulated trafficking of
-arrestin2-GFP. -Arrestin2-GFP and HA- 2AR were
coexpressed in HEK-293 cells. After stimulation or not (NS)
with 1 µM isoproterenol, cells were fixed using
paraformaldehyde. HA epitope was stained with a monoclonal antibody,
12CA5, followed by a secondary antibody to mouse IgG conjugated to
Texas Red. The proteins were visualized by confocal microscopy,
-arrestin (green channel), 2AR
(red channel). Colocalization (yellow) of the two
proteins is seen in the overlay panels. The images are
representative of three similar experiments. All bars equal
10 µm. D, AVP-stimulated trafficking of GFP- -arrestin2.
-Arrestin2-GFP and HA-V2R were coexpressed in HEK-293 cells. After
stimulation or not with 1 µM AVP, cells were fixed using
paraformaldehyde. Antibody staining and protein visualization followed
the same procedure as in C. All bars represent 10 µm.
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A striking correlation can be drawn between ubiquitination and
intracellular trafficking patterns of
-arrestins with
2AR and V2R. As shown in Fig. 1, C and
D, GFP-
-arrestin2 (green, top row)
is uniformly distributed in the cytoplasm in unstimulated cells. The
receptors (red, middle row) are seen at the
plasma membrane. Within 1 min of respective stimuli,
-arrestin2
translocates to the plasma membrane in both cases. However, at 15 min
after stimulation, in the case of
2AR, the receptor
alone is seen in the endocytic vesicles, with the
-arrestin still at
the plasma membrane, whereas in the case of V2R both
-arrestin and
receptor are seen colocalized in endocytic vesicles (yellow,
bottom row). Thus, the ubiquitination and deubiquitination
time course of
-arrestin parallel its association with and
dissociation from the two types of receptor.
Interaction of
-Arrestin with GPCR Tail Residues Governs Both
Intracellular Trafficking and Ubiquitination Patterns of
-Arrestin--
The differing affinity of a class A
versus a class B GPCR for
-arrestin has been attributed
to the phosphorylation by G protein-coupled receptor kinases of
specific serine clusters in the tail region of the GPCR (18). Moreover,
the exchange of the cytoplasmic tail residues between the two receptor
classes leads to reversal of the patterns of
-arrestin affinity for
the receptors as well as intracellular trafficking (16). As shown in
Fig. 2A, a chimeric
2ARV2CT with the first 341 amino acids of the
2AR (Met1-Cys341) fused to the
last 29 amino acids of the V2R (Ala343-Ser371)
interacts with
-arrestin more stably, and hence after 15 min of
isoproterenol stimulation, both the receptor and
-arrestin are
colocalized in endosomes. On the other hand the chimera
V2R
2CT, which contains the first 342 amino acids of the
V2R (Met1-Cys342) fused to the last 72 amino
acids of the
2AR
(Leu342-Leu413) shows the transient
-arrestin binding pattern of the
2AR, and hence
-arrestin remains at the plasma membrane, and receptor alone
traffics to endosomes (Fig. 2B).

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Fig. 2.
The GPCR tail residues govern both
trafficking and ubiquitination patterns of
-arrestin2. Trafficking of GFP- -arrestin2
(GFP- arr2) with the chimeric GPCRs 2ARV2CT
(A) and V2R 2CT (B) is shown.
HEK-293 cells were transiently transfected with GFP- -arrestin2 and
HA-tagged receptor. After agonist stimulation or not (NS),
cells were fixed, permeabilized, and stained with 12CA5 and Texas
Red-conjugated anti-mouse antibody. Shown are confocal images of
receptor immunofluorescence (red) and GFP- -arrestin
fluorescence (green). Colocalization of -arrestin and
receptor is seen (yellow) in the overlay.
Confocal images are representative of three similar experiments. All
bars represent 10 µm. ISO, isoproterenol.
C, 2ARV2CT-dependent -arrestin
ubiquitination. COS-7 cells (left panel) and HEK-293 cells
(right panel) overexpressing 2ARV2CT and
-arrestin2-FLAG were stimulated or not with 10 µM
isoproterenol for the indicated times, and -arrestin was
immunoprecipitated (IP) with anti-FLAG affinity beads. The
immunoprecipitate was probed (IB) with Ub antibody to detect
ubiquitinated forms of -arrestin. The blots are representative of
three independent experiments in both cell types. D, time
course of -arrestin ubiquitination dependent on
V2R 2CT stimulation. COS-7 cells (left panel)
and HEK-293 cells (right panel) overexpressing
V2R 2CT and -arrestin2-FLAG were stimulated or not
with 1 µM AVP. -Arrestin was immunoprecipitated, and
the immunoprecipitate was probed with Ub antibody. The blots are
representative of three independent experiments in each cell
type.
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If the ability of
-arrestin to remain ubiquitinated is dependent on
its interaction with the GPCR, one might expect that the receptor
cytoplasmic tail residues would govern the pattern of ubiquitination.
To test this hypothesis, we determined the agonist-promoted
ubiquitination patterns of
-arrestin for the 2 chimeric receptors.
As shown in Fig. 2C, a 1-min isoproterenol stimulation of
COS-7 cells (left) or HEK-293 cells (right)
expressing
2ARV2CT leads to a marked increase in
-arrestin ubiquitination which does not decrease at 15 min. In
marked contrast, the stimulation of the chimera V2R
2CT
leads to a very robust increase in ubiquitination at 1 min of AVP
treatment, but the signal returns to almost basal levels at 15 min
(Fig. 2D). These results, together with the data shown in
Fig. 1, strongly suggest that the ubiquitinated form of
-arrestin
corresponds to the receptor-bound
-arrestin complex.
A Persistently Ubiquitinated Form of
-Arrestin2 Shows Enhanced
Binding to GPCR--
Our data suggest that
-arrestin2 translocates
to the plasma membrane and becomes ubiquitinated within 1 min of GPCR
stimulation but is deubiquitinated with differing kinetics depending
upon the receptor. However, it is unknown whether deubiquitination is a
cause or consequence of
-arrestin dissociation from the receptor. We
theorized that fusion of a ubiquitin moiety to the
-arrestin protein
would provide a persistently ubiquitinated form of
-arrestin. If
-arrestin2 deubiquitination was the trigger for dissociation from
the class A receptor, then we would expect that the chimeric protein
would not dissociate from the receptor and would show tighter binding
to the class A receptor than wild type
-arrestin because it cannot
be efficiently deubiquitinated. On the other hand, if deubiquitination
of
-arrestin occurs after its dissociation from the receptor then
there should be no alterations in
-arrestin-Ub binding or in its
dissociation kinetics.
Because ubiquitinated proteins are substrates for proteasomal
degradation (19), we tested the stability of a
-arrestin-Ub chimeric
protein. A comparison of the half-lives of YFP-
-arrestin2 and
YFP-
-arrestin2-Ub is shown in Fig.
3A. Wild type
-arrestin2 has a half-life of about 10-12 h in COS-7 cells as determined by
35S metabolic labeling. Not surprisingly, fusion of
ubiquitin in frame to
-arrestin decreased its half-life to about
2 h (Fig. 3A). Nonetheless the chimeric protein could
be over-expressed in sufficient amounts to be detected by antibody to
both
-arrestin (not shown) and YFP (Fig. 3, B and
C).

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Fig. 3.
Characterization of
YFP- -arrestin-Ub chimeric protein.
A, stability of the ubiquitin chimera in COS-7 cells.
Monolayers of cells overexpressing either YFP- -arrestin2 or
YFP- -arrestin2-Ub were labeled with 35S, and both
proteins were immunoprecipitated with GFP-agarose beads or A1CT
antibody beads as described under "Experimental Procedures." The
bands on the autorad were quantitated by fluoroimaging and are
represented graphically in the figure: , YFP- -arrestin2; *,
YFP- -arrestin2-Ub. The zero time point represents 100% protein. The
graph is an average of three independent experiments. B,
interaction of the -arrestin2-Ub chimera with 2AR.
FLAG- 2AR was coexpressed with either YFP- -arrestin2
or YFP- -arrestin2-Ub in COS-7 cells. After stimulation or not the
receptor was immunoprecipitated (IP) with anti-FLAG beads in
the absence (left panels) or presence (right
panels) of 10 mM NEM; the immunoprecipitate was
blotted (IB) for YFP- -arrestins with a monoclonal
antibody to GFP (top panel). The middle panel
shows the amount of receptor as detected by anti-FLAG antibody M2. The
bottom panel shows the expression levels of
YFP- -arrestins in whole cell extracts as detected by the
GFP-monoclonal antibody. The blots are representative of similar blots
from three independent experiments. Iso, isoproterenol.
C, FLAG- 2AR was coexpressed with either
YFP- -arrestin2 or YFP- -arrestin2-Ub in HEK-293 cells. After
stimulation or not the receptor was immunoprecipitated with anti-FLAG
beads in the presence of 10 mM NEM; the immunoprecipitate
was blotted for YFP- -arrestins with a monoclonal antibody to GFP
(top panel). The middle panel shows the amount of
receptor as detected by anti-FLAG antibody M2. The bottom
panel shows the expression levels of YFP- -arrestins in whole
cell extracts as detected by the GFP monoclonal antibody. The blots are
representative of similar blots from four independent
experiments.
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Several research groups including ours have demonstrated
coimmunoprecipitation (in the presence or absence of chemical
cross-linking reagents) of
-arrestin2 with
2AR.
However, under such experimental conditions only a modest increase of
the protein interaction is observed upon agonist treatment. In
contrast, as assessed by confocal microscopy with YFP- or GFP-tagged
-arrestin, within a few seconds of isoproterenol treatment, one can
observe a major portion of the cytosolic
-arrestin move to the
plasma membrane and colocalize with the receptor. If
-arrestin
ubiquitination contributes to the stability of the GPCR-
-arrestin
interaction, then coimmunoprecipitation done under conditions that
preserve the ubiquitination of
-arrestin, such as treatment with NEM
(which inactivates deubiquitinating enzymes; see Ref. 20) should result
in a considerable increase in the amount of
-arrestin that
coprecipitates with the receptor. This indeed is the case as shown in
Fig. 3B, which compares the amount of
-arrestin that is
coimmunoprecipitated with
2AR in the presence and
absence of NEM. The laddering of bands as detected by the antibody to
YFP-
-arrestin represents the multimerized as well as ubiquitinated
forms of the
-arrestin protein. NEM treatment produced identical
receptor-
-arrestin complexes in coimmunoprecipitations done in
HEK-293 cells (Fig. 3C). Note that these bands are not
detectable at 15 min of agonist treatment with the wild type
YFP-
-arrestin2 but are detectable with the chimeric protein,
YFP-
-arrestin2-Ub. This is probably because the chimeric protein is
not efficiently deubiquitinated as is the wild type and hence is
detectable as the receptor-bound form of
-arrestin2. The 90 kDa band
that corresponds to monoubiquitinated YFP-
-arrestin2 is indicated in
Fig. 3B.
Expression of
-Arrestin-Ub Chimera Transforms a Class A Receptor
to Class B with Respect to Intracellular Trafficking--
To determine
whether
-arrestin-Ub chimeric protein influences its trafficking we
utilized confocal microscopy to examine the fluorescent proteins in
live cells. Fig. 4A
illustrates the trafficking of wild type YFP-
-arrestin2 in HEK-293
cells transiently overexpressing
2AR. In unstimulated
cells, YFP-
-arrestin2 is distributed homogeneously in the cytoplasm.
Within 2 min of isoproterenol treatment,
-arrestin is seen to
concentrate at the plasma membrane. The distribution of
YFP-
-arrestin is unaltered at 30 min of isoproterenol treatment
(Fig. 4A, upper panels). The punctate
fluorescence of YFP-
-arrestin2 at the membrane is probably because
of its localization in clathrin-coated pits at the plasma membrane. A
cytosolic distribution of YFP-
-arrestin2-Ub in unstimulated and
plasma membrane localization in 2-min stimulated cells are shown in
Fig. 4A, lower panels. However, at 15 min,
YFP-
-arrestin-Ub is seen to redistribute from the plasma membrane to
endocytic vesicles. Longer agonist treatment results in a further
increase in number as well as size of YFP-
-arrestin-Ub-containing
endocytic vesicles.

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Fig. 4.
Intracellular trafficking patterns of
YFP- -arrestin and
YFP- -arrestin-Ub. HEK-293 cells were
transiently transfected with either YFP- -arrestin2 or
YFP- -arrestin2-Ub along with 2AR (A) or
V2R 2CT (B). NS, not stimulated.
Cells were starved for 1 h in serum-free medium. The distribution
of YFP- -arrestin2 and YFP- -arrestin2-Ub was visualized before and
after treatment with 10 µM isoproterenol (ISO;
A) or 1 µM AVP (B). Shown are
representative confocal images of YFP fluorescence followed in the same
HEK-293 cells treated for 2, 15, and 30 min at 37 °C. All
bars represent 10 µm.
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V2R
2CT is a chimeric receptor in which the class B
pattern of trafficking and ubiquitination is converted to that of a
class A receptor (Fig. 2, B and D). Stimulation
of V2R
2CT with AVP leads to the redistribution of
YFP-
-arrestin2 to the plasma membrane in a punctate pattern like the
2AR (Fig. 4B, upper panels) at 2 min. The YFP-
-arrestin2 remains at the plasma membrane at longer time points. On the other hand, YFP-
-arrestin-Ub, which translocates to the plasma membrane within 2 min of agonist, moves into endocytic vesicles as early as 15 min of AVP treatment (Fig. 4B,
lower panels). The addition of tail residues of
2AR (class A) to V2R (class B) confers class A
trafficking pattern to the V2R
2CT chimeric receptor. As
a further step, overexpression of YFP-
-arrestin2-Ub reverts the
trafficking pattern back to that of class B. Thus a "double
reversal" of
-arrestin trafficking pattern is seen with respect to
V2R
2CT.
To determine whether the YFP-
-arrestin2-Ub in endocytic vesicles
colocalizes with the GPCR, we examined the distribution of both
2AR and
-arrestin after 15 min of isoproterenol
treatment (Fig. 5). In unstimulated cells
receptor is seen at the plasma membrane, and either YFP-
-arrestin or
YFP-
-arrestin-Ub is seen uniformly distributed in the cytoplasm
(data not shown). Stimulation of cells with isoproterenol for 15 min
resulted in the redistribution of
2AR (red)
and YFP-
-arrestin2 (green) into small puncta at the
plasma membrane, which likely represent colocalization of
2AR and YFP-
-arrestin in clathrin-coated pits at the
membrane (Fig. 5A). Internalized receptors, seen as
red vesicles in the cytoplasm, do not, however, contain any
YFP-
-arrestin2, suggesting that
-arrestin2 dissociates from the
receptor during or shortly after vesicle formation. Fig. 5B
shows the distribution of
2AR and YFP-
-arrestin2-Ub
after 15 min of isoproterenol treatment. In contrast to what was seen
with wild type YFP-
-arrestin2, the YFP-
-arrestin-Ub chimera is
seen to colocalize with the internalized
2AR. Further,
very little staining of either protein is seen at the plasma
membrane. Thus, a ubiquitinated form of
-arrestin2 can
transform a class A receptor such as the
2AR into a
class B receptor with respect to its pattern of internalization.

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Fig. 5.
YFP- -arrestin2-Ub
but not YFP- -arrestin colocalizes in endocytic
vesicles with 2AR. HEK-293
cells were transiently transfected with HA- 2AR and
YFP- -arrestin2 (A) or YFP- -arrestin2-Ub
(B). After isoproterenol (ISO) treatment or not
the cells were fixed, permeabilized, and stained for receptor with the
primary antibody 12CA5 and secondary antibody anti-mouse IgG conjugated
to Texas Red. Shown are confocal images where the receptor is
visualized in the red channels and -arrestin in the
green channels. Colocalization of two proteins is seen as
yellow in the overlay. The results are representative of
three independent experiments. All bars represent 10 µm.
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Effect of
-Arrestin-Ub on the Internalization and Degradation of
2AR--
Because
-arrestin2-Ub can form stable
complexes with
2AR, we tested whether this interaction
had any effect on agonist-stimulated receptor internalization. As shown
Fig. 6A,
2AR
internalization, as measured in HEK-293 cells after a 30-min
isoproterenol treatment, was 25 ± 1.8%. Upon overexpression of
-arrestin2 and
-arrestin2-Ub the internalization increased to
33 ± 1.6% and 44 ± 3.5%, respectively. Pretreatment of
cells with 200 µM monodansylcadaverine, a known inhibitor
of receptor movement to clathrin-coated pits (21), reduced this
internalization by 58% (Fig. 6C), suggesting that the
-arrestin-Ub promoted internalization proceeds via clathrin-coated vesicles.

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Fig. 6.
Effect of chimeric
-arrestin-Ub on sequestration of GPCRs.
HEK-293 cells were transiently transfected with FLAG- 2AR
(A) and HA-V2R (B). In each case the receptor was
cotransfected with vector plasmid (Mock), -arrestin2, or
-arrestin2-Ub. After serum starvation cells were treated with 10 µM isoproterenol (ISO; A) or 1 µM AVP (B) for 30 min at 37 °C. Cell
surface receptors before and after agonist treatment were determined by
flow cytometry. Data in A are the mean ± S.E. of seven
independent experiments done in triplicate. Data in B are
the mean ± S.E. of five independent experiments done in
triplicate. # p < 0.05 versus
-arrestin2; ** p < 0.001 versus Mock,
one-way analysis of variance with Tukey multiple comparison.
C, effect of monodansylcadaverine on 2AR
internalization with and without overexpression of -arrestin-Ub
chimera. HEK-293 cells were transiently transfected with
FLAG- 2AR with vector plasmid or -arrestin2-Ub. After
serum starvation, cells were incubated or not with 200 µM
monodansylcadaverine (MDC) for 30 min followed by
stimulation or not with 10 µM isoproterenol for 30 min at
37 °C. Cell surface receptors before and after agonist treatment
were determined by flow cytometry. Data represent the average ± S.E. of four independent experiments done in triplicate.
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Overexpression of
-arrestin-Ub did not, however, result in a similar
enhancement of internalization of V2R over wild type
-arrestin as
measured after 30 min of AVP stimulation (Fig. 6B). This
result is expected because the ubiquitination of
-arrestin is stable
with this receptor.
A short term isoproterenol treatment of cells expressing
2AR results in a decrease in the number of cell surface
receptors without changing total cellular receptor levels. However,
prolonged exposure to isoproterenol, for hours or days, results in a
decline in the total receptor number, measurable by radioligand
binding. In HEK-293 cells, after 24 h of isoproterenol treatment,
overexpressed
2AR levels decrease by about 25% (Fig.
7A). The amount of degradation increased with
-arrestin overexpression by about 6-7%. On the other hand, the amount of receptor degradation was doubled when
-arrestin2-Ub was coexpressed with the receptor. For comparison, we
measured the degradation of the
2ARV2CT chimera, which
essentially mimics the properties of V2R with respect to endocytosis.
However, no change in the degradation of
2ARV2CT was
observed with
-arrestin2-Ub overexpression (Fig. 7B).
These data suggest that the ubiquitination status of
-arrestin
influences both the rate at which a given receptor is sequestered from
the plasma membrane as well as the likelihood that it will be sorted to
lysosomes.

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Fig. 7.
Effect of chimeric
-arrestin-Ub on degradation of GPCRs. HEK-293
cells were transiently transfected with FLAG- 2AR
(A) and HA- 2ARV2CT (B). In each
case the receptor was cotransfected with vector plasmid
(Mock), -arrestin2, or -arrestin2-Ub. After serum
starvation cells were treated with 10 µM isoproterenol
(ISO) for 24 h at 37 °C. [125I]CYP
binding was done on whole cells as described under "Experimental
Procedures" to determine the receptor numbers with and without
agonist treatment. The results are the mean ± S.E. of three to
five experiments. * p < 0.01 versus Mock, # p < 0.05 versus -arr2, one-way analysis
of variance with Tukey multiple comparison.
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DISCUSSION |
Binding of
-arrestins to activated receptors leads to
homologous receptor desensitization as well as to the recruitment of specialized endocytic machinery to internalize receptors. Recently, we
have shown that both
-arrestin and the
2AR undergo
agonist-dependent ubiquitination (15). In the case of yeast
-factor receptor, ubiquitination appears to serve as an
internalization signal (9). However,
2AR ubiquitination,
which requires
-arrestin as an adaptor to recruit ubiquitination
enzymes, is necessary for targeting of the receptors to lysosomes
for degradation rather than for their internalization.
Interestingly,
-arrestin ubiquitination catalyzed by Mdm2, a
RING-type E3 ubiquitin ligase, is required for
2AR internalization.
The
2AR and the V2R are seven-membrane-spanning
receptors, which typify two very different patterns of receptor
trafficking and
-arrestin interaction. Importantly,
2AR stimulation leads to a transient pattern of
-arrestin ubiquitination, whereas V2R stimulation leads to stable
-arrestin ubiquitination, which parallels the intracellular
trafficking interaction of
-arrestin with these two receptors.
Exchanging the C-terminal domains of these two receptors reverses the
ubiquitination patterns of
-arrestin. Agonist stimulation of the
2AR leads to the recruitment of
-arrestin to the cell
membrane within a few seconds, where it gets ubiquitinated.
2ARs then move to clathrin-coated pits and are
internalized into vesicles within 15 min. This event is accompanied by
the dissociation of
-arrestin from the receptor after its
deubiquitination. This order of events is supported by the experiments
done with a chimeric
-arrestin with ubiquitin fused to its C
terminus. Stimulation of the
2AR with isoproterenol
leads to the redistribution of this chimera to the plasma membrane
where it colocalizes with the receptor. However, unlike its wild type
counterpart, the ubiquitin chimera does not dissociate from the
receptor but is seen to colocalize with the receptor in endocytic
vesicles. If deubiquitination occurred after
-arrestin dissociation,
the ubiquitin chimera would have nevertheless dissociated from the
receptor. Thus, stable ubiquitination forces
-arrestin to move into
endocytic vesicles with the receptor. However, this movement could also
be the result of its tighter association with the receptor as seen in
the coimmunoprecipitation experiments done in the presence of
inhibitors of deubiquitinating enzymes. Thus, our data indicate that
when
-arrestin interacts with a class A receptor it is prone to
deubiquitination by an unknown mechanism, and this causes it to
dissociate from the receptor at the plasma membrane. The reason for
sustained ubiquitination with a class B receptor is not known.
Previously, we found that inhibiting
-arrestin ubiquitination either
by use of a dominant negative form of its E3 ligase Mdm2 or by using
Mdm2-null cell lines did not inhibit degradation of the
2AR. However, in the present work, a persistently
ubiquitinated
-arrestin2 is observed to be associated with not only
more robust
2AR sequestration but also increased
receptor degradation. How can these two sets of observations be
reconciled? Previous findings suggest that very slow rates of
2AR sequestration can support robust receptor
degradation, especially as monitored at late time points such as
24 h of agonist exposure (15). It appears that even the small
amount of receptor sequestration remaining when we inhibit
-arrestin
ubiquitination is likely sufficient to support receptor degradation. On
the other hand, in the presence of the
-arrestin-Ub chimera the
2ARs sequester more robustly and do not recycle readily.
As noted above, this is now typical of a class B receptor rather than
the normal class A receptor behavior of the
2AR. Our
data suggest that such non- or slowly recycling
2ARs may
be more subject to entry into degradative pathways and hence show
increased down-regulation. In other words, the persistently ubiquitinated
-arrestin likely fundamentally alters the relationship between the rate of receptor sequestration and the ultimate
down-regulation of the receptor. Presumably, the presence of
ubiquitinated
-arrestin in complex with internalized receptors
somehow leads to more efficient sorting of these internalized receptors
to lysosomes. Whether this is the result of inhibition of receptor
dephosphorylating enzymes, blocking the interaction of recycling
proteins such as N-ethylmaleimide-sensitive fusion protein
(NSF) (22), enhancing the interaction with sorting proteins such
as the sorting nexins (23, 24), or some other mechanism remains to be determined.
Our data thus reveal an interesting and previously unappreciated
connection between the ubiquitin modification of
-arrestin2 and its
trafficking pattern with distinct types of receptors. Recently,
ubiquitination of adaptor proteins has been implicated as a trigger for
downstream signaling pathways (25). It was shown that stimulation of
interleukin-1 receptors leads to recruitment, dimerization, and
auto-ubiquitination of TRAF6 at the membrane, which in turn triggers
the kinase TAK1, which can phosphorylate I
kinase and MKK6
leading to the activation of nuclear factor-
B and JNK-p38 kinase
pathways (25). We have observed that agonist stimulation of receptors
such as angiotensin AT1a and vasopressin V2 lead to sustained
ubiquitination of
-arrestins. These receptors, when activated, are
also known to engage pools of
-arrestin and active ERK complexes in
the cytoplasm (26). Whether
-arrestin ubiquitination forms a
foundation for scaffolding of these signaling complexes on endosomes is
another tantalizing question for future research.