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INTRODUCTION |
G protein-coupled receptors
(GPCRs),1 a large cellular
membrane receptor family comprised of more than a thousand members, mediate many physiological functions. Agonist binding to GPCRs results
in both the initiation of cellular signaling and the receptor phosphorylation by G protein-coupled receptor kinases. Arrestins were
originally discovered to bind to the phosphorylated GPCRs and function
in regulation of the desensitization and internalization of these
receptors (1, 2). To date, at least four members of the arrestin gene
family have been identified: visual arrestin, cone arrestin, and two
-arrestins (
-arrestin1 and
-arrestin2). Visual arrestin and
cone arrestin have specialized functions due to their limited
localization to the retina, whereas
-arrestins are ubiquitously
expressed in various tissues (3-6).
-Arrestin1 and
-arrestin2
are two highly homologous proteins (sharing 78% identity in amino acid
composition), and both of them rapidly translocate from the cytoplasm
to cell plasma membrane when GPCRs are stimulated. Binding of either
-arrestin to the activated GPCRs can disassociate G proteins from
the receptors and thus quench the receptor signaling. In addition, both
-arrestins effectively regulate the internalization of many GPCRs by
direct interaction with AP2 and clathrin (7, 8).
Although
-arrestin1 and
-arrestin2 perform similar functions in
the regulation of GPCR signaling, some differences between them have
been reported such as in their binding affinity to different classes of
GPCRs (9) and in their association with different binding partners
(10). One of the well established differences is their subcellular
localization. It is reported that when expressed in HEK293 and HeLa
cells,
-arrestin1 is localized in the cytoplasm and nucleus, but
-arrestin2 is predominantly distributed in the cytoplasm (9, 11). In
addition, a very recent study (11) shows that two
-arrestins shuttle
differentially between the nucleus and cytoplasm due to the presence of
a two-leucine nuclear export signal (NES) in
-arrestin2 that is
absent in
-arrestin1. However, the molecular determinant of their
nuclear import remains to be further investigated.
Novel functions of
-arrestins, such as regulation of the
cytoskeletal reorganization via association with Ral guanine
nucleotide dissociation stimulator (Ral-GDS) (12) and enhancement of
the CXCR4-mediated chemotaxis (13), have been reported recently. One of
the most exciting findings is that
-arrestins can serve as a
scaffold protein to regulate the functions of ERK and JNK3 cascades by
directly interacting with these kinases. For example,
-arrestin2 can
simultaneously associate with ERK and its upstream Raf or JNK3 and its
upstream ASK1 and thus effectively promotes GPCR-mediated activation of
these kinases. Moreover, the interaction of
-arrestin2 with its
binding partners such as ERKs and JNK3 can lead to the relocalization
of the kinases in the cytoplasm (14). Consequently, the cytoplasmic
retention of ERKs inhibits activation of the transcription factor Elk
(15). Recently, Mdm2, a well known ubiquitin-protein isopeptide ligase
(E3) for p53, has been shown to regulate the trafficking of
2-adrenergic receptor via its direct interaction with
-arrestin2
(16). However, whether the interaction of Mdm2/
-arrestin2 affects
the subcellular distribution of Mdm2 remains unclear.
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EXPERIMENTAL PROCEDURES |
Materials--
Leptomycin B (LMB) and DAPI were obtained from
Sigma. Mouse anti-hemagglutinin (HA) monoclonal antibody (12CA5),
recognizing influenza HA epitope, was purchased from Roche Molecular
Biochemicals. Texas Red-conjugated goat anti-mouse IgG secondary
antibody was purchased from Molecular Probes.
Protein-A-Sepharose was from Amersham Biosciences. The
anti-
-arrestin polyclonal antibody was produced in our lab as
described (18).
Plasmid Constructs--
Construction of
-arrestin1 and
-arrestin2 expression vectors was described previously (18). pEGFP-N
(GFP fused to the N terminus of the targeted protein) was generated by
inserting the EGFP coding sequence into HindIII and
BamHI sites of pcDNA3. pEGFP-C (EGFP fused to the C
terminus of the targeted protein) was generated by inserting the EGFP
coding sequence into XhoI and XbaI sites of
pcDNA3. All GFP fusion proteins used were constructed using these
two plasmids as vector.
arr2-GFP was generated by subcloning the
full-length cDNA fragment of human
-arrestin2 into the
BamHI and NotI sites of pEGFP-N.
arr1-GFP was
created by subcloning full-length human
-arrestin1 cDNA
fragments into the BamHI and XhoI sites of
pEGFP-N. HA-tagged
-arrestins were generated by subcloning the
full-length human
-arrestins cDNA fragments into the
pcDNA3-containing coding sequence of HA epitope.
Mutant constructs were made by mutating Ile-386, Val-387, Phe-388,
Phe-391, Leu-394, Leu-396, and Met-399 of
-arrestin2 to Ala by a
PCR-based strategy. To detect the subcellular localization of
different parts of
-arrestins, each fragment was amplified by PCR
using specific primers that contained recognition sequences for
specific restriction enzymes. The PCR products were purified and
digested with restriction enzymes and inserted into the
BamHI-NotI sites of pEGFP-C.
The GFP-Mdm2 construct was created by inserting the mouse Mdm2 cDNA
fragment (amplified from mouse brain by reverse transcription-PCR (RT-PCR) strategy) into EcoRI and XhoI sites
of pEGFP-N. An NES mutant of Mdm2 was made by mutating two
critical leucines of its NES (Leu-190 and Leu-192) to alanine by
PCR-based strategy. GFP-JNK3 construct was made by inserting the JNK3
cDNA fragment into BamHI and XhoI sites of
pEGFP-N. All constructs were verified by sequencing.
Cell Culture and Transfection--
Human Saos2 (osteosarcomal)
and HeLa cells were cultured in Dulbecco's modified Eagle's medium
plus 10% (v/v) heat-inactivated fetal bovine serum. Human embryonic
kidney (HEK293) cells were grown in Eagle's minimal essential medium
with Earle's salt supplemented with 10% (v/v) heat-inactivated fetal
bovine serum. All cells were cultured at 37 °C in a humidified 5%
CO2 incubator. Plasmids were transiently transfected in the
cells using the calcium phosphate method as described previously
(19).
Subcellular Localization Analyses and
Immunofluorescence--
For the subcellular localization studies of
the different GFP fusion proteins, cells were plated onto coverslips in
12-well dishes 16 h before transfection. The cells were
transfected with 1-2 µg of expression vectors for the different GFP
fusions. To examine the subcellular localization by DAPI staining, the
cells were fixed and permeabilized in 4% paraformaldehyde, 0.1%
Triton X-100 for 20 min at 4 °C, and the DNA was stained with 0.5 µg/ml DAPI for 2 min at room temperature. HA-tagged arrestins were
detected using a monoclonal anti-HA antibody. 24 h after
transfection, and the cells were fixed in 4% paraformaldehyde and
permeabilized with 0.1% Triton X-100. The fixed cells were incubated
with 2% bovine serum albumin in phosphate-buffered saline for 1 h
at room temperature before incubation with the anti-HA antibody
(diluted 1:200 in blocking solution) for 2 h at room temperature.
The immunostaining was developed using Texas Red-conjugated goat
anti-mouse IgG secondary antibody (diluted 1:100 in blocking solution).
Visualization was with an Olympus microscope (×60 oil or ×100 oil
immersion lens) equipped for epifluorescence. Images were captured with
a CCD camera and analyzed with the Spot Advanced software.
Coimmunoprecipitation--
Various HA-
-arrestin constructs
were cotransfected with GFP-Mdm2 plasmid. 48 h after transfection,
the cells were lysed in Lysis buffer (50 mM Tris-HCl, pH
7.5, 1 mM EDTA, 150 mM NaCl, 20 mM
NaF, 0.5% Nonidet P-40, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride) for 1.5 h at 4 °C. Cell extracts
were cleared by centrifugation at 12,000 × g for 10 min, and supernatants were incubated at 4 °C with 1 µg of anti-HA
antibody for 2 h. Immune complexes were immobilized on
protein-A-Sepharose beads for 3 h, washed three times with Lysis
buffer, and heated in SDS sample buffer in 50 °C water bath for 20 min.
Western Blotting Analysis--
Lysates from cells (48 h after
transfection) were boiled for 5 min in Sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, and 50 mM dithiothreitol). Aliquots containing 30 µg of protein
were subjected to 10% SDS-PAGE, and the proteins resolved were
electroblotted onto nitrocellulose membrane. The membrane was probed
with anti-HA primary and peroxidase-conjugated secondary antibodies.
The immune complexes were visualized using enhanced chemiluminescence
detection (Amersham Biosciences) according to the manufacturer's protocol.
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RESULTS |
Expression of
-Arrestin2 but not
-Arrestin1 Relocalized Mdm2
from the Nucleus to the Cytoplasm--
The full length of human
-arrestin1 and
-arrestin2, respectively, fused to the GAL4
DNA-binding domain was used as bait in a yeast two-hybrid system to
screen a mouse fetal brain complementary DNA library, and Mdm2 was thus
identified as a binding partner of both
-arrestin1 and
-arrestin2
(data not shown). Coimmunoprecipitation assay demonstrated the
abilities of both
-arrestin1 and
-arrestin2 to bind Mdm2 in
intact cells (Fig. 1A),
consistent with the recent report (16). To investigate the potential
biological significance of Mdm2/
-arrestin interaction, Mdm2 was
fused to GFP at the N terminus (GFP-Mdm2), and the effects of
Mdm2/
-arrestin interaction on the subcellular localization of Mdm2
is examined. GFP-Mdm2 was predominantly localized in the nucleus when
expressed in Saos2 cells (Fig. 1B);
-arrestin1 was
distributed in both the cytoplasm and nucleus, whereas
-arrestin2
was mainly localized in the cytoplasm (data not shown). No change in
the subcellular localization of Mdm2 was observed when Mdm2 was
coexpressed with
-arrestin1. However, in strong contrast,
coexpression of Mdm2 with
-arrestin2 resulted in a dramatic
redistribution of Mdm2 from the nucleus to the cytoplasm (Fig.
1C).

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Fig. 1.
Expression of
-arrestin2 but not
-arrestin1 relocalized Mdm2 from the nucleus to the
cytoplasm. GFP-Mdm2 was coexpressed with (A and
C) or without (B) HA- -arrestin1 or
HA- -arrestin2 in Saos2 cells. As shown in A, the cell
lysates were probed with anti-GFP antibody or immunoprecipitated with
anti-HA antibody and blotted with anti-GFP or anti-arrestin antibodies
following gel electrophoresis as indicated. As shown in B
and C, the cells were stained with anti-HA antibody to
detect -arrestins (red). Green fluorescence
represents GFP-Mdm2. The cells were counterstained with DAPI to
visualize nuclei (blue). The subcellular localization of
Mdm2 and -arrestins was analyzed by fluorescent microscopy.
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The molecular determinant of the differential effects of
-arrestin1
and
-arrestin2 on the subcellular localization of Mdm2 was analyzed
next. At first, we tested whether the relocalization of Mdm2 was a
result of the enhancement of the Mdm2 export from the nucleus by its
interaction with
-arrestin2. Mutant Mdm2 with L190A/L192A
substitutions (Mdm2NES
), which greatly impairs export of
Mdm2 from the nucleus (20), was used in this study. Mdm2NES
was mainly localized in the nucleus when
expressed alone (data not shown). However, coexpression with
-arrestin2 caused the significant relocalization of the mutated Mdm2
from the nucleus to cytoplasm (Fig. 2).
This result clearly indicates that the relocalization of Mdm2 by
-arrestin2 was independent of the nuclear export of Mdm2. Next, we
tested whether the relocalization of Mdm2 by expression of
-arrestin2 is affected by LMB, an antifungal antibiotic, which binds
directly and irreversibly to CRM1 and inhibits NES-mediated active
nuclear export (21, 22). As shown in Fig. 2, Mdm2 was accumulated in
the cells coexpressing
-arrestin2 and Mdm2 following LMB treatment
since
-arrestin2 was detained in the nucleus by the LMB treatment
(Fig. 2). This result strongly suggests that interaction of
-arrestin2 and Mdm2 modulates the nucleus/cytoplasm equilibrium of
Mdm2.

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Fig. 2.
Relocalization of Mdm2 by
-arrestin2 was independent of the
nucleocytoplasmic shuttling of Mdm2. 36 h after transfection,
Saos2 cells co-transfected with plasmids encoding HA- -arrestin2 and
GFP-Mdm2 or GFP-Mdm2NES were treated with or without 5 ng/ml LMB overnight. The cells were stained with anti-HA antibody to
detect -arrestins (red) and DAPI to visualize nuclei
(blue). Green fluorescence represents GFP-Mdm2.
The subcellular localization of Mdm2 and -arrestins was analyzed by
fluorescent microscopy.
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Difference between
-Arrestin2 and
-Arrestin1 in the
Nucleocytoplasmic Shuttling--
To further study the
nucleocytoplasmic shuttling of
-arrestins, we fused GFP to the C
termini of
-arrestin1 (
arr1-GFP) and
-arrestin2 (
arr2-GFP).
When transiently transfected into Saos2,
arr1-GFP was distributed in
both cytoplasm and nucleus, whereas
arr2-GFP was excluded from the
nucleus in most of the transfected cells. Treatment with 5 ng/ml LMB
induced the significant nuclear accumulation of
arr2-GFP fusion
protein. In contrast, LMB exerted no significant effect on the
subcellular distribution of
arr1-GFP (Fig.
3A). A similar result was also
obtained using HA-
-arrestins in HEK293 cells (data not shown). These
results suggest that
-arrestin2 but not
-arrestin1 shuttles
between the nucleus and cytoplasm in an LMB-sensitive manner.

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Fig. 3.
-Arrestin2 but not
-arrestin1 shuttled between the nucleus and
cytoplasm in an LMB-sensitive manner. The subcellular localization
of -arrestins was analyzed by fluorescent microscopy. As shown in
A, Saos2 cells were transfected with GFP- -arrestin1 or
GFP- -arrestin2 vectors. 36 h after transfection, the cells were
treated with or without 5 ng/ml LMB overnight, and the subcellular
localization of GFP- -arrestins was analyzed by fluorescent
microscopy. As shown in B, Hela cells treated with or
without LMB overnight were fixed, and the endogenous -arrestins were
detected with -arrestin specific antiserum.
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In addition, the nucleocytoplasmic shuttling of native
-arrestins in
HeLa cells was also investigated through immunostaining with the
specific anti-
-arrestin antibody that recognizes both
-arrestins.
Under resting conditions, endogenous
-arrestins were distributed
throughout the HeLa cells with a preferential cytoplasmic localization.
LMB treatment induced significant nuclear accumulation of
-arrestins
(Fig. 3B), indicating that endogenous
-arrestins
constitutively shuttle between the nucleus and cytoplasm by a mechanism
involving a functional cis-acting, LMB-sensitive NES or via an
NES-containing interaction partner.
Presence of a Typical NES Motif at the C Terminus of
-Arrestin2
but Not
-Arrestin1--
Usually, nuclear export in an LMB-sensitive
manner depends on a functional NES, and a typical NES possesses a
leucine- or other hydrophobic residue-rich motif (23). When we compared the amino acid sequence of
-arrestin2 with the reported functional NES sequences (Fig. 4A), a
10-residue region (residues 385-396) in
-arrestin2 was revealed to
constitute a hydrophobic motif similar to the reported NES. To test
whether this region is a functional NES of
-arrestin2, we attached
the C-terminal peptide (residues 384-409) of
-arrestin2 containing
this putative NES to the C terminus of GFP (GFP-C26). As shown in Fig.
4B, GFP, when expressed alone, was distributed throughout
the nucleus and cytoplasm with slightly more accumulation in the
nucleus. In contrast, GFP-C26 showed an evident nuclear exclusion of
fluorescence in the cells, and this was reversed by the LMB treatment
(Fig. 4B). Moreover, deletion of the C terminus (residues
384-409) of
-arrestin2 resulted in a significant nuclear
accumulation of
-arrestin2 (data not shown).

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Fig. 4.
Identification of the NES in
-arrestin2. A, alignments of the
putative NES in arrestins from different species with the
previously characterized NESs of MAPKK, protein kinase inhibitor (PKI),
p53, and Mdm2. Important hydrophobic residues are in bold.
As shown in B, Saos2 cells were transiently transfected with
GFP or GFP-C26 and treated with or without 5 ng/ml LMB and analyzed
under a fluorescent microscope. C, Ala replacement of the
critical hydrophobic residues in the putative NES of -arrestin2.
WT, wild type. C, C terminus; N, N
terminus. As shown in D, -arrestin2 mutants as indicated
were transiently transfected into Saos2 cells. 36 h after
transfection, the cells were fixed, and the subcellular localization of
-arrestin2 mutants was examined under a fluorescent
microscope.
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To define the critical residues in the NES motif of
-arrestin2 in
addition to the two leucine residues known (11), each of seven
hydrophobic residues in the putative NES was replaced with alanine
(Fig. 4C). The
-arrestin2 mutant with Phe-391, Leu-394, or Leu-396 replaced failed to shuttle to the cytoplasm, whereas replacement of Ile-386, Phe-388, or Met-399 produced no change in
localization of
-arrestin2 (Fig. 4D). The V387A mutant
behaved in between the above two mutant groups, showing a partial
impairment of the NES function (Fig. 4D). In addition, the
nucleocytoplasmic shuttling of
-arrestin2 mutants R393A and R395A
and the clathrin-binding-deficient
-arrestin2 mutant of AAEA (24)
was not altered (data not shown). These data strongly support the
notion that the whole hydrophobic motif
VXXXFXXLXL serves as the functional
NES in
-arrestin2.
Differential Nucleocytoplasmic Shuttling of
-Arrestin1 and
-Arrestin2 Was a Result of a Single Amino Acid Difference--
The
above results showed that residue Leu-394 was critical for the
extranuclear localization of
-arrestin2, and the sequence inspection
revealed that the Leu-394 residue is the only different residue among
the 7 hydrophobic residues between
-arrestin2 and
-arrestin1 in
the region. In
-arrestin1 and visual arrestin, Leu is substituted by
Gln at the corresponding site. To determine whether this single amino
acid difference determines the differential nucleocytoplasmic shuttling
of
-arrestin1 and
-arrestin2, we generated
arr2L394Q and
arr1Q394L mutants. In contrast to the wild type
-arrestin2,
arr2L394Q was accumulated in the nucleus, and its distribution was
insensitive to LMB treatment (Fig. 5). In
contrast to the wild type
-arrestin1,
arr1Q394L was mainly localized in the cytoplasm, and LMB treatment induced its nuclear accumulation (Fig. 5). Thus, the exchange of a single amino acid between
-arrestin2 and
-arrestin1 can totally convert their subcellular localization, and this result is consistent with the very
recent report (11) that Leu/Gln difference at the C terminus of
-arrestin plays a critical role in differential shuttling of
-arrestin2 and
-arrestin1.

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Fig. 5.
Differential nucleocytoplasmic shuttling
of -arrestin1 and
-arrestin2 was due to a single amino acid residue
difference. Saos2 cells expressing GFP -arrestins and their
mutants were treated with 5 ng/ml LMB overnight, and the subcellular
localization of these -arrestins was analyzed by fluorescent
microscopy.
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The Intact N Domain of
-Arrestins Was Required for Their Nuclear
Import--
Proteins larger than 40-60 kDa cannot enter into the
nucleus through the nuclear pore complex by passive diffusion.
GFP-
-arrestins have an apparent molecular mass of about 82 kDa and
are apparently too large to diffuse into the nucleus. Thus, it seems
logical to assume that
-arrestins may contain a functional nuclear
localization signal (NLS) or may be transported via interaction with a
partner protein containing an NLS. To test the assumption, a series of deletion mutants of
-arrestin2 was created via a GFP fusion protein (Fig. 6A), and expression of
those GFP-fused proteins was confirmed by Western blot analysis
(data not shown). The
-arrestin2 mutants with deletion of its N
terminus (residues 1-185) were primarily present in the cytoplasm
whether with (data not shown) or without a functional NES (Fig.
6B). In strong contrast, the N-terminal fragment (residues
1-185) of
-arrestin2, which contains the intact N domain of
-arrestin2, was exclusively distributed in the nucleus, clearly
indicating that the N terminus of
-arrestin2 contains an essential
and sufficient structure for its nuclear localization. The results from
-arrestin1 mutants (
-arrestin11-184 and
-arrestin1185-418) also supported the notion (Fig.
6C). The further attempt to map down the NLS in the region,
however, failed to define a classical NLS structure with a motif
no larger than 20 amino acids (Fig. 6B). The mutations of
several Lys and Arg residues in the region did not provide any detailed
information about the putative NLS (data not shown).

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Fig. 6.
The intact N domain of
-arrestins was required for nuclear import of
-arrestins. A, schematic
representation of GFP-fused -arrestin2 and its deletion mutants.
C, C terminus; N, N terminus. B and
C, HEK293 cells transiently expressing GFP or GFP-fused
-arrestins. As shown in D, Saos2 cells transfected with
GFP- arr2V54D mutant were treated with or without LMB overnight. The
cells were fixed and examined by fluorescent microscopy.
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We then tested the subcellular distribution of mutant
-arrestin2
with a V54D point mutation in the N domain (
arr2V54D), a widely used
dominant negative mutant that inhibits the internalization of GPCRs.
Although
arr2V54D was predominantly distributed in the cytoplasm in
Saos2 cells, the LMB treatment exerted no effect on its subcellular
distribution even after longer treatment as compared with the wild type
-arrestin2 (Fig. 6D). This result not only supports that
the structural integrity of N domain is important for the nuclear
import of
-arrestin2 but also implies that the N domain of
-arrestin2 may interact with other partner proteins to facilitate
its nuclear import.
Subcellular Localization of
-Arrestin2 Modulated the
Distribution of Its Binding Partner Mdm2 and JNK3--
Whether
differential nucleocytoplasmic shuttling of two
-arrestins
contributes to their different effects on the localization of Mdm2 was
further examined. Unlike its wild type counterpart,
arr2L394Q was
localized in the nucleus; coexpression of
arr2L394Q with Mdm2
resulted in accumulation of Mdm2 in the nucleus, in contrast to what
was observed following coexpression of the wild type
-arrestin2
(Fig. 7A). This is unlikely to
be a result of the impairment of the interaction between
arr2L394Q
and Mdm2 since their interaction was not affected by the mutation (data not shown). In addition, the coexpression of
arr1Q394L with Mdm2 also evidently relocalized Mdm2 in the cytoplasm in contrast to the
wild type
-arrestin1 (Fig. 7A).

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Fig. 7.
Subcellular localization of Mdm2 and JNK3 was
affected by -arrestin distribution. As
shown in A, HA-tagged -arrestin or its mutant
( arr2L394Q or arr1Q394L) was cotransfected with GFP-Mdm2 into
Saos2 cells. As shown in B, HA-tagged -arrestin2 or its
mutant ( arr2L394Q or arr2V54D) was coexpressed with GFP-JNK3 in
HEK293 cells. The cells were fixed and stained with anti-HA antibody to
detect -arrestins (red). Green fluorescence
was used to determine subcellular distribution of GFP-Mdm2 or JNK3. The
samples were examined under a fluorescent microscope.
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It was reported that the binding to
-arrestin2 keeps JNK3 outside
the nucleus (14). We therefore studied the effect of the nuclear import
and export of
-arrestin2 on the subcellular localization of JNK3.
When expressed alone, JNK3 was distributed evenly in the nucleus and
cytoplasm, and coexpression with
-arrestin2 caused the significant
extranuclear localization of JNK3 (Fig. 7B), in good
agreement with a previous report (14). When co-expressed with
arr2V54D, JNK3 translocated to the cytoplasm, where it co-localized with
arr2V54D, whereas coexpression of
arr2L394Q did not change the JNK3 localization (Fig. 7B). Thus, our data indicate
that the nucleocytoplasmic shuttling of
-arrestins modulates
subcellular distribution of its binding partner Mdm2 and JNK3.
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DISCUSSION |
-Arrestin1 and
-arrestin2 play a critical role in the
regulation of the signaling of GPCRs, and accumulating evidence
demonstrates that the functions of
-arrestins depend on their
nucleocytoplasmic shuttling. The current study clearly demonstrated
that
-arrestins, either endogenous or exogenous, could effectively
enter the nucleus, and this is likely mediated by its interaction with
an NLS-containing partner since no classical nuclear import sequence
was identified in
-arrestins. A similar mechanism has been reported
for other regulatory proteins. For example,
-catenin is imported
into the nucleus via its interaction with the lymphoid enhancer
factor/T-cell factor (LEF/TCF) transcription factor (25, 26). In
addition, our study also established that the N terminus of
-arrestins is essential and sufficient for their nuclear
localization. Therefore, it can be speculated that the assumed partner
with a functional NLS binds to the N terminus of
-arrestins, and
thus, the complex is imported into the nucleus. However, these partners
of
-arrestins remain to be identified.
The crystal structures of visual arrestin and
-arrestin1 show that a
typical arrestin molecule has a central polar core flanked by the N and
C domains and a C-terminal tail connecting the two domains (27, 28).
Although the crystal structure of
-arrestin2 is not yet available,
it is very likely to possess a similar conformation since two arrestins
share a high homology in their amino acid sequences. Our current study
has shown that the N terminus of
-arrestins, which contains the
intact N domain (residues 6-172 in
-arrestin1), is fully
responsible for their nuclear localization. It has been shown that the
N domain is more flexible and shares a higher degree of homology than
other domains or whole proteins among different arrestins. Moreover,
the N domain contains the binding sites for several
-arrestin
partners, including a GPCR-binding site. For example, c-Src has been
demonstrated to bind to the proline-rich regions at the N domain of
-arrestins (29). In addition, our recent data revealed that Mdm2
also binds to the N domain of
-arrestins (data not shown). Thus, the
structural integrity of the N domain within the N terminus of
-arrestins is important for their subcellular localization.
Results from our current study and the very recent report from another
laboratory show that
-arrestin2, but not
-arrestin1, contains a functional NES at its C terminus (42). It is well established that a typical NES is typically comprised of a
10-amino-acid motif with at least 4 conserved hydrophobic residues. In
this study, we identified the hydrophobic motif
VXXXFXXLXL as a functional NES for
-arrestin2 in addition to the two leucines reported previously (11).
More interestingly, the alignment of arrestin amino acid sequences from
different species clearly reveals that the typical NES in
-arrestin2
is highly conserved not only in human and rat
-arrestin2 but also in
fruit fly, trout, and locust in which only one nonvisual arrestin
exists (Fig. 4A). This suggests the nucleocytoplasmic
shuttling is very important for the functions of these arrestins.
-Arrestins are originally identified as a negative regulator of GPCR
signaling involved in the desensitization and internalization of the
receptors. Recent findings of new binding partners of
-arrestins had
expanded our knowledge about their functions (14). Although the
function of
-arrestins as endocytic proteins in the nucleus is still
unclear, several other endocytic proteins, such as Epsin1, the clathrin
assembly lymphoid myeloid leukemia, and
-adaptin, have been reported
to shuttle between the nucleus and cytoplasm and play a direct or
indirect role in transcriptional regulation (30). Moreover, it has been
reported that some of GPCRs such as angiotensin receptor,
prostaglandin ubiquitin carrier protein (E2) receptor, and
muscarinic receptor reside on the nuclear membrane (31-34) and that
some other GPCR signal molecules such as G
protein, G
5, and
regulators of G protein are imported into the nucleus (35-38). Thus,
it is conceivable that
-arrestins may modulate the transcriptional
activity directly via interacting with transcriptional factors or
indirectly via regulating the nuclear GPCR signaling.
Subcellular localization of proteins is vital to their functions. The
nuclear localization of Mdm2 has been known to be important for its
ability to degrade p53. Impairment of nuclear import of Mdm2, such as
in the cases of the mutant or the alternative spliced form of Mdm2,
greatly reduces its ability to degrade p53 (39). However, the
enhancement of Mdm2 nuclear targeting, such as phosphorylation of Mdm2,
can increase its function (40). In the present study,
-arrestin2 was
found to be able to alter the subcellular localization of Mdm2 from the
nucleus to the cytoplasm via its nucleocytoplasmic shuttling, and thus,
it is anticipated that
-arrestin2 likely modulates the functions of
Mdm2 as well as p53. In fact, our recent data (41) indeed provided the
evidence for the expectation and demonstrated that
-arrestin2
reduces the Mdm2-mediated ubiquitination and degradation of p53 and
subsequently enhances p53-induced apoptosis. Moreover, it has been
reported (16) that
-arrestin2 but not
-arrestin1 can mediate
ubiquitination of
2AR. From the results of the current
study, the specificity of the effect of
-arrestins can be reasonably
explained since only
-arrestin2 can shift the subcellular
localization of Mdm2 from nucleus to cytoplasm although both
-arrestins interact well with Mdm2.