beta -Arrestin 2 Functions as a G-Protein-coupled Receptor-activated Regulator of Oncoprotein Mdm2*

Ping WangDagger §, Hua GaoDagger §, Yanxiang NiDagger , Beibei WangDagger , Yalan WuDagger , Lili JiDagger , Linhua QinDagger , Lan Ma, and Gang PeiDagger ||

From the Dagger  Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, People's Republic of China and the  National Laboratory of Medical Neurobiology, Fudan University Medical Center, Shanghai 200032, People's Republic of China

Received for publication, October 9, 2002, and in revised form, December 2, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Oncoprotein Mdm2 is a master negative regulator of the tumor suppressor p53 and has been recently shown to regulate the ubiquitination of beta -arrestin 2, an important adapter and scaffold in signaling of G-protein-coupled receptors (GPCRs). However, whether beta -arrestin 2 has any effect on the function of Mdm2 is still unclear. Our current results demonstrated that the binding of Mdm2 to beta -arrestin 2 was significantly enhanced by stimulation of GPCRs. Activation of GPCRs led to formation of a ternary complex of Mdm2, beta -arrestin 2, and GPCRs and thus recruited Mdm2 to GPCRs at plasma membrane. Moreover, the binding of beta -arrestin 2 to Mdm2 suppressed the self-ubiquitination of Mdm2 and consequently reduced the Mdm2-mediated p53 degradation and ubiquitination. Further experiments revealed that overexpression of beta -arrestin 2 enhanced the p53-mediated apoptosis while suppression of endogenous beta -arrestin 2 expression by RNA interference technology considerably attenuated the p53-mediated apoptosis. Our study thus suggests that beta -arrestin 2 may serve as a cross-talk linker between GPCR and p53 signaling pathways.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The tumor suppressor protein p53 is a potent inhibitor of cell growth that can induce growth arrest and apoptosis (1-3). Oncoprotein Mdm2 is a key negative regulator of p53 function, which has been demonstrated by many studies including allelic knockout mice. Mice possessing homozygous deletions of Mdm2 die at around day 5 of embryogenesis; whereas mice possessing a homozygous deletion of both Mdm2 and p53 are viable and develop normally (4, 5). On the other hand, overexpression of Mdm2 leads to the loss of p53 and tumor development (6). Transcription of the Mdm2 gene is strongly stimulated by the activated p53. The Mdm2 protein, in turn, binds to p53 and conceals its transactivation domain. Moreover, Mdm2 functions as a RING finger-dependent E3 ubiquitin ligase and promotes the ubiquitination and degradation of p53 by the proteasome (6, 7). Thus, Mdm2 and p53 form an autoregulatory feedback loop between the expression and function of p53 and Mdm2. It has been well established that the feedback loop connected Mdm2 and p53 is significantly activated in response to stress such as DNA damage or activation of oncogenes (6, 8). However, other important signaling may also potentially influence the Mdm2/p53 pathway.

Mdm2 interacts with several cellular proteins in addition to p53 and some of them can regulate the Mdm2/p53 feedback loop by a change in the cellular distribution of Mdm2, alternation of the stability of Mdm2, or inhibition the E3 ligase activity of Mdm2 (6-9). The distribution and shuttling of Mdm2 between the nucleus and the cytoplasm (10-12) are greatly modulated by its association with the regulators, such as ARF, which assists the sequestering of Mdm2 in nucleoli and prevents its export to the cytoplasm (13, 14). Moreover, Mdm2 regulates its own stability by autoubiquitination via the interaction with its partner. For example, the direct binding of Tsg101 or MdmX to Mdm2 leads to the inhibition of the degradation and autoubiquitination of Mdm2 (6, 9, 15). Recently, beta -arrestin 2 has been identified as a novel partner of Mdm2, which targets beta -arrestin 2 for ubiquitination (16). But whether beta -arrestin 2 has any effect on the function of Mdm2 is still to be investigated.

G-protein-coupled receptors (GPCRs),1 a large superfamily of seven transmembrane cell-surface receptors, are responsible for many physiological processes. Stimulation with agonist causes the phosphorylation of the receptor by G-protein-coupled receptor kinases (GRKs) (17). Then beta -arrestin translocates from cytoplasm to cell membrane and binds to the phosphorylated GPCRs. Binding of beta -arrestin to GPCRs causes receptor desensitization and internalization and thus regulates signaling of GPCRs (18, 19). Recent discoveries indicate that the beta -arrestin also plays important roles as scaffold-linking GPCRs to mitogen-activated protein kinase (MAPK) cascades, such as JNK3 (20). beta -Arrestin 2 recruits ASK1 and JNK3 together and enhances the GPCR-mediated activation of JNK3, which has been reported to be tightly associated with cell apoptosis (21). More interestingly, it is demonstrated that the stable recruitment of visual arrestin to the activated rhodopsin in the fly leads to retinal cell apoptosis (22, 23). These experiments suggest that arrestin may function as a potential linker of GPCR signaling to apoptotic pathways, which, however, remains to be further explored. In the current study, we demonstrate that activation of GPCRs stimulates the association of beta -arrestin 2 with oncoprotein Mdm2 and thus regulates the function of Mdm2. Our results suggest that beta -arrestin 2 play a potential role in mediating GPCR signaling to p53 pathway.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Yeast Two-hybrid System-- The ProQuest two-hybrid system and the mouse fetal brain cDNA library were purchased from Invitrogen. The bait for library screening was the full-length of human beta -arrestin 2. Two-hybrid screening was carried out according to the protocol provided by the manufacturer.

Cell Culture and Transfection-- Human embryonic kidney (HEK)293 cells were cultured in minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum. Normal liver L02 and HepG2 cells were cultured in 1640 supplemented with 10% fetal bovine serum. p53-null human osteosarcoma Saos2 and lung adenocarcinoma H1299 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. All cells were transfected using the calcium phosphate-DNA co-precipitation method as described previously (24). For all transfection experiments, CMV-beta -gal was used to compensate the total DNA input.

Plasmids-- Full-length Mdm2, MdmX, and p53 were cloned from mouse brain into modified pcDNA3 vector in-frame with HA or FLAG tag at the N terminus. GFP-Mdm2 and the Mdm2 mutants were subcloned by PCR from HA-Mdm2 into pcDNA3 with the GFP tag at the N terminus. beta -Arrestin 2 and its mutant V54D clones were generated as described (24). Ubiquitin plasmid was obtained from Dr. Dirk Bohmann (University of Rochester Medical Center). The authenticity of the DNA sequences was confirmed by sequencing. Construction of RNAi plasmid for beta -arrestin 2 was performed as described (25). The BS/U6 vector was a generous gift from Dr. Yang Shi (Department of Pathology, Harvard Medical School, Boston, MA). A 22-nt oligonucleotide (oligo1) corresponding to nucleotides 216-237 of the beta -arrestin 2 coding region was first inserted into the BS/U6 vector with ApaI and XhoI. The inverted motif that contains the 6-nt spacer and 5 Ts (oligo2) was then subcloned into the XhoI and EcoRI sites of the intermediate plasmid to generate BS/U6/beta -arrestin 2.

Adenovirus-- Recombinant adenoviruses encoding beta -galactosidase or beta -arrestin 2 were prepared as described previously (26). Briefly, beta - galactosidase or beta -arrestin 2 was cloned into shuttle vector pAdTrack-CMV using standard cloning protocol, and the shuttle vectors were recombined with pAdEasy-1, which harbors a CMV-driven GFP, to form the viral constructs. The adenoviral plasmids were transfected into HEK293 cells to generate recombinant adenovirus. The L02 or Saos2 cells were infected with the adenovirus for 12 h, and then the virus was removed to let cells recover. The infection efficiency was determined by observing GFP fluorescence, and the quantity of virus that afforded at least 80% transfection efficiency was selected for the experiments.

Immunoprecipitation and Western Blotting-- 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 48 h after transfection. Cell extracts were cleared, and the supernatant was incubated at 4 °C with anti-HA (1 µg; Roche Molecular Biochemicals), anti-FLAG (1 µg, Santa Cruz Biotechnology) antibody or anti-beta -arrestin egg yolk IgY produced with recombinant beta -arrestin 2 from chicken (1:1000) for 2 h. Immune complexes were immobilized on protein A (Amersham Biosciences) or anti-chicken IgY Sepharose beads (Promega) for 3 h, washed three times with Lysis Buffer, heated in SDS sample buffer in a 50 °C water bath for 20 min. Western analysis was performed as previously described (24). HA-tagged proteins were detected with anti-HA antibody and GFP-tagged protein with anti-GFP antibody. Endogenous Mdm2 was detected with antibody specific to Mdm2 (Ab3, Oncogene Research Products) or SIMP14 antibody (Santa Cruz Biotechnology).

Immunofluorescence Microscopy-- Cells were grown on glass coverslips and transfected with indicated plasmids. 24 h after transfection, the cells were fixed with 4% polyformaldehyde for 20 min and incubated with phosphate-buffered saline containing 0.1% Triton X-100 for 5 min. HA-tagged fusion proteins were detected with anti-HA antibody and a fluorescein isothiocyanate-conjugated secondary antibody (green) (Jackson ImmunoResearch). Endogenous Mdm2 were detected with anti-Mdm2 SIMP14 antibody and a Texas Red-conjugated IgG (red). Coverslips were analyzed on Leica TCSNT laser confocal scanning microscope.

Ubiquitination Assay-- 44 h after transfection, the cells were treated with the cell-permeable proteasomal inhibitor, MG132 (10 µM, Sigma), for 4 h and then lysed in Lysis Buffer. The Mdm2-ubiquitin and p53-ubiquitin complexes were immunoprecipitated using anti-GFP antibody and anti-FLAG antibody and immunoblotted with anti-HA antibody to detect ubiquitinated proteins. Endogenous Mdm2 were immunoprecipitated with anti-Mdm2 antibody (H-221, Santa Cruz Biotechnology) and p53 with anti-p53 antibody (FL-393, Santa Cruz Biotechnology). Immunoprecipitates were then subjected to Western blotting analysis with ubiquitin-specific antibody (Ub P4D1, Santa Cruz Biotechnology).

Apoptosis and Cell Death-- Saos2 cells were cultured in 6-well plates and transfected with indicated plasmids along with the plasmid of CMV-EGFP. After 48 h, the cultures were analyzed under a fluorescence microscope, and the transfected cells were marked by the presence of green fluorescence. Apoptotic cells were identified and accounted by their rounded morphology, in contrast to the spreaded-out morphology of nonapoptotic cells (27). The assay for cell death was performed by trypan blue staining (28). DNA fragmentation analysis was performed as described (29). Briefly, 48 h after infection with indicated virus, the cells were treated with UV (10 J/m2) and cultured for additional 24 h. Both floating and adherent cells were harvested and lysed with buffer containing 5 mM Tris-HCl, pH 8.0, 10 mM EDTA, and 0.5% Triton X-100, and left on ice overnight at 4 °C. After centrifugation at 13,000 × g for 20 min at 4 °C, the fragmented DNA in the supernatant fraction was extracted twice with phenol/chloroform/isopropyl alcohol (25:24:1, v/v) and once with chloroform and then precipitated overnight at -20 °C. The DNA fragments were separated by 1.4% agarose gel electrophoresis and visualized by ethidium bromide staining.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activation of GPCRs Enhanced the Binding of Mdm2 to beta -Arrestin 2-- The full-length human beta -arrestin 2 was fused to the GAL4 DNA-binding domain (DBD) as a bait in the yeast two-hybrid system to screen a mouse fetal brain complementary DNA library, and Mdm2 was thus identified as a binding partner of beta -arrestin 2. Their interaction in mammalian cells was confirmed by coimmunoprecipitation (Fig. 1). These results were consistent with the recent report by Shenoy et al. (16). The interaction between beta -arrestin 2 and MdmX, a Mdm2-related protein, was also examined in expressed HEK293 cells. As shown in Fig. 1, beta -arrestin 2 interacted well with Mdm2 whereas the interaction between MdmX and beta -arrestin 2 was hardly detected under the same conditions. Moreover, the presence of MdmX did not interfere with the interaction between Mdm2 and beta -arrestin 2. When the presence Mdm2, MdmX could be coimmunoprecipitated with beta -arrestin 2, indicating beta -arrestin 2, Mdm2, and MdmX can form a ternary complex.


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Fig. 1.   beta -Arrestin 2 interacted with Mdm2 but not MdmX. HEK293 cells were cotransfected with GFP-Mdm2, GFP-MdmX, or HA-beta -arrestin 2 as indicated, and then the cells were subjected to immunoprecipitation with anti-HA antibody. Coimmunoprecipitated Mdm2 or MdmX was detected with anti-GFP antibody. beta arr, beta -arrestin; IP, immunoprecipitate; IB, immunoblot.

Agonist stimulation of the beta 2-adrenergic receptor could increase the association of beta -arrestin 1 and its partner Src (30). Therefore, the effect of GPCR activation on the interaction between beta -arrestin 2 and Mdm2 was examined. The HEK293 cells coexpressing beta -arrestin 2, Mdm2, and delta  opioid receptor (DOR), which has been shown to interact with beta -arrestin 2 well (31), were stimulated with DOR-specific agonist DPDPE, and then beta -arrestin 2 was immunoprecipited. As shown in Fig. 2A, Mdm2 was present in the beta -arrestin 2 immunoprecipitates, and an increase in beta -arrestin 2-bound Mdm2 was clearly detected after 2 min of agonist exposure and peaked at 5 min. Stimulation of another GPCR, the beta 2 adrenergic receptor, could also lead to the increase in the association of these two proteins (data not shown).


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Fig. 2.   Activation of GPCRs enhanced the interaction of beta -arrestin 2 with Mdm2. A, HEK293 cells cotransfected with DOR, HA-beta -arrestin 2, and GFP-Mdm2, were challenged with 1 µM DPDPE for the indicated time, and then the cells were subjected to immunoprecipitation with anti-HA antibody. Coimmunoprecipitated Mdm2 was detected with anti-GFP antibody. Quantitative data are represented as fold values of the basal coimmunoprecipitated Mdm2 and means ± S.E. of three separate experiments. B, SK-N-SH cells were treated with or without 100 µM naloxone for 20 min prior to stimulation with 1 µM DPDPE for 10 min, and then the cells were subjected to immunoprecipitation with anti-beta -arrestin antibody. C, HepG2 cells were treated with 1 µM bradykinin, and cells were subjected to coimmunoprecipitation analysis.

Then we tested the effect of endogenous GPCR activation on the interaction between the endogenous beta -arrestin 2 and Mdm2. As shown in Fig. 2B, DPDPE treatment apparently increased the association of endogenous Mdm2 and beta -arrestin in neuroblastoma SK-N-SH cells and pretreatment with DOR antagonist naloxone blocked the effect of DPDPE. Stimulation with bradykinin, which stimulates the endogenous bradykinin receptors, for 30 min also enhanced the interaction of endogenous Mdm2 and beta -arrestin in HepG2 cells (Fig. 2C). These results provide clear evidence that stimulation of GPCRs can regulate the interaction between beta -arrestin 2 and Mdm2.

beta -Arrestin 2 Recruited Mdm2 to the Activated GPCRs-- Then we asked whether beta -arrestin 2 could bring Mdm2 to the activated receptors through its binding to Mdm2. DOR was coexpressed with Mdm2 in the presence or absence of beta -arrestin 2 in HEK293 cells. As shown in Fig. 3, A and B, in the absence of coexpressed beta -arrestin 2, DOR could not coprecipitate Mdm2 (this may be due to a low level of endogenous beta -arrestin 2 in HEK293 cells). In the presence of beta -arrestin 2 but without stimulation of DOR, only weak binding of Mdm2 to DOR was observed. This binding was significantly enhanced after a 10-min exposure to DOR agonist. These data demonstrate that beta -arrestin 2 can recruit Mdm2 to DOR, and suggest that DOR, beta -arrestin 2, and Mdm2 form a ternary complex in an agonist-dependent manner.


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Fig. 3.   Mdm2 was recruited to the activated GPCR. A, recruitment of Mdm2 to the activated DOR in a beta -arrestin 2-dependent manner. HEK293 cells were cotransfected with HA-tagged DOR and GFP-Mdm2 in the presence or absence of beta -arrestin 2. After treated with 1 µM DPDPE for 10 min, the cells were lysed and immunoprecipitated with anti-HA antibody. The presence of Mdm2 was detected using anti-GFP antibody. B, quantitative data are represented as fold values of the basal coimmunoprecipitated Mdm2. C, colocalization of endogenous Mdm2 with the activated DOR at plasma membrane. HEK293 cells transfected with HA-DOR were treated with or without 100 µM naloxone for 20 min prior to stimulation with 1 µM DPDPE for 10 min. Receptors were detected with anti-HA antibody (green), and Mdm2 was detected with its specific antibody (red).

We further examined whether Mdm2 could colocalize with the activated GPCRs under more physiological conditions. When DOR was expressed in HEK293 cells, endogenous Mdm2 was predominantly distributed in nucleus and scarcely colocalized with DOR at the plasma membrane. After the cells were exposed to DPDPE, a proportion of Mdm2 was found to translocate to the plasma membrane where it colocalized with DOR (Fig. 3C). Similar results were also obtained when the beta 2 adrenergic receptor was stimulated (data not shown). Moreover, pretreatment with naloxone completely blocked the translocation of Mdm2 induced by DPDPE (Fig. 3C). Thus, our data suggest that the subcellular distribution of endogenous Mdm2 can be modulated by GPCR activation.

The N Terminus of beta -Arrestin 2 Interacted with the Central Region of Mdm2-- To map the Mdm2 interaction region in beta -arrestin 2, we used beta -arrestin 2 deletion mutants in coimmunoprecipitation experiments. The wild type or mutant beta -arrestin 2 was expressed in HEK293 cells along with Mdm2, and coimmunoprecipitation of Mdm2 with beta -arrestin 2 was monitored by immunoblotting. Deletion of amino acids 1-185 from the N terminus of beta -arrestin 2 resulted in the complete loss of Mdm2 binding. Conversely, a beta -arrestin 2 fragment containing amino acids 1-185 associated with Mdm2 as efficiently as the full-length beta -arrestin 2, strongly indicating that Mdm2 binds to the beta -arrestin 2 N terminus (Fig. 4A). Interestingly, an N-terminal point mutation of beta -arrestin 2, Val54 right-arrow Asp (V54D), which inhibits the internalization of GPCRs (32), also failed to interact with Mdm2 (Fig. 4B). This further supports that the N terminus of beta -arrestin 2 contains the Mdm2 binding sites.


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Fig. 4.   Mapping the interaction region between beta -arrestin 2 and Mdm2. A, HEK293 cells were cotransfected with GFP-Mdm2 and full-length or truncated HA-tagged beta -arrestin 2. Full-length and truncated beta -arrestin 2 were immunoprecipitated with anti-HA antibody, and the presence of Mdm2 in the immunoprecipitate was detected using anti-GFP antibody. B, HEK293 cells were cotransfected with GFP-Mdm2 and HA-tagged wild type or V54D mutated beta -arrestin 2 (beta 2V54D). beta -Arrestin 2 was immunoprecipitated with anti-HA antibody. The immunoblot was detected with anti-GFP antibody for Mdm2 and anti-HA antibody for beta -arrestin 2. C, schematic representation of wild type and mutants of Mdm2. NLS, nuclear localization signal; NES, nuclear export signal; RING, RING finger domain; p53, p53 binding domain; beta Arr2, beta -arrestin 2 binding domain. D, HEK293 cells were cotransfected with GFP-beta -arrestin2 and full-length or truncated HA-tagged Mdm2. Full-length and truncated Mdm2 were immunoprecipitated with anti-HA antibody, and the presence of beta -arrestin 2 in the immunoprecipitate was detected using anti-beta -arrestin antibody. Immunoprecipitated Mdm2 was detected with an anti-HA antibody.

Because the shortest Mdm2 clone identified in our yeast two-hybrid system-encoded fragment comprised amino acids from 358 to 489 in the C terminus of Mmd2, we further tested the importance of this region to the Mdm2 binding to beta -arrestin 2. A series of Mdm2 deletion mutants (Fig. 4C) were used in coimmunoprecipitation assays, and the results showed that among them the mutants of Mdm2-(1-358) and Mdm2-(1-383) lost their ability to bind to beta -arrestin 2 (Fig. 4D). In contrast, Mdm2-(1-410) and Mdm2-(1-435) still associated with beta -arrestin 2 to an extent comparable to the full-length Mdm2 (Fig. 4D). These data indicate that the region from 383 to 410 of Mdm2 is essential for beta -arrestin 2 binding.

Binding of beta -Arrestin 2 with Mdm2 Negatively Regulated the Function of Mdm2-- Mdm2 is a RING finger-dependent E3 ubiquitin ligase for p53 and targets p53 for ubiquitination and degradation in a ubiquitin-dependent pathway. It has been reported that degradation of p53 mediated by Mdm2 is regulated by several binding partners of Mdm2 (13, 15). We therefore questioned whether binding of beta -arrestin 2 to Mdm2 could interfere with Mdm2-directed p53 degradation. In a standard assay that tests the potential effect of Mdm2 partner on Mdm2/p53 feedback loop, coexpression of Mdm2 resulted in a significant reduction of p53 protein level in Saos2 cells, which lack p53 (Fig. 5, A and B). Further coexpression of increasing amounts of beta -arrestin 2 could effectively restore the protein level of p53 (Fig. 5, A and B), indicating the reduction of the Mdm2-mediated p53 degradation by beta -arrestin 2. Furthermore, the coexpression of beta -arrestin 2-(1-185), which binds to Mdm2, could restore p53 level while beta -arrestin 2-(186-409), which does not bind to Mdm2, failed to do so (Fig. 5D). The above data demonstrate that binding of beta -arrestin 2 with Mdm2 functionally regulates Mdm2-mediated p53 degradation. In support of this notion, our results showed that coexpression of beta -arrestin 2 or beta -arrestin 2-(1-185) but not beta -arrestin 2-(186-409) also increased the protein level of Mdm2 since the binding of beta -arrestin 2 with Mdm2 regulates the stability of Mdm2 (Fig. 5, A and C).


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Fig. 5.   beta -Arrestin 2 reduced the degradation of Mdm2 and p53. A, stabilization of p53 and Mdm2 by beta -arrestin 2. Saos2 cells were transfected with 100 ng of FLAG-p53 expression vector alone or together with 2 µg of HA-Mdm2 expression vector in the absence or presence of 2, 4, or 6 µg of full-length beta -arrestin 2 expression vector. 100 ng of CMV-GFP expression vector was cotransfected to examine transfection efficiency, and CMV-beta -gal vector was used to compensate total DNA input. Cell lysates were analyzed by Western blotting with anti-FLAG, anti-HA, or anti-GFP antibody. B, quantitative data of the protein level of p53. Data are means of three independent experiments. C, quantitative data of the protein level of Mdm2. Data are represented as the fold values of the control. D, stabilization of Mdm2 and p53 by beta -arrestin 2-(1-185) fragment. Wild type and mutant beta -arrestin 2 were transfected into Saos2 cells with GFP-Mdm2 at a 3:1 ratio.

In all cases, the expression levels of the control protein GFP cotransfected were not affected by cotransfection of other plasmids, beta -arrestin 2, Mdm2, and p53 (Fig. 5A), indicating that coexpression of beta -arrestin 2 unlikely interferes with the protein synthesis of Mdm2 or p53. To further investigate beta -arrestin 2-mediated stabilization of the Mdm2 and p53, the effect of beta -arrestin 2 on ubiquitination of Mdm2 and p53 was analyzed. As shown in Fig. 6, A and B, coexpression of beta -arrestin 2 significantly reduced the self-ubiquitination of Mdm2 and the Mdm2-mediated p53 ubiquitination. In contrast, coexpression of V54D beta -arrestin 2 mutant, which does not bind to Mdm2, had no effect on Mdm2-mediated p53 ubiquitination (Fig. 6B). In L02 cells, a normal liver cell line that expresses wild type p53 (32), infection with adenovirus expressing beta -arrestin 2 could similarly reduce the ubiquitination of both endogenous Mdm2 and p53 as compared with that with the control virus (Fig. 6, C and D). Taken together, these data indicate that beta -arrestin 2 negatively regulates self-ubiquitination of Mdm2 and Mdm2-mediated ubiquitination of p53 via its binding with Mdm2.


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Fig. 6.   beta -Arrestin 2 reduced the ubiquitination of Mdm2 and p53. A, reduction of Mdm2 self-ubiquitination. Saos2 cells were transfected with GFP-Mdm2, HA-ubiquitin, and full-length beta -arrestin 2 in various combinations. Cells were pretreated with 10 µM MG132 for 4 h, and Mdm2 was immunoprecipitated with anti-GFP antibody. The Mdm2-ubiquitin complex was detected with anti-HA antibody. B, reduction of Mdm2-mediated p53 ubiquitination by beta -arrestin 2. Saos2 cells were transfected with plasmids in combination as indicated. p53 was immunoprecipitated with anti-FLAG antibody, and the p53-ubiquitin complex was examined with anti-HA antibody. C, reduction of the ubiquitination of endogenous Mdm2 by beta -arrestin 2. L02 cells infected with control adenovirus or beta -arrestin 2 adenovirus were treated with 10 µM MG132 for 4 h, and Mdm2 was immunoprecipited with anti-Mdm2 antibody. The ubiquitinated Mdm2 was detected with ubiquitin-specific antibody. D, reduction of the ubiquitination of endogenous p53 by beta -arrestin 2. L02 cells infected with control adenovirus or beta -arrestin2 adenovirus were treated with 10 µM MG132 for 4 h and p53 was immunoprecipitated with p53-specific antibody. The ubiquitinated p53 was detected with ubiquitin-specific antibody.

beta -Arrestin 2 Enhanced p53-mediated Apoptosis-- Since overexpression of beta -arrestin 2 could effectively reduce the Mdm2-mediated p53 degradation, its consequence on the p53 function was next examined in Saos2 cells. As shown in Fig. 7A, introduction of p53 into Saos2 cells resulted in apoptosis in 60% of the transfected cells and coexpression of Mdm2 significantly inhibited the apoptotic function of p53. Further coexpression of beta -arrestin 2 strongly restored p53-mediated apoptosis (Fig. 7A), consistent with its reduction of p53 degradation. Similar results were also observed in another p53-null cell line H1299 (data not shown).


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Fig. 7.   beta -Arrestin 2 enhanced p53-mediated apoptosis. A, reduction of the anti-apoptotic function of Mdm2 on p53-mediated apoptosis by beta -arrestin 2. Saos2 cells were transfected with plasmids in various combinations. A GFP expression vector was included in all the combinations to mark the transfected cells. The dishes 48 h after transfection were scored under fluorescence microscopy for the appearance of cells with distinct apoptotic morphology. Mean values were derived from three experiments. B, suppression of the endogenous expressed beta -arrestin 2 using RNA interference technology in Saos2 cells. C, inhibition of the expression of beta -arrestin 2 attenuated the p53-induced apoptosis. Saos2 cells were transfected with GFP with beta -arrestin 2 RNAi plasmids or control vector. The apoptosis was performed as in A. D, induction of cell death by overexpression of beta -arrestin 2. After infection with indicated adenovirus, the cell death of L02 cells was examined with trypan blue staining at indicated time. Mean values were derived from three experiments. E, enhancement of apoptosis in L02 but not in Saos2 cells by overexpression of beta -arrestin 2. L02 and Saos2 cells were infected with adenovirus expressing beta -arrestin 2 or beta -galactosidase. 48 h after infection, cells were exposed to UV or left unexposed. Apoptosis induction was monitored 24 h after irradiation. F, restoration of p21 protein level by beta -arrestin 2. 48 h after transfection with indicated plasmids, Saos2 cells were lysed and subjected to Western blotting analysis with anti-p21 antibody and anti-actin antibody.

Then we used double-stranded RNA-mediated interference (RNAi), which has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms, to test the effect of endogenous beta -arrestin 2 on p53-induced apoptosis. We transfected Saos2 cells with RNAi plasmid of beta -arrestin 2 or the control vector, and the expression of endogenous beta -arrestin 2 was remarkably reduced compared with the control level as detected by Western blotting (Fig. 7B), while the expression of actin was not affected under the same conditions. Further experiments showed that when beta -arrestin 2 expression was reduced by RNAi, the p53-mediated apoptosis was also attenuated (Fig. 7C).

The effect of beta -arrestin 2 overexpression was examined using another independent assay that measures cell death. In L02 cells that express wild type p53, the infection with beta -arrestin 2 adenovirus caused a significantly higher ratio of cell death than that with the comparable level of beta -galactosidase adenovirus (Fig. 7D). It has been known that Mdm2/p53 loop is regulated by UV irradiation, and our results from DNA fragmentation assays showed that overexpression of beta -arrestin 2 significantly enhanced the apoptosis in both UV-irradiated or control L02 cells. In contrast, overexpression of beta -arrestin 2 exerted no effect on apoptosis in p53-null Saos2 cells (Fig. 7E). These data clearly show that beta -arrestin 2 affects cell apoptosis in a p53-dependent manner.

To further test the effect of beta -arrestin 2 expression on p53-mediated apoptosis, the expression level of p21, which is strongly transactivated by p53, was analyzed under the same conditions. In Saos2 cells, transfection of p53 strongly induced the expression of endogenous p21, and this could be inhibited by cotransfected Mdm2. Further coexpression of beta -arrestin 2 was able to restore the protein levels of p21 transactivated by p53 while V54D beta -arrestin 2 mutant failed to do so, as shown in Fig. 7F.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

beta -Arrestin 2 has been established as an important modulator of GPCR signaling and Mdm2 as the master regulator of p53 function, but little is known regarding cross-talk between GPCR pathways and p53 apoptotic apparatus. Under normal circumstances, the cellular concentration of Mdm2 is maintained at low levels and most are distributed in the nucleus to control the function of p53 (33, 34). Mdm2 in the cytoplasm is present at much lower levels compared with the nucleus, and little Mdm2 is detected at the plasma membrane to regulate GPCRs (35). In this study, we demonstrated that under physiological conditions beta -arrestin 2 not only physically interacted with oncoprotein Mdm2 but also functionally modulated the subcellular distribution of Mdm2, thus providing a solid basis for the functional consequence of beta -arrestin 2-Mdm2 interaction. Our study further revealed that the association of beta -arrestin and Mdm2 was greatly promoted by activation of GPCRs and thus led to reduction of Mdm2 function in the feedback loop of Mdm2/p53. According to the results of this study and Shenoy et al. (16), which demonstrates that Mdm2 functionally regulates GPCR trafficking by interacting with and ubiquitinating beta -arrestin 2, a bi-directional regulatory model of beta -arrestin 2/Mdm2 interaction can be proposed as following. Activation of GPCRs by various extracellular signals significantly increases the binding of beta -arrestin 2 and Mdm2 and thus effectively shifts the equilibrium of Mdm2 subcellular distribution from nucleus to plasma membrane. The functional consequences of the enhanced beta -arrestin 2/Mdm2 interaction are in one direction to promote ubiquitination of beta -arrestin 2 and assist internalization of GPCRs and in the other direction to modulate Mdm2/p53 feedback loop and reduce the ubiquitination and degradation of p53. In summary, the current study strongly suggests a potential role for GPCR signaling in modulation of p53-mediated apoptosis and clearly indicates that beta -arrestin 2 may serve as a signaling linker between the GPCR and p53 pathways.

Accumulating evidence indicates that residues 220-437 of Mdm2 are critical for its function since the mutant of Mdm2 lacking this region is incapable of mediating p53 degradation (36). Many partners of Mdm2 are known to bind to this region of Mdm2 and thus exert their indirect effects on p53 (6). For instance, Rb and ARF proteins, both of which bind to Mdm2 in this region, are able to inhibit the ability of Mdm2 to target p53 for degradation though different mechanisms may exist (6, 9, 37, 38). A recent study (39) shows that ATM kinase phosphorylates Mdm2 at serine 395 in this region in response to DNA damage and reduces the capability of Mdm2 to promote p53 degradation. Our current results demonstrate that the beta -arrestin 2 binding site on Mdm2 (residues 383-410) was also located in this important region, and the interaction of beta -arrestin with this small segment was critical to functionally inhibit Mdm2-mediated p53 degradation as in the case of Rb (Mdm2-(272-320)) or ARF (Mdm2-(210-244)). The beta -arrestin 2 binding site on Mdm2 identified in this study is a little different from the beta -arrestin 2 binding site on Mdm2 reported previously (16) (which may be due to the different source of Mdm2), but both these sites are in this important region. Furthermore, it is worth pointing out that the beta -arrestin 2 binding site is adjacent to the RING finger domain of Mdm2 (435-489), which is necessary for its ubiquitin ligase activity for itself and p53 (40). One of the Mdm2 regulators, Mdmx, is demonstrated to bind to the RING finger domain of Mdm2 and to stabilize both p53 and Mdm2 (41). Thus one of the molecular mechanisms for beta -arrestin 2 to inhibit Mdm2 function could be hypothesized as that the binding of beta -arrestin 2 to Mdm2 somehow interferes with its ubiquitin ligase activity and thus consequently attenuates the self-ubiquitination of Mdm2 and ubiquitination of p53, as observed in this study. Moreover, our previous report shows that the -(S/T)X4-5(S/T)- motif is critical for beta -arrestin 2 to bind to DOR (42). Interestingly, the beta -arrestin 2 binding region in Mdm2 identified in this study also contains several such motifs, and this may provide the structural basis for beta -arrestin 2/Mdm2 interaction.

MdmX is structurally homologous to Mdm2 and has been demonstrated to function as another critical negative regulator of p53 function in vivo. Loss of MdmX results in p53-dependent embryonic lethality in mice (43, 44). MdmX can directly bind to the RING finger domain of Mdm2 and thus stabilizes both Mdm2 and p53 via inhibition of the E3 ubiquitin ligase activity of Mdm2 (40). In addition, MdmX can directly interact with p53 and inhibit the acetylation of p53 induced by CBP/p300 (45) and inhibit the p53-mediated transcription. Recent evidence shows that MdmX is also a ubiquitin ligase and mediates the ubiquitination of p53 (46). Although MdmX has similar structure and function with Mdm2, our results showed that the interaction between MdmX and beta -arrestin 2, if any, should be much weaker than that between Mdm2 and beta -arrestin 2. Moreover, our data also revealed that the presence of MdmX did not interfere with the interaction between the beta -arrestin 2 and Mdm2. Taken together, the current study indicates that MdmX is unlikely involved in the beta -arrestin 2 regulation of Mdm2 function.

Several lines of evidence from this study clearly demonstrated that the association of beta -arrestin 2 and Mdm2 was significantly enhanced by specific stimulation of GPCRs. The enhancement of beta -arrestin 2/Mdm2 interaction by GPCR activation has greatly shifted the distribution equilibrium of Mdm2 from nucleus to cytoplasm and thus effectively reduces the Mdm2-mediated p53 degradation. There exist at least two possible explanations on how beta -arrestin 2/Mdm2 interaction is enhanced by GPCR activation. It has been well established that stimulation of GPCRs leads to the phosphorylation of receptors and the subsequent enhancement of beta -arrestin binding to the phosphorylated GPCRs. Moreover, the association of beta -arrestin with its partner, such as dishevelled protein (Dvl), is also significantly augmented by phosphorylation of Dvl (47). Since Mdm2 can be phosphorylated by several protein kinases such as ATM or Akt, and importantly Akt can be activated efficiently by GPCR stimulation (48). So one possibility may be that the potential phosphorylation of Mdm2 stimulated by GPCR signaling enhances the beta -arrestin 2/Mdm2 interaction. In addition, the current study demonstrated that Mdm2, mediated by beta -arrestin 2, could be recruited to the activated GPCRs at cytoplasm membrane where they were colocalized and that the activated receptor, beta -arrestin 2, and Mdm2 could be precipitated together in an agonist-dependent manner. Therefore, the other possibility may be that after stimulation of GPCRs the activated receptor, beta -arrestin 2, and Mdm2 form a ternary complex that greatly enhances the association between beta -arrestin 2 and Mdm2. This may also explain why stimulation of either Gi-, Gs-, or Gq-coupled receptors can enhance the beta -arrestin 2-Mdm2 interaction. However, the actual scenario, in which both mechanisms described above may work simultaneously or other unknown ones may be involved, remains to be further investigated.

    ACKNOWLEDGEMENTS

We thank Dr. D. Bohmann for the HA-ubiquitin construct and Dr. Yang Shi for BS/U6 vector. We thank Jiyong Wang, Yue Sun, Jiali Li, and Shunmei Xin for their technical assistance, and Xin Ge, Yaya Wang, Yujie Li, Xiaohui Zhao, and Peihua Wu for kind help. We also thank Wenbo Zhang and Nanjie Xu for helpful discussions.

    FOOTNOTES

* This work was supported by Grants from the Ministry of Science and Technology (G1999053907 and G1999054003), Chinese Academy of Sciences (KSCX2-2 and KSCX2-SW), and the National Natural Science Foundation of China (30021003).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.

§ These authors contributed equally to this work.

|| To whom correspondence should be addressed: Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, People's Republic of China. Tel.: 86-21-64716049; Fax: 86-21-64718563; E-mail: gpei@sibs.ac.cn.

Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M210350200

    ABBREVIATIONS

The abbreviations used are: GPCR, G-protein-coupled receptor; HEK, human embryonic kidney; HA, hemagglutinin; CMV, cytomegalovirus; GFP, green fluorescent protein; DOR, delta opioid receptor; nt, nucleotide; RNAi, RNA-mediated interference.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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