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J. Biol. Chem., Vol. 282, Issue 51, 36971-36979, December 21, 2007
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From the
||Department of Pharmacology and Experimental Therapeutics and the Program in Molecular Biology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153, the Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7365, and the ¶Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Received for publication, June 20, 2007 , and in revised form, September 20, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, also termed CXCL12, play an important role in several biological processes such as development of the heart and brain, leukocyte chemotaxis, and stem cell homing (1–3). Although CXCR4 dysregulation has been linked to several pathologies, especially cancer, the molecular mechanisms regulating CXCR4 remain poorly understood (4, 5). Activated CXCR4 is targeted for lysosomal degradation through a pathway involving ubiquitination of carboxyl-terminal tail lysine residues mediated by the E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4) (6, 7). AIP4 belongs to the neural precursor cell-expressed developmentally down-regulated gene 4-like family of homologous to E6-AP carboxyl-terminal domain E3 ubiquitin ligases, which interact with their target proteins either directly or indirectly via their WW domains or possibly other domains (8, 9).
In addition, AIP4 regulates endosomal sorting of activated CXCR4 by targeting the receptor to the endosomal sorting complex required for transport pathway (7), which is a complex network of proteins that recognize and sort ubiquitinated cargo into the multivesicular body (10). Entry into this pathway requires the action of HRS, a ubiquitin-binding protein localized to flat clathrin lattices on endosomes adjacent to invaginating domains, where it sequesters ubiquitinated cargo destined for entry into the multivesicular body for subsequent degradation (11, 12). CXCR4 and AIP4 localize to HRS-positive microdomains on endosomes (7). In addition, AIP4 mediates CXCR4-dependent ubiquitination of HRS, an action that likely plays a role in the sorting function of HRS (7). Whether additional proteins play a role in the endosomal sorting of CXCR4, however, remains to be determined.
Nonvisual arrestins, arrestin-2 and arrestin-3 (also termed β-arrestin-1 and β-arrestin-2, respectively), function in GPCR desensitization and also serve as endocytic adaptors to recruit phosphorylated receptors for internalization through clathrin-coated pits (13). Arrestins have also been implicated in ubiquitination of GPCRs. Ubiquitination of the β2-adrenergic receptor appears to require arrestin-3 and a yet to be identified E3 ubiquitin ligase (14). Arrestins have also been implicated in CXCR4 desensitization (15, 16) and are thought to play a role in CXCR4 internalization (15, 17), but whether they function in ubiquitination and degradation of activated CXCR4 remains unknown. Therefore, to gain further insight into the molecular mechanisms mediating CXCR4 down-regulation, we examined the role of nonvisual arrestins in targeting CXCR4 to the degradative pathway.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(also called CXCL12) was from PeproTech (Rocky Hill, NJ). Degradation Assay—Agonist-promoted degradation of HA-tagged CXCR4 in HEK293 cells and endogenous CXCR4 in HeLa cells was detected by immunoblot analysis, as previously described (7). Briefly, the cells were treated with vehicle (DMEM + 0.5% FBS) or CXCL12 (10 nM) in the presence of 50 µg/ml cycloheximide (to prevent protein synthesis) for 3 h. We have found that 10 nM CXCL12 produces the same level of CXCR4 degradation as 100 nM CXCL12 (20). The cells were directly lysed in 2x sample buffer, and receptor levels were detected as we have previously shown (6, 7, 20, 21). For RNA interference experiments, HeLa cells were transfected with control siRNA or siRNA directed against arrestin-2 (100 nM), arrestin-3 (100 nM), or arrestin-2 and arrestin-3 together (50 nM each) using Lipofectamine 2000, according to the manufacturer's instructions. For studies in HEK293 cells, the cells were transfected with siRNA as described for HeLa cells, followed by transfection with HA-CXCR4 the following day using FuGENE 6 transfection reagent, according to the manufacturer's instructions.
Detection of Ubiquitinated CXCR4—HEK293 cells grown on 10-cm dishes were transfected with arrestin-2, arrestin-3, arrestin-2/3, and control siRNA as described above, followed by co-transfection of HA-CXCR4 and FLAG-ubiquitin the next day. Detection of ubiquitinated CXCR4 was as previously described (7).
Measuring Receptor Internalization—HEK293 cells grown on 10-cm dishes were transfected with siRNA for arrestin-2 and control siRNA as described above, followed by co-transfection of HA-CXCR4 the next day. Receptor internalization was measured by cell surface enzyme-linked immunosorbent assay, as previously described (6).
Co-immunoprecipitation Studies—HEK293 cells stably expressing HA-tagged CXCR4 were serum-starved in DMEM supplemented with 0.5% FBS overnight (14–16 h) followed by treatment with the same medium without or with 30 nM CXCL12 for 15, 30, and 60 min. After stimulation, the cells were placed on ice, lysed in ice-cold immunoprecipitation buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, phosphatase inhibitor cocktails I and II (Sigma), and protease inhibitors (10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A)). The lysates were clarified by centrifugation at 21,000 x g for 20 min at 4 °C, and equal amounts of clarified lysates were processed. An anti-AIP4 monoclonal antibody (G11; Santa Cruz) was used to immunoprecipitate endogenously expressed AIP4 in HEK293 cells, essentially as previously described (7), followed by SDS-PAGE and immunoblotting to detect endogenous arrestins.
GST Pulldowns—Escherichia coli BL21 cells transformed with GST fusion constructs were grown overnight at 37 °C. The following day, the cultures were diluted 1:50 and grown to an A600 
0.4–0.5 at 37 °C and then induced with 500 µM isopropyl-1-thio-β-D-galactopyranoside for 3 h at 18 °C. The cells were pelleted, resuspended in binding buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 1 mM dithiothreitol, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 10 µg/ml aprotinin), and sonicated on ice. The lysates were clarified by centrifugation for 20 min at 21,000 x g at 4 °C followed by incubation with glutathione-Sepharose 4B beads overnight at 4 °C while rocking. The samples were washed and resuspended in binding buffer. For binding experiments using transfected cells, 1–3 µg of GST fusion proteins were incubated with cell lysates prepared from HEK293 cells expressing the protein of interest for 12–16 h. The samples were washed three times in binding buffer, eluted in 2x sample buffer by boiling for 10 min, and analyzed by SDS-PAGE and Western blotting. For binding experiments using purified proteins, 1 µg of GST fusion protein was incubated with 300 ng of purified arrestin-2 for 1 h and processed in the same manner.
Immunofluorescence Microscopy—HEK293 cells were transfected with arrestin-2 siRNA followed by transfection of HA-CXCR4 tagged with yellow fluorescent protein at its carboxyl-terminal end (HA-CXCR4-YFP). The cells were treated with CXCL12 for 3 h, followed by fixation, permeabilization, and immunostaining for EEA1 and LAMP2. For AIP4 and arrestin-2 co-localization experiments, HEK293 cells transiently co-transfected with HA-CXCR4, mCh-AIP4, and YFP-arrestin-2 were serum-starved for 14–16 h in DMEM containing 0.5% FBS and treated with vehicle or 30 nM CXCL12 for 30 min followed by permeabilization and fixation. The images were acquired using a Zeiss LSM 510 laser scanning confocal imaging system with a 63x W Apochromat objective at 512 x 512 resolution. The acquired images were analyzed using Image J 1.3v software (NIH).
Statistical Analysis—The data were analyzed and subjected to statistical analyses using Prism 4.0 software (GraphPad Software, Inc.).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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We next examined the role of arrestin-2 in agonist-promoted CXCR4 degradation in HEK293 cells, a model cell line used previously to study CXCR4 trafficking (6, 7). We found that degradation of HA-tagged CXCR4 induced by agonist was significantly inhibited in arrestin-2-depleted cells as compared with control siRNA transfected cells (Fig. 1, C and D), consistent with that observed with endogenous CXCR4 in HeLa cells. We have previously shown that internalization is a prerequisite for degradation (6), and because arrestins are thought to play a role in CXCR4 internalization (15, 17), we next determined whether inhibition of internalization could account for the attenuated degradation observed in the arrestin-2 siRNA-treated cells. HEK293 cells were transfected with siRNA targeting arrestin-2 or control siRNA together with HA-tagged CXCR4, and receptor internalization was assessed by cell surface enzyme-linked immunosorbent assay, as previously described (6). As shown in Fig. 1E, agonist-promoted internalization of CXCR4 in arrestin-2-depleted cells was similar to that in control siRNA-transfected cells, suggesting that arrestin-2 is not involved in agonist-promoted internalization of CXCR4.
Direct ubiquitination of CXCR4 at the plasma membrane is required for its sorting to the degradative pathway but not for its internalization (6, 7). Because arrestin-2 also appears to be required for CXCR4 degradation but not internalization, we next assessed whether the effect on CXCR4 degradation is due to a role on CXCR4 ubiquitination. HEK293 cells transfected with siRNA targeted against arrestin-2 and arrestin-3 or control siRNA plus HA-tagged CXCR4 and FLAG-tagged ubiquitin were stimulated with or without agonist for 30 min, followed by receptor immunoprecipitation and immunoblotting to detect the incorporation of tagged ubiquitin, as previously described (7). As shown in Fig. 1F, ubiquitination of CXCR4 was not affected in arrestin-2 or arrestin-3 siRNA depleted cells as compared with control siRNA-treated cells, even though arrestins were substantially depleted (Fig. 1F, bottom panel). In addition, the ubiquitination status of CXCR4 was not altered in cells that were transfected with a siRNA that targets both arrestin-2 and arrestin-3 with respect to mock transfected cells (Fig. 1G), further suggesting that arrestins are not involved in CXCR4 ubiquitination. Taken together, these data indicate that arrestin-2 is not involved in CXCR4 ubiquitination and internalization and suggest that its role in CXCR4 degradation is likely due to a post-internalization function such as endosomal sorting into the degradative pathway.
We next examined the distribution of CXCR4 in arrestin-2-depleted HEK293 cells that were treated with or without CXCL12 using confocal immunofluorescence microscopy. In the absence of stimulation, CXCR4 was localized predominantly to the plasma membrane in both control and arrestin-2 siRNA-treated cells, although a portion of CXCR4 was also localized to intracellular puncta that partially overlapped with the early endosomal marker EEA1 (Fig. 2A, top panels, control and arrestin-2) or late endosomal/lysosomal marker LAMP2 (Fig. 2B, top panels, control and arrestin-2), suggesting that CXCR4 undergoes constitutive internalization and trafficking to early endosomes and lysosomes. Upon treatment with CXCL12 for 3 h, in the control treated cells, CXCR4 mostly co-localized with the late endosomal/lysosomal marker LAMP2 (Fig. 2B), but not with the early endosomal marker EEA1 (Fig. 2A), suggesting that CXCR4 traffics efficiently to lysosomes upon stimulation with CXCL12. In contrast, upon stimulation with CXCL12 in the arrestin-2 siRNA-treated cells, CXCR4 mostly co-localized with EEA1 and not LAMP2, suggesting that transit from endosomes to lysosomes is impaired as compared with control treated cells. Taken together, these data suggest that arrestin-2 is required for proper CXCR4 trafficking from early endosomes to lysosomes.
We previously showed that AIP4 is localized to endosomes and mediates endosomal sorting of CXCR4 (7); therefore, we next asked whether arrestins facilitate AIP4-mediated trafficking of CXCR4. To address this, we initially determined whether arrestins interact with AIP4 in cells. HEK293 cells were transiently transfected with HA-tagged arrestin-2 or arrestin-3 and FLAG-tagged AIP4 or empty vector (pCMV). Whole cell lysates were subject to immunoprecipitation using a FLAG antibody followed by immunoblotting to detect arrestin-2 or arrestin-3 in the immunoprecipitates. As shown in Fig. 3A, arrestin-2 and arrestin-3 co-immunoprecipitation was observed in cells transfected with FLAG-tagged AIP4 but not in vector-transfected cells, suggesting that arrestins associate with AIP4 in cells.
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WW domains interact with proline-rich motifs in various contexts, and because arrestins contain several proline-rich sequences, we decided to use truncation mutagenesis to determine the AIP4-binding regions on arrestin-2. Various arrestin-2 truncation mutants used in these studies are shown schematically in Fig. 4A. Initially, we examined two truncation mutants encompassing the amino-terminal (amino acids 1–260) and carboxyl-terminal (amino acids 261–418) regions of arrestin-2. Whole cell lysates of HEK293 cells expressing HA-tagged arrestin-2 truncation mutants were incubated with glutathione beads coated with WW domains I–IV fused to GST or GST alone. As shown in Fig. 4B, GST-WW-I-IV bound to arrestin-2-(1–260) but not arrestin-2-(261–418). Further truncation of arrestin-2-(1–260) to residues 179 and 161 revealed that binding to AIP4 was most likely mediated by residues located between residues 161 and 260. Interestingly, this region contains a proline-rich stretch within the hinge region (174–181) that connects the amino- and carboxyl-terminal domains of arrestin-2 (23). Mutation of two proline residues to alanines (arrestin-2-(P180A/P182A)) and deletion of 8 amino acids (
175–182) encompassing the proline residues within this region in the context of the 1–260 construct failed to abrogate binding to GST-WW-I-IV (data not shown), suggesting that these residues are not important for binding to AIP4 and that other regions may be involved.
To gain further insight into the mechanism by which arrestins function in AIP4-dependent degradation of CXCR4, we examined whether receptor activation promotes the interaction between AIP4 and arrestin-2. Initially, we compared the ability of AIP4 to bind to wild-type arrestin-2 and arrestin-2-R169E. The arrestin-2-R169E mutant is thought to mimic the form of arrestin when it is bound to activated and phosphorylated GPCRs and is thus considered to be "constitutively active" (24). HEK293 cells were transiently transfected with either arrestin-2 wild-type or R169E mutant together with FLAG-tagged AIP4 or empty vector (pCMV). Whole cell lysates were subject to immunoprecipitation using the FLAG antibody followed by immunoblotting to detect arrestin-2 in the immunoprecipitates. As shown in Fig. 5A, a single band corresponding to arrestin-2 was observed in cells transfected with FLAG-tagged AIP4 but not in cells transfected with empty vector. Interestingly, arrestin-2-R169E showed enhanced binding to FLAG-tagged AIP4 as compared with wild-type arrestin-2 (Fig. 5A), suggesting that receptor activation may promote arrestin-2 binding to AIP4.
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To determine whether AIP4 and arrestin-2 co-localize within the same subcellular compartment, we employed immunofluorescence confocal microscopy on HEK293 cells co-expressing HA-CXCR4, YFP-arrestin-2, and mCh-AIP4. The cells were treated with agonist for 30 min, permeabilized, fixed, and observed using a confocal microscope. As shown in Fig. 5C, in the vehicle-treated cells, arrestin-2 and AIP4 were mostly diffuse, although AIP4 was also found in puncta, consistent with its localization to endosomes (7). In contrast, in the CXCL12-treated cells, arrestin-2 became punctate and showed strong co-localization with AIP4. Taken together, these data suggest that arrestin-2 co-localizes with AIP4 on endosomes upon CXCR4 activation, which may facilitate its ability to mediate sorting of CXCR4 into the degradative pathway.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Although the interaction between AIP4 and arrestin-2 in cells is regulated by activation of CXCR4, we can detect basal binding between the proteins when overexpressed in the absence of activated receptor (Fig. 3). It may be possible that the AIP4-binding site is only partially accessible in the inactive conformation and that arrestin-2 activation makes it more accessible leading to greater AIP4 binding. Alternatively, it is possible that there are multiple AIP4-binding sites in arrestin-2. For example, a low affinity binding site may be accessible when arrestin-2 is in the inactive conformation, and a second, high affinity site may become accessible upon arrestin-2 activation, leading to a stronger interaction with AIP4. Nevertheless, our data indicate that AIP4 and arrestin-2 interact directly in vitro, and their association in cells is regulated by CXCR4 activation. In addition, we have identified the amino-terminal region of arrestin-2 and AIP4 WW domains I and II as the binding regions that may facilitate the interaction in cells.
Typically, arrestin binding to activated and phosphorylated GPCRs occurs at the plasma membrane, which is thought to promote a conformational change in arrestin exposing the buried carboxyl-terminal end that binds to partners such as clathrin and β-adaptin (13). Because CXCR4 activation promotes AIP4 binding to arrestin-2, arrestin-2 may be recruited to phosphorylated CXCR4 before it interacts with AIP4. However, because the AIP4-binding site maps to the amino-terminal half of arrestin-2, we believe the mechanism of the interaction will be distinct from that for clathrin and β-adaptin, although a conformational change in arrestin-2 is likely required.
Interestingly, the arrestin-2-AIP4 interaction is maximal after 15–30 min of agonist treatment, when both proteins co-localize on endocytic vesicles. Thus, it is possible that arrestin-2 binds to phosphorylated CXCR4 and AIP4 at the plasma membrane, and all three proteins co-traffic as a complex to endosomes. Arrestins have been shown to form stable complexes with certain GPCRs and several signaling molecules that persist for some time on endosomes (25), but whether this is true for CXCR4, AIP4, and arrestin-2 remains to be determined. It is also possible that arrestin-2 interacts with phosphorylated CXCR4 at the plasma membrane, which co-internalize as a complex and once on endosomes somehow interacts with the endosomal pool of AIP4. This may also explain why arrestin-2 is not important for ubiquitination of the receptor at the plasma membrane. We show that in addition to not being involved in the ubiquitination of CXCR4, arrestin-2 is not involved in its internalization. It is possible that arrestin-2 mediates desensitization of CXCR4, although this function has been previously assigned to arrestin-3 (16), which possibly also mediates CXCR4 internalization (15, 17). Alternatively, arrestin-2 may instead be directly recruited to endosomes by activated CXCR4, where it carries out its endosomal sorting function. Regardless of the mechanism, our data reveal that arrestin-2 co-localizes with AIP4 on endocytic vesicles, where it likely functions to mediate lysosomal targeting of CXCR4.
In conclusion, we describe a novel function for arrestin-2 as an endosomal sorting molecule together with AIP4 to mediate CXCR4 entry into a degradative pathway. This function deviates from the general paradigm that arrestins serve as adaptors for clathrin-dependent internalization of GPCRs at the plasma membrane and suggests that arrestins may also serve as endosomal adaptor proteins required for sorting GPCRs into the degradative pathway.
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FOOTNOTES |
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1 Supported by a predoctoral fellowship from the American Heart Association.
2 To whom correspondence should be addressed: Dept. of Pharmacology, Loyola University Chicago, Stritch School of Medicine, 2160 S. 1st Ave., Bldg.101/Rm. 2721, Maywood, IL 60153. Tel.: 708-216-3456; Fax: 708-216-6596; E-mail: amarchese{at}lumc.edu.
3 The abbreviations used are: GPCR, G protein-coupled receptor; AIP4, atrophin-interacting protein 4; GST, glutathione S-transferase; E3, ubiquitin-protein isopeptide ligase; siRNA, small interfering RNA; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; HA, hemagglutinin; WCL, whole cell lysates; HRS, hepatocyte growth factor-regulated tyrosine kinase substrate; IP, immuno precipitation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1 µg) were incubated with purified arrestin-2 (300 ng) followed by SDS-PAGE and immunoblotting with an anti-arrestin antibody to detect arrestin-2. The input represents 10 ng of purified arrestin-2. A Coomassie-stained gel is shown to indicate the levels of the GST fusion proteins used in the binding experiment. The extra bands in the GST-AIP4 lanes represent degradation products co-purified with full-length GST-AIP4. Shown are representative blots from three independent experiments. The molecular weights are indicated. D, the WW domains of AIP4 mediate the interaction with arrestin-2. WCL from HEK293 cells expressing HA-arrestin-2 or pcDNA were incubated with GST-AIP4, GST-WW-I-IV, or GST alone followed by SDS-PAGE and immunoblotting to detect HA-arrestin-2 binding. The input equals 2% of the lysate that was used in the binding experiment. A Coomassie-stained gel is shown to indicate the levels of the GST fusion proteins used in the binding experiment. The extra bands in the GST-AIP4 lanes represent degradation products co-purified with full-length GST-AIP4. Shown are representative blots from three independent experiments. The molecular weights are indicated. E, WW domains I and II interact with arrestin-2. WCL from HEK293 cells expressing HA-arrestin-2 were incubated with GST-AIP4, GST-WW-I-IV, GST fused to each of the individual four WW domains, or GST alone followed by SDS-PAGE and immunoblotting to detect HA-arrestin-2 binding. The input equals 0.5% of the lysate that was used in the binding experiment and approximately one-half of bound sample was immunoblotted. The upper blot was stained with Ponceau S to show the levels of the GST fusion proteins used in the binding experiment. Shown are representative blots from three independent experiments. The molecular weights are indicated.
