Ubiquitination of β-Arrestin Links Seven-transmembrane Receptor Endocytosis and ERK Activation
2007; Elsevier BV; Volume: 282; Issue: 40 Linguagem: Inglês
10.1074/jbc.m700852200
ISSN1083-351X
AutoresSudha K. Shenoy, Larry S. Barak, Kunhong Xiao, Seungkirl Ahn, Magali Berthouze, Arun K. Shukla, Louis M. Luttrell, Robert J. Lefkowitz,
Tópico(s)Neuropeptides and Animal Physiology
Resumoβ-Arrestin2 and its ubiquitination play crucial roles in both internalization and signaling of seven-transmembrane receptors (7TMRs). To understand the connection between ubiquitination and the endocytic and signaling functions of β-arrestin, we generated a β-arrestin2 mutant that is defective in ubiquitination (β-arrestin20K), by mutating all of the ubiquitin acceptor lysines to arginines and compared its properties with the wild type and a stably ubiquitinated β-arrestin2-ubiquitin (Ub) chimera. In vitro translated β-arrestin2 and β-arrestin20K displayed equivalent binding to recombinant β2-adrenergic receptor (β2AR) reconstituted in vesicles, whereas β-arrestin2-Ub bound ∼4-fold more. In cellular coimmunoprecipitation assays, β-arrestin20K bound nonreceptor partners, such as AP-2 and c-Raf and scaffolded phosphorylated ERK robustly but displayed weak binding to clathrin. Moreover, β-arrestin20K was recruited only transiently to activated receptors at the membrane, did not enhance receptor internalization, and decreased the amount of phosphorylated ERK assimilated into isolated β2AR complexes. Although the wild type β-arrestin2 formed ERK signaling complexes with the β2AR at the membrane, a stably ubiquitinated β-arrestin2-Ub chimera not only stabilized the ERK signalosomes but also led to their endosomal targeting. Interestingly, in cellular fractionation assays, the ubiquitination state of β-arrestin2 favors its distribution in membrane fractions, suggesting that ubiquitination increases the propensity of β-arrestin for membrane association. Our findings suggest that although β-arrestin ubiquitination is dispensable for β-arrestin cytosol to membrane translocation and its "constitutive" interactions with some cytosolic proteins, it nevertheless is a prerequisite both for the formation of tight complexes with 7TMRs in vivo and for membrane compartment interactions that are crucial for downstream endocytic and signaling processes. β-Arrestin2 and its ubiquitination play crucial roles in both internalization and signaling of seven-transmembrane receptors (7TMRs). To understand the connection between ubiquitination and the endocytic and signaling functions of β-arrestin, we generated a β-arrestin2 mutant that is defective in ubiquitination (β-arrestin20K), by mutating all of the ubiquitin acceptor lysines to arginines and compared its properties with the wild type and a stably ubiquitinated β-arrestin2-ubiquitin (Ub) chimera. In vitro translated β-arrestin2 and β-arrestin20K displayed equivalent binding to recombinant β2-adrenergic receptor (β2AR) reconstituted in vesicles, whereas β-arrestin2-Ub bound ∼4-fold more. In cellular coimmunoprecipitation assays, β-arrestin20K bound nonreceptor partners, such as AP-2 and c-Raf and scaffolded phosphorylated ERK robustly but displayed weak binding to clathrin. Moreover, β-arrestin20K was recruited only transiently to activated receptors at the membrane, did not enhance receptor internalization, and decreased the amount of phosphorylated ERK assimilated into isolated β2AR complexes. Although the wild type β-arrestin2 formed ERK signaling complexes with the β2AR at the membrane, a stably ubiquitinated β-arrestin2-Ub chimera not only stabilized the ERK signalosomes but also led to their endosomal targeting. Interestingly, in cellular fractionation assays, the ubiquitination state of β-arrestin2 favors its distribution in membrane fractions, suggesting that ubiquitination increases the propensity of β-arrestin for membrane association. Our findings suggest that although β-arrestin ubiquitination is dispensable for β-arrestin cytosol to membrane translocation and its "constitutive" interactions with some cytosolic proteins, it nevertheless is a prerequisite both for the formation of tight complexes with 7TMRs in vivo and for membrane compartment interactions that are crucial for downstream endocytic and signaling processes. The multifunctional adaptor proteins β-arrestins (β-arrestin1 and -2) were originally identified as desensitizing molecules that prevent the coupling between seven-transmembrane receptors (7TMRs) 3The abbreviations used are:7TMRseven-transmembrane receptorβ2ARβ2-adrenergic receptorAP-2adaptin protein subunit 2ERKextracellular signal-regulated kinasepERKphosphorylated ERKMAPKmitogen-activated protein kinasePMAphorbol 12-myristate 13-acetateUbubiquitinGFPgreen fluorescent proteinHAhemagglutininPBSphosphate-buffered salineDTMEdithiobis-maleimidoethaneWTwild typeIPimmunoprecipitationE3ubiquitin-protein isopeptide ligase.3The abbreviations used are:7TMRseven-transmembrane receptorβ2ARβ2-adrenergic receptorAP-2adaptin protein subunit 2ERKextracellular signal-regulated kinasepERKphosphorylated ERKMAPKmitogen-activated protein kinasePMAphorbol 12-myristate 13-acetateUbubiquitinGFPgreen fluorescent proteinHAhemagglutininPBSphosphate-buffered salineDTMEdithiobis-maleimidoethaneWTwild typeIPimmunoprecipitationE3ubiquitin-protein isopeptide ligase. and G proteins (1Benovic J.L. Kuhn H. Weyand I. Codina J. Caron M.G. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8879-8882Crossref PubMed Scopus (355) Google Scholar, 2Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (897) Google Scholar, 3Attramadal H. Arriza J.L. Aoki C. Dawson T.M. Codina J. Kwatra M.M. Snyder S.H. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar). More recently, however, it was found that β-arrestin binding to receptors not only stops G protein-mediated second messenger signaling but also engages several novel signaling pathways, including mitogen-activated protein kinase (MAPK) cascades (4Lefkowitz R.J. Shenoy S.K. Science. 2005; 308: 512-517Crossref PubMed Scopus (1408) Google Scholar, 5Gurevich V.V. Gurevich E.V. Structure. 2003; 11: 1037-1042Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Furthermore, β-arrestins have also been shown to bind and regulate cell surface receptors other than 7TMRs, and their signaling has been implicated in regulating the actin cytoskeleton, chemotaxis, antiapoptosis, and metastasis (6Lefkowitz R.J. Rajagopal K. Whalen E.J. Mol Cell. 2006; 24: 643-652Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar).β-Arrestins serve as endocytic adaptors that bind clathrin and adaptin protein subunit 2 (AP-2) and facilitate receptor internalization via clathrin-coated vesicles (7Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1153) Google Scholar, 8Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (520) Google Scholar, 9Ferguson S.S. Downey 3rd, W.E. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (839) Google Scholar). The differing affinity and trafficking patterns of GFP-β-arrestins induced by several 7TMRs have led to the classification of receptors into two groups, Class A and Class B (10Oakley R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. J. Biol. Chem. 2000; 275: 17201-17210Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar). Class A receptors (e.g. β2-adrenergic, α1b-adrenergic, μ-opioid, endothelin 1A, and dopamine D1A receptors) show higher affinity for β-arrestin2 than β-arrestin1 and recruit GFP-β-arrestins only to the plasma membrane. Class B receptors (e.g. vasopressin V2, angiotensin AT1a, neurotensin1, thyrotropin-releasing hormone, and neurokinin NK-1 receptors) bind to both β-arrestin1 and -2 with equal affinity and cotraffic and colocalize with GFP-β-arrestin in endocytic vesicles. Thus, complexes formed between β-arrestin and Class A receptors are transient and exist only at the membrane, whereas those formed between β-arrestin and Class B receptors are stable and persist after receptor endocytosis (10Oakley R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. J. Biol. Chem. 2000; 275: 17201-17210Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar). These differential patterns of β-arrestin2 recruitment correlate with the amplitude of β-arrestin-bound phosphorylated ERK1/2 (pERK). Class B receptors, such as the angiotensin 1a and the V2 vasopressin receptors activate a β-arrestin-bound pool of ERK more persistently than Class A receptors, such as the β2-adrenergic receptor (β2AR) and the α1b-adrenergic receptor (11Tohgo A. Choy E.W. Gesty-Palmer D. Pierce K.L. Laporte S. Oakley R.H. Caron M.G. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2003; 278: 6258-6267Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar).β-Arrestins also become ubiquitinated (attachment of ubiquitin (Ub) on lysine residues) upon agonist stimulation of various 7TMRs. Upon β2AR stimulation, Mdm2 (mouse double minute2), a RING (really interesting new gene) type E3 ligase, ubiquitinates β-arrestin2, and this modification is required for rapid internalization of the receptor (12Shenoy S.K. McDonald P.H. Kohout T.A. Lefkowitz R.J. Science. 2001; 294: 1307-1313Crossref PubMed Scopus (706) Google Scholar). The pattern of β-arrestin ubiquitination correlates with the stability of receptor-β-arrestin interaction (i.e. transient interaction (Class A) is associated with transient ubiquitination, and persistent interaction (Class B) is associated with sustained ubiquitination) (13Shenoy S.K. Lefkowitz R.J. J. Biol. Chem. 2003; 278: 14498-14506Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 14Perroy J. Pontier S. Charest P.G. Aubry M. Bouvier M. Nat. Methods. 2004; 1: 203-208Crossref PubMed Scopus (135) Google Scholar). Exchanging the carboxyl-terminal amino acid residues of these two types of receptors reverses the patterns of β-arrestin trafficking as well as the time course of ubiquitination and the extent of β-arrestin-bound ERK activation (11Tohgo A. Choy E.W. Gesty-Palmer D. Pierce K.L. Laporte S. Oakley R.H. Caron M.G. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2003; 278: 6258-6267Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 13Shenoy S.K. Lefkowitz R.J. J. Biol. Chem. 2003; 278: 14498-14506Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 15Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). Additionally, translational fusion of ubiquitin to the C terminus of β-arrestin (β-arrestin2-Ub) leads to its cotrafficking and colocalization with the β2AR (Class A) in endocytic vesicles, thus mimicking a Class B trafficking pattern (13Shenoy S.K. Lefkowitz R.J. J. Biol. Chem. 2003; 278: 14498-14506Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar).Interestingly, specific lysine residues are targeted for modification in response to agonist stimulation of a particular 7TMR. For example, angiotensin 1a receptor (AT1aR)-dependent sustained β-arrestin ubiquitination occurs primarily at lysines 11 and 12 in β-arrestin2 (16Shenoy S.K. Lefkowitz R.J. J. Biol. Chem. 2005; 280: 15315-15324Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Mutation of these lysines to arginine residues leads to the reversal of angiotensin II-stimulated β-arrestin ubiquitination from a sustained to a transient pattern, with a corresponding reversal of AT1aR-β-arrestin binding from stable endosome-localized complexes to transiently associated complexes seen only at the plasma membrane.In an attempt to understand the role of ubiquitination in the regulation of the endocytic and signaling functions of β-arrestin, we generated a β-arrestin2 mutant (β-arrestin20K) that is defective in ubiquitination by mutating all of the ubiquitin acceptor lysines in β-arrestin2 to arginines and compared it with the wild type and a stably ubiquitinated form in its ability to interact with 7TMRs and nonreceptor partners as well as its capability to facilitate receptor internalization and signaling.EXPERIMENTAL PROCEDURESCell Lines, Reagents, and Plasmids—COS-7 and HEK-293 cells were obtained from the American Type Culture Collection. COS-7 cells were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and transiently transfected with Lipofectamine 2000 reagent (Invitrogen). HEK-293 cells were maintained in minimal essential medium supplemented with fetal bovine serum and transiently transfected with FuGene 6 reagent (Roche Applied Science). M2 anti-FLAG affinity-agarose beads, isoproterenol, arginine-vasopressin peptide, anti-FLAG M1 and M2 antibodies, fluorescein isothiocyanate-anti-mouse secondary IgG, and N-ethylmaleimide were from Sigma. Ubiquitin antibody FK2 was from Biomol. Monoclonal antibody 12CA5 to HA epitope was from Roche Applied Science. Alexa 594 and Alexa 633, conjugated secondary antibodies, were from Invitrogen. Horseradish peroxidase-conjugated secondary antibodies were from GE/Amersham Biosciences. Detection of active ERK was with a rabbit polyclonal anti-phospho-p44/42 MAPK (1:2000 for Western blot and 1:200 for immunostaining; Cell Signaling Technology). Total ERK was detected with anti-MAPK1/2 (1:10,000 dilution for Western blots; Millipore). A1CT, a rabbit polyclonal antibody to the β-arrestin1 C terminus generated in the Lefkowitz laboratory was used to detect β-arrestin isoforms.Rat β-arrestin2/pEGFPN1 plasmid has been previously described (17Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (546) Google Scholar). A 1500-bp DNA fragment encoding β-arrestin2-Ub was subcloned into the KpnI and ApaI sites of pEGFPC1 vector to obtain the expression plasmid for GFP-β-arrestin2-Ub. The lysine residue at position 48 in Ub was replaced with arginine using a QuikChange® site-directed mutagenesis kit (Stratagene). GFP-β-arrestin2-Ub used in this work actually represents GFP-β-arrestin2-UbK48R.Five rounds of mutations accomplished the construction of β-arrestin20K, each mutagenesis step targeting 5-7 lysine residues. We used the QuikChange® multisite-directed mutagenesis kit (Stratagene) and followed the manufacturer's instructions for the design of oligonucleotides and PCR protocols. The DNA fragment encoding β-arrestin20K was later cloned into pEGFP-N1 to yield β-arrestin20K-GFP. All DNA constructs were verified by sequencing. HA-β2AR plasmid was a gift from Dr. Neil Freedman (Duke University); HA-V2R plasmid was provided by Dr. Marc Caron (Duke University). Myc-c-Raf and RFP-ERK2 have been previously reported (18Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (696) Google Scholar).To achieve equivalent expression of β-arrestin2 WT and lysine mutants (Fig. 1), we transfected cells on a 100-mm dish with 1 μg of DNA for the WT and -7K, 3 μg of DNA for -14K, -19K, and -26K, and 2.5 μg for -31K. For the GFP-tagged plasmids (Fig. 5), 1 μg was used for the WT and -7K, and 2 μg was used for the rest.FIGURE 5Translocation of GFP-tagged β-arrestin2 lysine mutants to activated β2ARs. HEK-293 cells were transfected with HA-β2AR and the indicated β-arrestin2 plasmid. Cells were stimulated with isoproterenol for 1 min, fixed, and immunostained for the β2AR. Confocal images shown represent one of three similar experiments. β2AR detection is shown in red, β-arrestin fluorescence is shown in green, and overlay panels depict colocalization (yellow).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Immunoprecipitation and Immunodetection—β-Arrestin2-FLAG/pCDNA3, β-arrestin20K-FLAG/pCDNA3.1 or FLAG-β-arrestin2-Ub/pCDNA3.1 were used to immunoprecipitate β-arrestins. To detect active ERK in receptor immunoprecipitates, FLAG-β2AR was co-expressed with GFP-tagged β-arrestin constructs and RFP-ERK2. Cells were serum-starved for 2 h (COS-7) or 4 h (HEK-293) and then stimulated or not for the indicated times with the appropriate agonists. Cells were solubilized in a lysis buffer containing 50 mm HEPES (pH 7.5), 0.5% Nonidet P-40, 250 mm NaCl, 2 mm EDTA, 10% (v/v) glycerol, 1 mm sodium orthovanadate, 1 mm sodium fluoride, 1 mm phenylmethylsulfonyl fluoride, leupeptin (5 μg/ml), aprotinin (5 μg/ml), pepstatin A (1 μg/ml), benzaminidine (100 μm), and 10 mm N-ethylmaleimide. The use of N-ethylmaleimide in lysis buffers in coimmunoprecipitation procedures is an important technical feature, since it stabilizes ubiquitinated species by preventing their deubiquitination. Soluble extracts were mixed with FLAG M2 affinity beads and rotated at 4 °C overnight. Nonspecific binding was eliminated by repeated washes with lysis buffer, and bound protein was eluted with sample buffer containing SDS. The proteins were separated on a gradient gel (4-20%; Invitrogen) and transferred to nitrocellulose membrane for Western blotting. Chemiluminescence detection was performed using SuperSignal® West Pico reagent (Pierce). pERK and β-arrestin signals were quantified by densitometry using GeneTools software.In Vitro Translation of β-Arrestins and β-Arrestin-Recombinant β2 AR Binding—[35S]β-arrestins were in vitro translated using a TNT® T7 quick coupled transcription/translation system (catalog number L1170; Invitrogen) according to the manufacturer's recommended procedure. Briefly, reactions were assembled by mixing appropriate amounts of TNT® Quick Master Mix, [35S]methionine (catalog number AG1094; Amersham Biosciences), and pCDNA3.1-β-arrestin 2 wild type, β-arrestin20K, or β-arrestin2-Ub plasmids in 0.5-ml microcentrifuge tubes. The reactions were incubated at 30 °C for 90 min, and the in vitro translated [35S]β-arrestins were stored at -80 °C before performing binding experiments.To study receptor binding, the in vitro translated [35S]β-arrestins were incubated in 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 2 mm EDTA with 14.6 pmol (0.7 μg) of β2AR reconstituted in phospholipid vesicles at room temperature for 1 h. Purified GRK2 (0.5 μg), 80 μm ATP, 50 μm isoproterenol, or 50 μm propranolol were added to the reaction mixture where indicated. After the incubation period, an aliquot of the reaction was set aside to determine input levels of [35S]β-arrestins, and the remaining samples were diluted with ice-cold buffer and centrifuged at 85,000 rpm for 30 min with a bench top Optima TLX ultracentrifuge. After ultracentrifugation, the supernatants were removed, and the pellets were washed with 0.5 ml of 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, and 2 mm EDTA. The samples were centrifuged again, and the wash was repeated five times. Finally, 30 μl of SDS-PAGE buffer were added to each sample, and proteins were separated by 4-20% gel. The gels were dried, and the amounts of β-arrestins bound to the β2AR were determined by autoradiography. Control experiments were performed by the same experimental procedure, except that empty vesicles were used in the place of receptor-containing vesicles. Bands were quantified by densitometry, and the amount of each β-arrestin was normalized to its input levels.Cross-linking—COS-7 cells were transiently transfected with FLAG-β2AR along with pEGFP or β-arrestin20K-GFP. 30 h post-transfection, cells in 100-mm dishes were stimulated at 37 °C in phosphate-buffered saline (PBS) containing 10 mm HEPES (pH 7.4), with vehicle or agonist. Stimulations were terminated by the addition of dithiobis-maleimidoethane (DTME; Pierce) to a final concentration of 2 mm, and plates were rocked for 40 min at room temperature. Cells were washed three times with PBS/HEPES to remove unreacted DTME and lysed in radioimmune precipitation buffer (150 mm NaCl, 50 mm Tris, pH 8.0, 5 mm EDTA, 1% Nonidet P-40, 0.5% deoxycholate), and receptors were immunoprecipitated.Phospho-ERK Time Course—HEK-293 cells stably expressing the β2AR, transfected with vector, β-arrestin2-FLAG, FLAG-β-arrestin2-Ub, or β-arrestin20K-FLAG on 12-well plates were starved for at least 4 h in serum-free medium prior to stimulation. After stimulation, cells were solubilized by directly adding 2× SDS-sample buffer, followed by boiling at 100 °C for 5 min. For each transfection, an equal portion of the cells was set aside for protein determination (modified Bradford protocol). Equal amounts (μg) of cellular extracts were separated on 4-20% Tris-glycine polyacrylamide gels (Invitrogen) and transferred to nitrocellulose membranes for immunoblotting. Phosphorylated ERK1/2, total ERK1/2, and β-arrestins were detected by immunoblotting with rabbit polyclonal anti-phospho-p44/42 MAPK (1:2000; Cell Signaling), anti-MAPK1/2 (1:10,000; Millipore), and anti-β-arrestin (1:3000; A1CT) antibodies, respectively. Chemiluminiscence detection was performed using the SuperSignal West Pico reagent (Pierce), and phosphorylated ERK1/2 immunoblots were quantified using Gene-Tools software.Confocal Microscopy—HEK-293 cells have a favorable morphology, such that sections of cytoplasm and nucleus can be simultaneously imaged; hence, they were used in these experiments. HEK-293 cells on 10-cm dishes were transiently transfected with HA-β2AR along with β-arrestin2-GFP, GFP-β-arrestin2-Ub, or βarrestin20K-GFP. Twenty-four hours post-transfection, cells were plated on collagen-coated 35-mm glass bottom plates. On the following day, cells were starved for at least 2 h in serum-free medium prior to stimulation. After stimulation, cells were fixed with 5% formaldehyde diluted in PBS containing calcium and magnesium. Fixed cells were permeabilized with 0.01% Triton in PBS containing 2% bovine serum albumin for 60 min and incubated at room temperature with appropriate primary antibody. The secondary antibody incubations were done for 1 h, followed by repeated washes using PBS. Confocal images were obtained on a Zeiss LSM510 laser-scanning microscope using multitrack sequential excitation (488, 568, and 633 nm) and emission (515-540 nm, GFP; 585-615 nm, Texas Red) filter sets. Live cell GFP images were acquired using a heated (37 °C) microscope stage and collected sequentially using single line excitation (488 nm).Receptor Internalization—FLAG or HA epitope-tagged receptors expressed in HEK-293 cells in 12-well dishes were incubated with or without agonist for 30 min in serum-free medium at 37 °C. Cell surface receptors were labeled with M1 FLAG or 12CA5 monoclonal antibody and with fluorescein isothiocyanate-conjugated goat antibody to mouse IgG as a secondary antibody. Receptor internalization was quantified as loss of cell surface receptors as measured by fluorescence-assisted cell sorting (Duke University flow cytometry facility).Subcellular Fractionation—Monolayers of COS-7 cells transfected with β-arrestin2 or β-arrestin2-Ub plasmids were gently scraped and collected in PBS containing protease inhibitors and 40 mm NaCl, subjected to two freeze-thaw cycles for lysis. Samples were centrifuged at 800 × g for 5 min to precipitate unlysed cells. The resulting supernatant was centrifuged at 100,000 × g to separate soluble and membrane components. 40 μg of each fraction was separated on SDS gels and analyzed by Western blotting.RESULTSA Ubiquitin Minus β-Arrestin2 Mutant—To obtain a β-arrestin2 mutant that is not ubiquitinated upon 7TMR stimulation, we made conservative changes of groups of lysines to arginines, overexpressed FLAG-tagged mutants in COS-7 cells, and tested the β-arrestin precipitates for the ubiquitination signal induced by 1-min isoproterenol stimulation (Fig. 1, A and B). Surprisingly, elimination of such a signal required replacement of all 31 lysine residues of β-arrestin2 (mutant β-arrestin20K Fig. 1B). When a FLAG epitope-tagged β-arrestin20K is overexpressed in HEK-293 cells, no ubiquitination smear is detected upon isoproterenol stimulation (Fig. 1C). Although these experiments indicate that β-arrestin20K can be expressed as a properly folded protein that is isolated and detected by the epitope tag, a concern still remains whether β-arrestin20K, despite its 31 lysine to arginine changes, is a bona fide form of β-arrestin.To test whether the basic folding and binding properties of β-arrestin20K are retained, we compared the binding of in vitro translated β-arrestin2 and β-arrestin20K to purified recombinant β2AR reconstituted in vesicles. We also tested β-arrestin2-Ub for receptor binding under the same conditions. In these in vitro assays, both WT and β-arrestin20K represent nonubiquitinated forms, and only the β-arrestin2-Ub chimera constitutes the ubiquitinated form. As shown in Fig. 2, A and B, β-arrestin20K bound to the β2AR to the same extent as β-arrestin2. However, the presence of a single ubiquitin moiety increased the binding by 4-fold (Fig. 2, A and B). These experiments suggest that although both nonubiquitinated forms of β-arrestin2 (i.e. WT and 0K) are equipotent for β2AR binding, there is more binding between the β2AR and the ubiquitinated form (i.e. β-arrestin2-Ub). When binding was performed in the presence of isoproterenol, a small increase was observed for all three β-arrestin forms (data not shown). We hypothesized that reconstituted β2AR was already in an activated conformation due to the presence of zinterol in purification buffers. If so, inclusion of an antagonist could alter the observed binding. When β-arrestin-receptor complex formation was tested in the presence of propranolol, we found a dramatic decrease in binding for all three β-arrestin forms (Fig. 2, A and B), suggesting that propranolol destabilizes but does not eliminate receptor-β-arrestin binding in these experiments. Moreover, when reconstituted receptor samples were probed with a β2AR-specific phosphoserine antibody (serines 355 and 356), a small amount of phosphorylation was detected (Fig. 2C, top). The addition of GRK2 leads to an increase in the phosphorylation signal, and isoproterenol augments it further (Fig. 2C). We observed a comparable increase in binding above basal conditions for all three β-arrestin forms upon GRK2 phosphorylation and isoproterenol treatment of the reconstituted β2AR (Fig. 2D). Collectively, these in vitro binding assays confirm that, although ubiquitinated β-arrestin2 forms a tight complex with the β2AR, nonubiquitinated β-arrestin2 can bind reconstituted β2AR and that the protein-protein interaction domain(s) between the receptor and β-arrestin20K is mostly unperturbed.FIGURE 2Binding of in vitro translated β-arrestins to recombinant β2ARs reconstituted in phospholipid vesicles. A, as described under "Experimental Procedures," in vitro translated, 35S-labeled β-arrestin2, β-arrestin20K, or β-arrestin2-Ub was incubated with either empty phospholipid vesicles or vesicles containing β2AR. The vesicles were then precipitated by repeated centrifugation and wash cycles. The final pellet was solubilized and separated on SDS gels, and the bound β-arrestins were detected by autoradiography. In the indicated lanes, propranolol (50 μm) was included in the reaction mixture. B, results from three separate binding experiments were quantified and shown as bar graphs. **, p < 0.001, β-arrestin2 or β-arrestin20K versus β-arrestin2-Ub, one-way analysis of variance, and Tukey's multiple comparison test; β-arrestin2 versus β-arrestin20K, no significant difference. C, reconstituted β2AR samples were probed with a phosphoserine antibody (serines 355 and 356 within the β2AR carboxyl tail) in the upper panel and with a β2AR antibody (H-20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in the lower panel. D, receptor-β-arrestin binding was tested as in A. GRK2 alone or GRK2 and isoproterenol (ISO;50 μm) were added to the reaction as indicated. The bar graph is a summarization of two independent experiments performed in duplicate. For each β-arrestin form, basal binding in the absence of GRK2 and isoproterenol was assigned as 1. IB, immunoblot; EV, empty vesicles.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine the isoproterenol-stimulated binding of β-arrestin20K to the β2AR in a cellular context, we employed immunoprecipitation assays utilizing chemical cross-linking with a sufhydryl-reactive compound, DTME (Fig. 3). We used COS-7 cells transiently transfected with FLAG-β2AR and β-arrestin20K-GFP, immunoprecipitated the receptors under nonstimulated or stimulated conditions (5 min, 1 μm isoproterenol), and detected β-arrestin20K-GFP by Western blotting (Fig. 3A). β-Arrestin20K-GFP binds to activated receptors with a 2-3-fold agonist-induced recruitment (Fig. 3B). In similar assays, the WT and β-arrestin2-Ub were recruited 10-12- and 12-15-fold, respectively (data not shown). These experiments further suggest that β-arrestin20K-GFP, albeit much weaker than the WT, nevertheless binds the β2AR upon agonist stimulation. Probably, the robust association of β-arrestin20K and the β2AR does not occur in cells due to a lack of β-arrestin2 ubiquitination, which helps to stabilize the complex.FIGURE 3β-arrestin20K binding to receptors demonstrated by chemical cross-linking. A, COS-7 cells transiently transfected with FLAG-β2AR with pEGFP vector or β-arrestin20K-GFP were stimulated with 10 μm isoproterenol (iso) for 5 min, and FLAG receptors were immunoprecipitated after chemical cross-linking with DTME. The IP was probed with a β-arrestin antibody (top) and a receptor-specific antibody, H-20 (bottom). B, the bar gra
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