Trafficking Patterns of beta -Arrestin and G Protein-coupled Receptors Determined by the Kinetics of beta -Arrestin Deubiquitination*

Sudha K. ShenoyDagger § and Robert J. LefkowitzDagger §||

From the Dagger  Howard Hughes Medical Institute, Departments of § Medicine and  Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, September 19, 2002, and in revised form, January 28, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Agonist-dependent internalization of G protein-coupled receptors via clathrin-coated pits is dependent on the adaptor protein beta -arrestin, which interacts with elements of the endocytic machinery such as AP2 and clathrin. For the beta 2-adrenergic receptor (beta 2AR) this requires ubiquitination of beta -arrestin by E3 ubiquitin ligase, Mdm2. Based on trafficking patterns and affinity of beta -arrestin, G protein-coupled receptors are categorized into two classes. For class A receptors (e.g. beta 2AR), which recycle rapidly, beta -arrestin directs the receptors to clathrin-coated pits but does not internalize with them. For class B receptors (e.g. V2 vasopressin receptors), which recycle slowly, beta -arrestin internalizes with the receptor into endosomes. In COS-7 and human embryonic kidney (HEK)-293 cells, stimulation of the beta 2AR or V2 vasopressin receptor leads, respectively, to transient or stable beta -arrestin ubiquitination. The time course of ubiquitination and deubiquitination of beta -arrestin correlates with its association with and dissociation from each type of receptor. Chimeric receptors, constructed by switching the cytoplasmic tails of the two classes of receptors (beta 2AR and V2 vasopressin receptors), demonstrate reversal of the patterns of both beta -arrestin trafficking and beta -arrestin ubiquitination. To explore the functional consequences of beta -arrestin ubiquitination we constructed a yellow fluorescent protein-tagged beta -arrestin2-ubiquitin chimera that cannot be deubiquitinated by cellular deubiquitinases. This "permanently ubiquitinated" beta -arrestin did not dissociate from the beta 2AR but rather internalized with it into endosomes, thus transforming this class A receptor into a class B receptor with respect to its trafficking pattern. Overexpression of this beta -arrestin ubiquitin chimera in HEK-293 cells also results in enhancement of beta 2AR internalization and degradation. In the presence of N-ethylmaleimide (an inhibitor of deubiquitinating enzymes), coimmunoprecipitation of the receptor and beta -arrestin was increased dramatically, suggesting that deubiquitination of beta -arrestin triggers its dissociation from the receptor. Thus the ubiquitination status of beta -arrestin determines the stability of the receptor-beta -arrestin complex as well as the trafficking pattern of beta -arrestin.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

G protein-coupled receptors (GPCRs)1are heptahelical receptors that couple to heterotrimeric G proteins. Upon agonist stimulation GPCRs initiate second messenger signaling cascades through activated G protein subunits. Agonist stimulation also leads to rapid receptor phosphorylation by serine/threonine kinases known as G protein-coupled receptor kinases. The receptor, thus phosphorylated, has increased affinity for cytosolic proteins called beta -arrestins. beta -Arrestin binding leads to the uncoupling of the receptor from its cognate G proteins, causing dampening or desensitization of GPCR signaling via the downstream second messenger molecules (1). Recently, novel adaptor and scaffold functions of beta -arrestins have been discovered. Thus, while terminating G protein signals, beta -arrestin binding can initiate new signal waves from GPCRs (2). For example, beta -arrestins serve as adaptors that bring nonreceptor tyrosine kinases such as Src to form signaling complexes with the internalizing receptor (3). beta -Arrestins function as GPCR-regulated scaffolds for mitogen-activated protein kinase modules such as ASK-MKK4-JNK3 and RAF-MEK-ERK1/2 (4, 5). In addition, beta -arrestins interact with proteins of the endocytic machinery such as clathrin, beta -adaptin subunit 2 of the AP2 complex, and Arf-6 and thus promote internalization of receptors via clathrin-coated vesicles (6-8).

Ubiquitination, a post-translational attachment of ubiquitin residues to the lysines of substrate proteins, has been implicated in the internalization of yeast pheromone receptors (9, 10), the endocytosis of several mammalian cell surface receptors (11-13), and sorting to the multivesicular bodies in yeast (14). The prototypic mammalian GPCR, beta 2AR is ubiquitinated in an agonist- and beta -arrestin-dependent manner (15). Interestingly, receptor ubiquitination is not crucial for its internalization but is essential for proper trafficking to lysosomes for degradation. On the other hand, beta 2AR internalization requires the agonist-promoted ubiquitin modification of the adaptor protein beta -arrestin2 catalyzed by a RING domain containing E3 ubiquitin ligase Mdm2 (15).

It is now evident that G protein-coupled receptor kinase phosphorylation and subsequent beta -arrestin binding promote internalization of numerous GPCRs. Furthermore, recently developed fluorescent-tagged beta -arrestins have aided in visualizing the translocation of cytosolic beta -arrestins to activated receptors at the cell surface using confocal microscopy (16). By employing such methods, two distinct patterns of beta -arrestin trafficking within the cell have been delineated resulting in the classification of GPCRs as follows: class A (e.g. beta 2AR, alpha 1b-adrenergic receptor, µ opioid receptor, endothelin 1A, and dopamine D1A receptors), where beta -arrestin interacts with the receptor at the cell surface but does not endocytose into vesicles, thus showing a transient interaction with the receptor; and class B (e.g. vasopressin receptor V2, angiotensin AT1a, neurotensin 1, thyrotropin-releasing hormone, and neurokinin NK-1 receptors), in which beta -arrestins and receptor traffic together from the cell membrane to endocytic vesicles (17). These two classes of receptor also differ with regard to their affinity for different beta -arrestin isoforms. Class A receptors preferentially bind beta -arrestin2, whereas class B receptors bind to beta -arrestin1 and beta -arrestin2 with equal affinity.

We now report that the distinct intracellular trafficking patterns of beta -arrestin with the two classes of receptors are a function of the differing pattern of beta -arrestin ubiquitination and deubiquitination, which they induce. The pattern of beta -arrestin ubiquitination is transient with beta 2AR (class A) and is sustained with V2R (class B), which parallels the intracellular trafficking of beta -arrestin2 with respect to these two receptors. Moreover a beta -arrestin-ubiquitin fusion protein changes the trafficking pattern such that a transient beta -arrestin binder such as beta 2AR now shows stable beta -arrestin interaction because beta -arrestin-Ub and beta 2AR traffic together into cells and colocalize in endocytic vesicles. Our data strongly suggest that beta -arrestin ubiquitination stabilizes the receptor-beta -arrestin complex and that the intracellular trafficking of beta -arrestin is determined by its ubiquitination status.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- LipofectAMINE was from Invitrogen. FuGENE 6 was from Roche Diagnostics. M2 anti-FLAG affinity agarose beads, monodansylcadaverine, isoproterenol, arginine vasopressin peptide, anti-FLAG M1 and M2 antibodies, and fluorescein isothiocyanate anti-mouse secondary IgG were from Sigma. pEYFPC1, anti-GFP monoclonal antibody (which recognizes YFP fusion proteins as well) were from Clontech. Ubiquitin antibody UbP4D1 was from Santa Cruz Biotechnology. Monoclonal antibody 12CA5 to HA epitope was from Roche Molecular Biochemicals. Easy tagTM express protein labeling mix, 35S- and 125I-labeled (-)iodocyanopindolol were from PerkinElmer Life Sciences. Horseradish peroxidase-conjugated secondary antibodies were from Amersham Biosciences. A1CT is a rabbit polyclonal antibody to beta -arrestin generated in the Lefkowitz Laboratory.

The expression plasmids for YFP-beta arrestin2-wild type, HA-beta 2ARV2CT, and HA-V2Rbeta 2CT (Refs. 16-18) were kindly provided by Dr. Marc Caron at Duke University.

The plasmids pcDNA3-beta -arrestin2-Ub and pEYFPC1-beta -arrestin2-Ub were constructed as follows. A 1250-bp DNA fragment encoding beta -arrestin2 was amplified to contain 5'-KpnI-HindIII and 3'-SalI ends; a 240-bp DNA fragment encoding ubiquitin was amplified to contain 5'-SalI and 3'-XbaI ends. The two fragments were ligated together with pcDNA3 with HindIII and XbaI ends to obtain beta -arrestin2-Ub expression plasmid. A 1500-bp DNA fragment encoding beta -arrestin2-Ub was subcloned into the KpnI and ApaI sites of pEYFPC1 vector to obtain the expression plasmid for YFP-beta -arrestin2-Ub. All constructs were confirmed by DNA sequencing.

Cell Culture and Transfection-- COS-7 and HEK-293 cells were obtained from American Type Culture Collection. COS-7 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin streptomycin and transiently transfected with LipofectAMINE reagent. HEK cells were maintained in minimal essential medium supplemented with fetal bovine serum and transiently transfected with FuGENE 6 reagent.

Immunoprecipitation and Immunoblotting-- To detect ubiquitinated beta -arrestin2, beta -arrestin2 with a C-terminal FLAG epitope was transiently transfected into COS-7 cells or HEK-293 cells. Cells were serum starved for at least 2 h and then stimulated or not for the stipulated times with the appropriate agonists. Cells were solubilized in a lysis buffer containing 50 mM HEPES (pH 7.5), 0.5% Nonidet P-40, 250 mM NaCl, 2 mM EDTA, 10% (v/v) glycerol, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 1 µg/ml pepstatin A, 100 µM benzaminidine, and 10 mM NEM. Soluble extracts were mixed with FLAG M2 affinity beads and rotated at 4 °C overnight. Nonspecific binding was eliminated by repeated washes with lysis buffer, and bound protein was eluted with sample buffer containing SDS. The proteins were separated on a gradient gel (4-20%, Invitrogen) and transferred to nitrocellulose membrane for Western blotting. Chemiluminescent detection was performed using SuperSignal® West Pico reagent (Pierce).

Metabolic Labeling-- COS-7 cells were transiently transfected with YFP-beta -arrestin2 or YFP-beta -arrestin2-Ub. Prior to labeling with isotope, the cells were washed twice with methionine/cysteine-free medium and incubated in this medium for 10 min. Cells were incubated with radiolabel solution (Dulbecco's modified Eagle's medium, 10 mM HEPES (pH 7.5), 2% dialyzed fetal bovine serum, 300 µCi/ml 35S) for 20 min at 37 °C, after which the cells were washed three times with regular medium and incubated in medium for 0, 2, 4, 6, 8, 10, and 12 h at 37 °C. At the end of incubation at each time point, the cells were lysed in glycerol lysis buffer (above), and immunoprecipitations of both constructs were done with either GFP beads (n = 2) or using A1CT antibody (n = 1).

Receptor Internalization-- FLAG or HA epitope-tagged receptors expressed in HEK-293 cells in 12-well dishes were treated with or without agonist for 30 min in serum-free medium at 37 °C. Cell surface receptors were labeled with M1 FLAG or 12CA5 monoclonal antibody and fluorescein isothiocyanate-conjugated goat antibody to mouse IgG as secondary antibody. Receptor internalization was quantified as loss of cell surface receptors as measured by flow cytometry (flow cytometry facility, Duke University).

Receptor Degradation-- Degradation assays were done with [125I](-)iodocyanopindolol ([125I]CYP) radioligand binding as reported before (15) on whole cells gently resuspended in Dulbecco's modified Eagle's medium buffered with 10 mM HEPES (pH 7.5). Binding was performed in triplicate with 400 pM [125I]CYP in the presence or absence of the hydrophobic antagonist propranolol (10 µM, to define nonspecific binding). Binding was terminated by rapid dilution and filtration on Whatman GFC glass fiber filters. For degradation assays incubation was at 30 °C for 30 min, and the receptor number (total specific [125I]CYP binding sites) was determined after 24 h of isoproterenol treatment and expressed as percent of receptor number assessed in nonstimulated cells.

Confocal Microscopy-- HEK-293 cells have a favorable morphology for examining sections of cytoplasm and nucleus simultaneously and hence were used in these experiments. HEK-293 cells on 10-cm dishes were transiently transfected with HA-beta 2AR, HA-V2R, HA-beta 2ARV2CT, or HA-V2Rbeta 2CT, along with beta -arrestin2-GFP, YFP-beta -arrestin2, or YFP-beta -arrestin2-Ub. 24 h post-transfection cells were plated on collagen-coated 35-mm glass bottom plates. Cells were starved for at least 2 h in serum-free medium prior to stimulation. After stimulation cells were fixed with 4% paraformaldehyde. For visualizing HA-tagged receptors, fixed cells were permeabilized with 0.01% Triton® X-100 in phosphate-buffered saline containing 2% bovine serum albumin for 20 min and incubated at room temperature with 12CA5 antibody at 1:500 dilution. The secondary antibody, anti-mouse IgG conjugated to Texas Red, followed this. Antibody incubations were done for 1 h followed by repeated washes using phosphate-buffered saline. Cells expressing low and equivalent levels of the fluorescent proteins were selected carefully to examine YFP-beta -arrestins. Confocal images were obtained on Zeiss LSM510 laser scanning microscope using dual excitation (488, 568 nm) and emission (515-540 nm GFP, YFP; 590-610 nm, Texas Red) filter sets. Live images were acquired using a heated (37 °C) microscope stage and collected sequentially using single line excitation (488 nm).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

beta -Arrestin Ubiquitination and Deubiquitination Correlate with the Stability of the GPCR-beta -Arrestin Complex during Agonist-promoted Internalization-- Stimulation of beta 2AR in COS-7 cells with the agonist isoproterenol can lead to robust ubiquitination of beta -arrestin2 at about 1 min after agonist treatment (Fig, 1A, left panel, and Ref. 15). However, this ubiquitination signal diminishes within 15 min. In contrast, stimulation of either V2 vasopressin receptors or angiotensin AT1a receptor leads to a stable ubiquitination pattern (Fig. 1B, left panel, and data not shown). With these receptors beta -arrestin2 is ubiquitinated robustly within 1 min, but the signal does not diminish at 15 min. Interestingly beta -arrestin2 is mostly polyubiquitinated (attachment of the first ubiquitin to the substrate lysine followed by formation of a polyubiquitin chain on the first ubiquitin) or possibly multiubiquitinated (ubiquitination of substrate at several lysines). Similar agonist-stimulated ubiquitination patterns were also observed for the beta -arrestin1 isoform with the aforementioned receptors (not shown). Identical ubiquitination time courses for beta -arrestin with the above receptors were seen in yet another cell system, namely, HEK-293 cells (Fig. 1, A and B, right panels). In addition, we found a higher level of basal ubiquitination in some experiments when the receptors were overexpressed.


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Fig. 1.   beta -Arrestin ubiquitination and deubiquitination parallel its association with and dissociation from GPCR. Endogenous or overexpressed beta 2AR (A) or overexpressed V2R (B) in COS-7 cells (left panels) and HEK-293 cells (right panels) were stimulated with the agonist 10 µM isoproterenol (ISO) or 1 µM AVP for the indicated times, and overexpressed beta -arrestin2-FLAG (beta -arr2 Flag) was immunoprecipitated (IP) with anti-FLAG beads. The immunoprecipitate was probed (IB) for ubiquitinated forms using Ub antibody (Santa Cruz). Horseradish peroxidase-linked anti-mouse IgG (Amersham Biosciences) was used as the secondary antibody. Unmodified beta -arrestin-FLAG has a mobility of ~51 kDa. The blots are representative of three independent experiments. C, isoproterenol stimulated trafficking of beta -arrestin2-GFP. beta -Arrestin2-GFP and HA-beta 2AR were coexpressed in HEK-293 cells. After stimulation or not (NS) with 1 µM isoproterenol, cells were fixed using paraformaldehyde. HA epitope was stained with a monoclonal antibody, 12CA5, followed by a secondary antibody to mouse IgG conjugated to Texas Red. The proteins were visualized by confocal microscopy, beta -arrestin (green channel), beta 2AR (red channel). Colocalization (yellow) of the two proteins is seen in the overlay panels. The images are representative of three similar experiments. All bars equal 10 µm. D, AVP-stimulated trafficking of GFP-beta -arrestin2. beta -Arrestin2-GFP and HA-V2R were coexpressed in HEK-293 cells. After stimulation or not with 1 µM AVP, cells were fixed using paraformaldehyde. Antibody staining and protein visualization followed the same procedure as in C. All bars represent 10 µm.

A striking correlation can be drawn between ubiquitination and intracellular trafficking patterns of beta -arrestins with beta 2AR and V2R. As shown in Fig. 1, C and D, GFP-beta -arrestin2 (green, top row) is uniformly distributed in the cytoplasm in unstimulated cells. The receptors (red, middle row) are seen at the plasma membrane. Within 1 min of respective stimuli, beta -arrestin2 translocates to the plasma membrane in both cases. However, at 15 min after stimulation, in the case of beta 2AR, the receptor alone is seen in the endocytic vesicles, with the beta -arrestin still at the plasma membrane, whereas in the case of V2R both beta -arrestin and receptor are seen colocalized in endocytic vesicles (yellow, bottom row). Thus, the ubiquitination and deubiquitination time course of beta -arrestin parallel its association with and dissociation from the two types of receptor.

Interaction of beta -Arrestin with GPCR Tail Residues Governs Both Intracellular Trafficking and Ubiquitination Patterns of beta -Arrestin-- The differing affinity of a class A versus a class B GPCR for beta -arrestin has been attributed to the phosphorylation by G protein-coupled receptor kinases of specific serine clusters in the tail region of the GPCR (18). Moreover, the exchange of the cytoplasmic tail residues between the two receptor classes leads to reversal of the patterns of beta -arrestin affinity for the receptors as well as intracellular trafficking (16). As shown in Fig. 2A, a chimeric beta 2ARV2CT with the first 341 amino acids of the beta 2AR (Met1-Cys341) fused to the last 29 amino acids of the V2R (Ala343-Ser371) interacts with beta -arrestin more stably, and hence after 15 min of isoproterenol stimulation, both the receptor and beta -arrestin are colocalized in endosomes. On the other hand the chimera V2Rbeta 2CT, which contains the first 342 amino acids of the V2R (Met1-Cys342) fused to the last 72 amino acids of the beta 2AR (Leu342-Leu413) shows the transient beta -arrestin binding pattern of the beta 2AR, and hence beta -arrestin remains at the plasma membrane, and receptor alone traffics to endosomes (Fig. 2B).


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Fig. 2.   The GPCR tail residues govern both trafficking and ubiquitination patterns of beta -arrestin2. Trafficking of GFP-beta -arrestin2 (GFP-beta arr2) with the chimeric GPCRs beta 2ARV2CT (A) and V2Rbeta 2CT (B) is shown. HEK-293 cells were transiently transfected with GFP-beta -arrestin2 and HA-tagged receptor. After agonist stimulation or not (NS), cells were fixed, permeabilized, and stained with 12CA5 and Texas Red-conjugated anti-mouse antibody. Shown are confocal images of receptor immunofluorescence (red) and GFP-beta -arrestin fluorescence (green). Colocalization of beta -arrestin and receptor is seen (yellow) in the overlay. Confocal images are representative of three similar experiments. All bars represent 10 µm. ISO, isoproterenol. C, beta 2ARV2CT-dependent beta -arrestin ubiquitination. COS-7 cells (left panel) and HEK-293 cells (right panel) overexpressing beta 2ARV2CT and beta -arrestin2-FLAG were stimulated or not with 10 µM isoproterenol for the indicated times, and beta -arrestin was immunoprecipitated (IP) with anti-FLAG affinity beads. The immunoprecipitate was probed (IB) with Ub antibody to detect ubiquitinated forms of beta -arrestin. The blots are representative of three independent experiments in both cell types. D, time course of beta -arrestin ubiquitination dependent on V2Rbeta 2CT stimulation. COS-7 cells (left panel) and HEK-293 cells (right panel) overexpressing V2Rbeta 2CT and beta -arrestin2-FLAG were stimulated or not with 1 µM AVP. beta -Arrestin was immunoprecipitated, and the immunoprecipitate was probed with Ub antibody. The blots are representative of three independent experiments in each cell type.

If the ability of beta -arrestin to remain ubiquitinated is dependent on its interaction with the GPCR, one might expect that the receptor cytoplasmic tail residues would govern the pattern of ubiquitination. To test this hypothesis, we determined the agonist-promoted ubiquitination patterns of beta -arrestin for the 2 chimeric receptors. As shown in Fig. 2C, a 1-min isoproterenol stimulation of COS-7 cells (left) or HEK-293 cells (right) expressing beta 2ARV2CT leads to a marked increase in beta -arrestin ubiquitination which does not decrease at 15 min. In marked contrast, the stimulation of the chimera V2Rbeta 2CT leads to a very robust increase in ubiquitination at 1 min of AVP treatment, but the signal returns to almost basal levels at 15 min (Fig. 2D). These results, together with the data shown in Fig. 1, strongly suggest that the ubiquitinated form of beta -arrestin corresponds to the receptor-bound beta -arrestin complex.

A Persistently Ubiquitinated Form of beta -Arrestin2 Shows Enhanced Binding to GPCR-- Our data suggest that beta -arrestin2 translocates to the plasma membrane and becomes ubiquitinated within 1 min of GPCR stimulation but is deubiquitinated with differing kinetics depending upon the receptor. However, it is unknown whether deubiquitination is a cause or consequence of beta -arrestin dissociation from the receptor. We theorized that fusion of a ubiquitin moiety to the beta -arrestin protein would provide a persistently ubiquitinated form of beta -arrestin. If beta -arrestin2 deubiquitination was the trigger for dissociation from the class A receptor, then we would expect that the chimeric protein would not dissociate from the receptor and would show tighter binding to the class A receptor than wild type beta -arrestin because it cannot be efficiently deubiquitinated. On the other hand, if deubiquitination of beta -arrestin occurs after its dissociation from the receptor then there should be no alterations in beta -arrestin-Ub binding or in its dissociation kinetics.

Because ubiquitinated proteins are substrates for proteasomal degradation (19), we tested the stability of a beta -arrestin-Ub chimeric protein. A comparison of the half-lives of YFP-beta -arrestin2 and YFP-beta -arrestin2-Ub is shown in Fig. 3A. Wild type beta -arrestin2 has a half-life of about 10-12 h in COS-7 cells as determined by 35S metabolic labeling. Not surprisingly, fusion of ubiquitin in frame to beta -arrestin decreased its half-life to about 2 h (Fig. 3A). Nonetheless the chimeric protein could be over-expressed in sufficient amounts to be detected by antibody to both beta -arrestin (not shown) and YFP (Fig. 3, B and C).


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Fig. 3.   Characterization of YFP-beta -arrestin-Ub chimeric protein. A, stability of the ubiquitin chimera in COS-7 cells. Monolayers of cells overexpressing either YFP-beta -arrestin2 or YFP-beta -arrestin2-Ub were labeled with 35S, and both proteins were immunoprecipitated with GFP-agarose beads or A1CT antibody beads as described under "Experimental Procedures." The bands on the autorad were quantitated by fluoroimaging and are represented graphically in the figure: , YFP-beta -arrestin2; *, YFP-beta -arrestin2-Ub. The zero time point represents 100% protein. The graph is an average of three independent experiments. B, interaction of the beta -arrestin2-Ub chimera with beta 2AR. FLAG-beta 2AR was coexpressed with either YFP-beta -arrestin2 or YFP-beta -arrestin2-Ub in COS-7 cells. After stimulation or not the receptor was immunoprecipitated (IP) with anti-FLAG beads in the absence (left panels) or presence (right panels) of 10 mM NEM; the immunoprecipitate was blotted (IB) for YFP-beta -arrestins with a monoclonal antibody to GFP (top panel). The middle panel shows the amount of receptor as detected by anti-FLAG antibody M2. The bottom panel shows the expression levels of YFP-beta -arrestins in whole cell extracts as detected by the GFP-monoclonal antibody. The blots are representative of similar blots from three independent experiments. Iso, isoproterenol. C, FLAG-beta 2AR was coexpressed with either YFP-beta -arrestin2 or YFP-beta -arrestin2-Ub in HEK-293 cells. After stimulation or not the receptor was immunoprecipitated with anti-FLAG beads in the presence of 10 mM NEM; the immunoprecipitate was blotted for YFP-beta -arrestins with a monoclonal antibody to GFP (top panel). The middle panel shows the amount of receptor as detected by anti-FLAG antibody M2. The bottom panel shows the expression levels of YFP-beta -arrestins in whole cell extracts as detected by the GFP monoclonal antibody. The blots are representative of similar blots from four independent experiments.

Several research groups including ours have demonstrated coimmunoprecipitation (in the presence or absence of chemical cross-linking reagents) of beta -arrestin2 with beta 2AR. However, under such experimental conditions only a modest increase of the protein interaction is observed upon agonist treatment. In contrast, as assessed by confocal microscopy with YFP- or GFP-tagged beta -arrestin, within a few seconds of isoproterenol treatment, one can observe a major portion of the cytosolic beta -arrestin move to the plasma membrane and colocalize with the receptor. If beta -arrestin ubiquitination contributes to the stability of the GPCR-beta -arrestin interaction, then coimmunoprecipitation done under conditions that preserve the ubiquitination of beta -arrestin, such as treatment with NEM (which inactivates deubiquitinating enzymes; see Ref. 20) should result in a considerable increase in the amount of beta -arrestin that coprecipitates with the receptor. This indeed is the case as shown in Fig. 3B, which compares the amount of beta -arrestin that is coimmunoprecipitated with beta 2AR in the presence and absence of NEM. The laddering of bands as detected by the antibody to YFP-beta -arrestin represents the multimerized as well as ubiquitinated forms of the beta -arrestin protein. NEM treatment produced identical receptor-beta -arrestin complexes in coimmunoprecipitations done in HEK-293 cells (Fig. 3C). Note that these bands are not detectable at 15 min of agonist treatment with the wild type YFP-beta -arrestin2 but are detectable with the chimeric protein, YFP-beta -arrestin2-Ub. This is probably because the chimeric protein is not efficiently deubiquitinated as is the wild type and hence is detectable as the receptor-bound form of beta -arrestin2. The 90 kDa band that corresponds to monoubiquitinated YFP-beta -arrestin2 is indicated in Fig. 3B.

Expression of beta -Arrestin-Ub Chimera Transforms a Class A Receptor to Class B with Respect to Intracellular Trafficking-- To determine whether beta -arrestin-Ub chimeric protein influences its trafficking we utilized confocal microscopy to examine the fluorescent proteins in live cells. Fig. 4A illustrates the trafficking of wild type YFP-beta -arrestin2 in HEK-293 cells transiently overexpressing beta 2AR. In unstimulated cells, YFP-beta -arrestin2 is distributed homogeneously in the cytoplasm. Within 2 min of isoproterenol treatment, beta -arrestin is seen to concentrate at the plasma membrane. The distribution of YFP-beta -arrestin is unaltered at 30 min of isoproterenol treatment (Fig. 4A, upper panels). The punctate fluorescence of YFP-beta -arrestin2 at the membrane is probably because of its localization in clathrin-coated pits at the plasma membrane. A cytosolic distribution of YFP-beta -arrestin2-Ub in unstimulated and plasma membrane localization in 2-min stimulated cells are shown in Fig. 4A, lower panels. However, at 15 min, YFP-beta -arrestin-Ub is seen to redistribute from the plasma membrane to endocytic vesicles. Longer agonist treatment results in a further increase in number as well as size of YFP-beta -arrestin-Ub-containing endocytic vesicles.


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Fig. 4.   Intracellular trafficking patterns of YFP-beta -arrestin and YFP-beta -arrestin-Ub. HEK-293 cells were transiently transfected with either YFP-beta -arrestin2 or YFP-beta -arrestin2-Ub along with beta 2AR (A) or V2Rbeta 2CT (B). NS, not stimulated. Cells were starved for 1 h in serum-free medium. The distribution of YFP-beta -arrestin2 and YFP-beta -arrestin2-Ub was visualized before and after treatment with 10 µM isoproterenol (ISO; A) or 1 µM AVP (B). Shown are representative confocal images of YFP fluorescence followed in the same HEK-293 cells treated for 2, 15, and 30 min at 37 °C. All bars represent 10 µm.

V2Rbeta 2CT is a chimeric receptor in which the class B pattern of trafficking and ubiquitination is converted to that of a class A receptor (Fig. 2, B and D). Stimulation of V2Rbeta 2CT with AVP leads to the redistribution of YFP-beta -arrestin2 to the plasma membrane in a punctate pattern like the beta 2AR (Fig. 4B, upper panels) at 2 min. The YFP-beta -arrestin2 remains at the plasma membrane at longer time points. On the other hand, YFP-beta -arrestin-Ub, which translocates to the plasma membrane within 2 min of agonist, moves into endocytic vesicles as early as 15 min of AVP treatment (Fig. 4B, lower panels). The addition of tail residues of beta 2AR (class A) to V2R (class B) confers class A trafficking pattern to the V2Rbeta 2CT chimeric receptor. As a further step, overexpression of YFP-beta -arrestin2-Ub reverts the trafficking pattern back to that of class B. Thus a "double reversal" of beta -arrestin trafficking pattern is seen with respect to V2Rbeta 2CT.

To determine whether the YFP-beta -arrestin2-Ub in endocytic vesicles colocalizes with the GPCR, we examined the distribution of both beta 2AR and beta -arrestin after 15 min of isoproterenol treatment (Fig. 5). In unstimulated cells receptor is seen at the plasma membrane, and either YFP-beta -arrestin or YFP-beta -arrestin-Ub is seen uniformly distributed in the cytoplasm (data not shown). Stimulation of cells with isoproterenol for 15 min resulted in the redistribution of beta 2AR (red) and YFP-beta -arrestin2 (green) into small puncta at the plasma membrane, which likely represent colocalization of beta 2AR and YFP-beta -arrestin in clathrin-coated pits at the membrane (Fig. 5A). Internalized receptors, seen as red vesicles in the cytoplasm, do not, however, contain any YFP-beta -arrestin2, suggesting that beta -arrestin2 dissociates from the receptor during or shortly after vesicle formation. Fig. 5B shows the distribution of beta 2AR and YFP-beta -arrestin2-Ub after 15 min of isoproterenol treatment. In contrast to what was seen with wild type YFP-beta -arrestin2, the YFP-beta -arrestin-Ub chimera is seen to colocalize with the internalized beta 2AR. Further, very little staining of either protein is seen at the plasma membrane. Thus, a ubiquitinated form of beta -arrestin2 can transform a class A receptor such as the beta 2AR into a class B receptor with respect to its pattern of internalization.


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Fig. 5.   YFP-beta -arrestin2-Ub but not YFP-beta -arrestin colocalizes in endocytic vesicles with beta 2AR. HEK-293 cells were transiently transfected with HA-beta 2AR and YFP-beta -arrestin2 (A) or YFP-beta -arrestin2-Ub (B). After isoproterenol (ISO) treatment or not the cells were fixed, permeabilized, and stained for receptor with the primary antibody 12CA5 and secondary antibody anti-mouse IgG conjugated to Texas Red. Shown are confocal images where the receptor is visualized in the red channels and beta -arrestin in the green channels. Colocalization of two proteins is seen as yellow in the overlay. The results are representative of three independent experiments. All bars represent 10 µm.

Effect of beta -Arrestin-Ub on the Internalization and Degradation of beta 2AR-- Because beta -arrestin2-Ub can form stable complexes with beta 2AR, we tested whether this interaction had any effect on agonist-stimulated receptor internalization. As shown Fig. 6A, beta 2AR internalization, as measured in HEK-293 cells after a 30-min isoproterenol treatment, was 25 ± 1.8%. Upon overexpression of beta -arrestin2 and beta -arrestin2-Ub the internalization increased to 33 ± 1.6% and 44 ± 3.5%, respectively. Pretreatment of cells with 200 µM monodansylcadaverine, a known inhibitor of receptor movement to clathrin-coated pits (21), reduced this internalization by 58% (Fig. 6C), suggesting that the beta -arrestin-Ub promoted internalization proceeds via clathrin-coated vesicles.


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Fig. 6.   Effect of chimeric beta -arrestin-Ub on sequestration of GPCRs. HEK-293 cells were transiently transfected with FLAG-beta 2AR (A) and HA-V2R (B). In each case the receptor was cotransfected with vector plasmid (Mock), beta -arrestin2, or beta -arrestin2-Ub. After serum starvation cells were treated with 10 µM isoproterenol (ISO; A) or 1 µM AVP (B) for 30 min at 37 °C. Cell surface receptors before and after agonist treatment were determined by flow cytometry. Data in A are the mean ± S.E. of seven independent experiments done in triplicate. Data in B are the mean ± S.E. of five independent experiments done in triplicate. # p < 0.05 versus beta -arrestin2; ** p < 0.001 versus Mock, one-way analysis of variance with Tukey multiple comparison. C, effect of monodansylcadaverine on beta 2AR internalization with and without overexpression of beta -arrestin-Ub chimera. HEK-293 cells were transiently transfected with FLAG-beta 2AR with vector plasmid or beta -arrestin2-Ub. After serum starvation, cells were incubated or not with 200 µM monodansylcadaverine (MDC) for 30 min followed by stimulation or not with 10 µM isoproterenol for 30 min at 37 °C. Cell surface receptors before and after agonist treatment were determined by flow cytometry. Data represent the average ± S.E. of four independent experiments done in triplicate.

Overexpression of beta -arrestin-Ub did not, however, result in a similar enhancement of internalization of V2R over wild type beta -arrestin as measured after 30 min of AVP stimulation (Fig. 6B). This result is expected because the ubiquitination of beta -arrestin is stable with this receptor.

A short term isoproterenol treatment of cells expressing beta 2AR results in a decrease in the number of cell surface receptors without changing total cellular receptor levels. However, prolonged exposure to isoproterenol, for hours or days, results in a decline in the total receptor number, measurable by radioligand binding. In HEK-293 cells, after 24 h of isoproterenol treatment, overexpressed beta 2AR levels decrease by about 25% (Fig. 7A). The amount of degradation increased with beta -arrestin overexpression by about 6-7%. On the other hand, the amount of receptor degradation was doubled when beta -arrestin2-Ub was coexpressed with the receptor. For comparison, we measured the degradation of the beta 2ARV2CT chimera, which essentially mimics the properties of V2R with respect to endocytosis. However, no change in the degradation of beta 2ARV2CT was observed with beta -arrestin2-Ub overexpression (Fig. 7B). These data suggest that the ubiquitination status of beta -arrestin influences both the rate at which a given receptor is sequestered from the plasma membrane as well as the likelihood that it will be sorted to lysosomes.


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Fig. 7.   Effect of chimeric beta -arrestin-Ub on degradation of GPCRs. HEK-293 cells were transiently transfected with FLAG-beta 2AR (A) and HA-beta 2ARV2CT (B). In each case the receptor was cotransfected with vector plasmid (Mock), beta -arrestin2, or beta -arrestin2-Ub. After serum starvation cells were treated with 10 µM isoproterenol (ISO) for 24 h at 37 °C. [125I]CYP binding was done on whole cells as described under "Experimental Procedures" to determine the receptor numbers with and without agonist treatment. The results are the mean ± S.E. of three to five experiments. * p < 0.01 versus Mock, # p < 0.05 versus beta -arr2, one-way analysis of variance with Tukey multiple comparison.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Binding of beta -arrestins to activated receptors leads to homologous receptor desensitization as well as to the recruitment of specialized endocytic machinery to internalize receptors. Recently, we have shown that both beta -arrestin and the beta 2AR undergo agonist-dependent ubiquitination (15). In the case of yeast alpha -factor receptor, ubiquitination appears to serve as an internalization signal (9). However, beta 2AR ubiquitination, which requires beta -arrestin as an adaptor to recruit ubiquitination enzymes, is necessary for targeting of the receptors to lysosomes for degradation rather than for their internalization. Interestingly, beta -arrestin ubiquitination catalyzed by Mdm2, a RING-type E3 ubiquitin ligase, is required for beta 2AR internalization.

The beta 2AR and the V2R are seven-membrane-spanning receptors, which typify two very different patterns of receptor trafficking and beta -arrestin interaction. Importantly, beta 2AR stimulation leads to a transient pattern of beta -arrestin ubiquitination, whereas V2R stimulation leads to stable beta -arrestin ubiquitination, which parallels the intracellular trafficking interaction of beta -arrestin with these two receptors. Exchanging the C-terminal domains of these two receptors reverses the ubiquitination patterns of beta -arrestin. Agonist stimulation of the beta 2AR leads to the recruitment of beta -arrestin to the cell membrane within a few seconds, where it gets ubiquitinated. beta 2ARs then move to clathrin-coated pits and are internalized into vesicles within 15 min. This event is accompanied by the dissociation of beta -arrestin from the receptor after its deubiquitination. This order of events is supported by the experiments done with a chimeric beta -arrestin with ubiquitin fused to its C terminus. Stimulation of the beta 2AR with isoproterenol leads to the redistribution of this chimera to the plasma membrane where it colocalizes with the receptor. However, unlike its wild type counterpart, the ubiquitin chimera does not dissociate from the receptor but is seen to colocalize with the receptor in endocytic vesicles. If deubiquitination occurred after beta -arrestin dissociation, the ubiquitin chimera would have nevertheless dissociated from the receptor. Thus, stable ubiquitination forces beta -arrestin to move into endocytic vesicles with the receptor. However, this movement could also be the result of its tighter association with the receptor as seen in the coimmunoprecipitation experiments done in the presence of inhibitors of deubiquitinating enzymes. Thus, our data indicate that when beta -arrestin interacts with a class A receptor it is prone to deubiquitination by an unknown mechanism, and this causes it to dissociate from the receptor at the plasma membrane. The reason for sustained ubiquitination with a class B receptor is not known.

Previously, we found that inhibiting beta -arrestin ubiquitination either by use of a dominant negative form of its E3 ligase Mdm2 or by using Mdm2-null cell lines did not inhibit degradation of the beta 2AR. However, in the present work, a persistently ubiquitinated beta -arrestin2 is observed to be associated with not only more robust beta 2AR sequestration but also increased receptor degradation. How can these two sets of observations be reconciled? Previous findings suggest that very slow rates of beta 2AR sequestration can support robust receptor degradation, especially as monitored at late time points such as 24 h of agonist exposure (15). It appears that even the small amount of receptor sequestration remaining when we inhibit beta -arrestin ubiquitination is likely sufficient to support receptor degradation. On the other hand, in the presence of the beta -arrestin-Ub chimera the beta 2ARs sequester more robustly and do not recycle readily. As noted above, this is now typical of a class B receptor rather than the normal class A receptor behavior of the beta 2AR. Our data suggest that such non- or slowly recycling beta 2ARs may be more subject to entry into degradative pathways and hence show increased down-regulation. In other words, the persistently ubiquitinated beta -arrestin likely fundamentally alters the relationship between the rate of receptor sequestration and the ultimate down-regulation of the receptor. Presumably, the presence of ubiquitinated beta -arrestin in complex with internalized receptors somehow leads to more efficient sorting of these internalized receptors to lysosomes. Whether this is the result of inhibition of receptor dephosphorylating enzymes, blocking the interaction of recycling proteins such as N-ethylmaleimide-sensitive fusion protein (NSF) (22), enhancing the interaction with sorting proteins such as the sorting nexins (23, 24), or some other mechanism remains to be determined.

Our data thus reveal an interesting and previously unappreciated connection between the ubiquitin modification of beta -arrestin2 and its trafficking pattern with distinct types of receptors. Recently, ubiquitination of adaptor proteins has been implicated as a trigger for downstream signaling pathways (25). It was shown that stimulation of interleukin-1 receptors leads to recruitment, dimerization, and auto-ubiquitination of TRAF6 at the membrane, which in turn triggers the kinase TAK1, which can phosphorylate Ikappa beta kinase and MKK6 leading to the activation of nuclear factor-kappa B and JNK-p38 kinase pathways (25). We have observed that agonist stimulation of receptors such as angiotensin AT1a and vasopressin V2 lead to sustained ubiquitination of beta -arrestins. These receptors, when activated, are also known to engage pools of beta -arrestin and active ERK complexes in the cytoplasm (26). Whether beta -arrestin ubiquitination forms a foundation for scaffolding of these signaling complexes on endosomes is another tantalizing question for future research.

    ACKNOWLEDGEMENTS

We thank Dr. Marc Caron for providing plasmid constructs, Dr. Louis Luttrell for critical reading, and Donna Addison and Julie Turnbough for excellent secretarial assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL16037 (to R. J. L.).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.

|| Investigator with the Howard Hughes Medical Institute. To whom correspondence should be addressed: Dept. of Medicine, Duke University Medical Center, Box 3821, Rm. 467, CARL Bldg., Durham, NC 27710. Tel.: 919-684-2974; Fax: 919-684-8875; E-mail: lefko001@receptor-biol.duke.edu.

Published, JBC Papers in Press, February 6, 2003, DOI 10.1074/jbc.M209626200

    ABBREVIATIONS

The abbreviations used are: GPCR(s), G protein-coupled receptor(s); AVP, arginine vasopressin; beta 2AR, beta 2-adrenergic receptor; E3, ubiquitin-protein isopeptide ligase; ERK1/2, extracellular signal-regulated kinases 1 and 2; GFP, green fluorescent protein; HA, hemagglutinin; HEK, human embryonic kidney; [125I]CYP, [125I](-)iodocyanopindolol; JNK3, c-Jun N-terminal kinase 3; NEM, N-ethylmaleimide; Ub, ubiquitin; V2R, vasopressin type 2 receptor; YFP, yellow fluorescent protein.

    REFERENCES
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ABSTRACT
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RESULTS
DISCUSSION
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