The Stability of the G Protein-coupled Receptor-beta -Arrestin Interaction Determines the Mechanism and Functional Consequence of ERK Activation*

Akira TohgoDagger , Eric W. ChoyDagger , Diane Gesty-PalmerDagger , Kristen L. Pierce§, Stephane Laporte, Robert H. Oakley, Marc G. Caron||**, Robert J. LefkowitzDagger §||**, and Louis M. LuttrellDagger Dagger Dagger §§

From the Departments of Dagger  Medicine, § Biochemistry and  Cell Biology, || The Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710 and Dagger Dagger  The Geriatrics Research, Education and Clinical Center, Durham Veterans Affairs Medical Center, Durham, North Carolina 27705

Received for publication, December 2, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

By binding to agonist-activated G protein-coupled receptors (GPCRs), beta -arrestins mediate homologous receptor desensitization and endocytosis via clathrin-coated pits. Recent data suggest that beta -arrestins also contribute to GPCR signaling by acting as scaffolds for components of the ERK mitogen-activated protein kinase cascade. Because of these dual functions, we hypothesized that the stability of the receptor-beta -arrestin interaction might affect the mechanism and functional consequences of GPCR-stimulated ERK activation. In transfected COS-7 cells, we found that angiotensin AT1a and vasopressin V2 receptors, which form stable receptor-beta -arrestin complexes, activated a beta -arrestin-bound pool of ERK2 more efficiently than alpha 1b and beta 2 adrenergic receptors, which form transient receptor-beta -arrestin complexes. We next studied chimeric receptors in which the pattern of beta -arrestin binding was reversed by exchanging the C-terminal tails of the beta 2 and V2 receptors. The ability of the V2beta 2 and beta 2V2 chimeras to activate beta -arrestin-bound ERK2 corresponded to the pattern of beta -arrestin binding, suggesting that the stability of the receptor-beta -arrestin complex determined the mechanism of ERK2 activation. Analysis of covalently cross-linked detergent lysates and cellular fractionation revealed that wild type V2 receptors generated a larger pool of cytosolic phospho-ERK1/2 and less nuclear phospho-ERK1/2 than the chimeric V2beta 2 receptor, consistent with the cytosolic retention of beta -arrestin-bound ERK. In stably transfected HEK-293 cells, the V2beta 2 receptor increased ERK1/2-mediated, Elk-1-driven transcription of a luciferase reporter to a greater extent than the wild type V2 receptor. Furthermore, the V2beta 2, but not the V2 receptor, was capable of eliciting a mitogenic response. These data suggest that the C-terminal tail of a GPCR, by determining the stability of the receptor-beta -arrestin complex, controls the extent of beta -arrestin-bound ERK activation, and influences both the subcellular localization of activated ERK and the physiologic consequences of ERK activation.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Homologous desensitization of most heptahelical, or G protein-coupled, receptors (GPCRs)1 results from the physical uncoupling of receptor and G protein as a consequence of arrestin binding. Agonist-occupied GPCRs are rapidly phosphorylated by specialized G protein-coupled receptor kinases (GRKs). Subsequent high affinity binding of arrestin to the GRK-phosphorylated receptor results in steric inhibition of receptor-G protein coupling. In addition, the two nonvisual arrestins, beta -arrestin 1 and beta -arrestin 2, function as adapter proteins, binding to clathrin and the beta 2 adaptin subunit of the AP-2 complex, and leading to targeting of GPCRs to clathrin-coated pits where they are internalized (1, 2).

Data obtained using green fluorescent protein (GFP)-tagged beta -arrestins and epitope-tagged GPCRs to visualize beta -arrestin and receptor trafficking in live cells have demonstrated that most GPCRs exhibit one of two characteristic patterns of agonist-induced beta -arrestin interaction that allow them to be separated into distinct classes (3). One class, represented by the beta 2 and alpha 1B adrenergic receptors (ARs), and the µ opioid, endothelin A, and dopamine D1A receptors, binds to beta -arrestin 2 with higher affinity than beta -arrestin 1. For these receptors, the interaction with beta -arrestin is transient. beta -Arrestin is recruited to the receptor at the plasma membrane and translocates with it to clathrin-coated pits. Upon internalization of the receptor, the receptor-beta -arrestin complex dissociates, such that, as the receptor proceeds into an endosomal pool, the beta -arrestin recycles to the cytosol. The second class, represented by the angiotensin AT1a receptor (AT1aR), vasopressin V2 receptor (V2R), and the neurotensin 1, thyrotropin-releasing hormone, and neurokinin NK-1 receptors, binds beta -arrestin 1 and beta -arrestin 2 with equal affinity. These receptors form stable complexes with beta -arrestin, such that the receptor-beta -arrestin complex internalizes as a unit that is targeted to endosomes. The stability of the receptor-beta -arrestin interaction influences the fate of the internalized GPCR. The beta 2AR, which binds beta -arrestin transiently, is rapidly dephosphorylated and recycled to the plasma membrane, whereas the V2R, which binds beta -arrestin stably, recycles slowly. Exchanging the C-terminal tails of these two receptors not only reverses the pattern of beta -arrestin binding, but also reverses the pattern of receptor dephosphorylation and recycling (4).

Recent data from yeast two-hybrid screens and from biochemical characterization of receptor-beta -arrestin complexes have indicated that beta -arrestins also have the ability to interact with proteins involved in signal transduction, suggesting that they may additionally function in the recruitment of signaling proteins to GPCRs (5-7). One potentially significant set of interactions is the binding of beta -arrestins to components of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinase 3 (JNK3) mitogen-activated protein kinase cascades, which allows them to act as scaffolds for localized mitogen-activated protein kinase activation (8-12). In KNRK cells, stimulation of the protease-activated receptor type 2 (PAR2) induces the assembly of multiprotein complexes that contain the internalized receptor, beta -arrestin 1, Raf-1, and activated ERK1/2 (9). The formation of these complexes, which are sufficiently stable that they can be isolated by both gel filtration and immunoprecipitation, also affects the cellular distribution of activated ERK. Because beta -arrestins are cytosolic proteins, the formation of stable complexes between beta -arrestin and ERK leads to cytosolic retention of ERK activated by PAR2 receptors. Qualitatively similar results have been obtained for the AT1aR expressed in HEK-293 and COS-7 cells (11, 12). AT1aR activation causes the formation of complexes containing the receptor, beta -arrestin 2, and the component kinases of the ERK cascade: cRaf-1, MEK1, and ERK2. Upon receptor internalization, activated ERK2 appears in the same endosomal vesicles that contain AT1aR-beta -arrestin complexes.

Thus, the beta -arrestins appear to play a dual role in GPCR signaling. They serve both to terminate G protein-dependent signals by precluding receptor-G protein coupling, and to confer novel signaling properties upon the receptor by acting as adapters or scaffolds for signaling proteins. Because of these divergent functions, we hypothesized that the stability of the receptor-beta -arrestin interaction might be a significant factor in determining both the mechanism of ERK activation employed by a GPCR and the functional consequences of ERK activation within the cell. To test this hypothesis, we have compared the relative efficiency with which GPCRs that form transient receptor-beta -arrestin complexes (alpha 1bAR and beta 2AR), and GPCRs that form stable receptor-beta -arrestin complexes (AT1aR and V2R), activate a beta -arrestin-bound pool of ERK. We also compared the signaling properties of chimeric receptors, in which the C-terminal tail domains have been exchanged so as to alter the stability of the receptor-beta -arrestin interaction (V2beta 2R and beta 2V2R). We tested whether these chimeric receptors exhibited altered activation of beta -arrestin-bound ERK, and whether changing the receptor-beta -arrestin interaction altered the cellular distribution of activated ERK, the ability of ERK to activate transcription of an Elk-1 reporter, and the ability of the receptor to produce a mitogenic response. We find that the formation of stable receptor-beta -arrestin complexes is associated with enhanced activation of beta -arrestin-bound ERK, cytosolic retention of active ERK, and a reduced transcriptional and mitogenic response to GPCR stimulation. Moreover, this phenotype can be reversed by exchange of the C-terminal tail domain. These data suggest that the stability of the GPCR-beta -arrestin interaction dictates the predominant mechanism of ERK activation and, thereby, the functional consequences of ERK activation following GPCR stimulation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
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Materials-- LipofectAMINE was from Invitrogen. FuGENE 6 was from Roche Molecular Diagnostics. Monoclonal M2 anti-FLAG affinity agarose was from Sigma. Monoclonal anti-hemagglutinin (HA) affinity agarose was from Covance. Anti-phospho-ERK1/2 antibody was from Cell Signaling and anti-ERK1/2 antibody was from Upstate Biotechnology. Goat polyclonal anti-lamin B antibody, rabbit polyclonal anti-actin, anti-retinoblastoma protein, anti-HA, and anti-FLAG antibodies were from Santa Cruz Biotechnology. Rhodamine-conjugated monoclonal anti-HA antiserum was from Molecular Probes. Horseradish peroxidase-conjugated donkey anti-rabbit antibody was from Amersham Biosciences. Horseradish peroxidase-conjugated rabbit anti-goat antibody was from Sigma. Rabbit polyclonal anti-beta -arrestin was prepared in the Lefkowitz laboratory.

cDNA Constructs-- PCDNA3.1 expression plasmids encoding GFP-tagged beta -arrestin 2, and HA-tagged AT1aR, V2R, alpha 1bAR, and beta 2AR, and the chimeric V2beta 2R and beta 2V2R were prepared in the Caron laboratory, as previously described (3, 4). The V2beta 2R chimera contains the first 342 amino acids of the V2R (Met-1 to Cys-342) fused to the last 72 amino acids of the beta 2AR (Leu-342 to Leu-413), whereas the beta 2V2R chimera contains the first 341 amino acids of the beta 2AR (Met-1 to Cys-341) fused to the last 29 amino acids of the V2R (Ala-343 to Ser-371). The pcDNA3.1 expression plasmid encoding FLAG epitope-tagged beta -arrestin 2 was created in the Lefkowitz laboratory. The pEGFP-N1 expression plasmid encoding GFP-ERK2 (9) was provided by K. A. DeFea and N. Bunnett (University of California, San Francisco). The pFR-Luc, GAL4-Elk-1, and pRL-tk-luc reporter plasmids were from Stratagene.

Cell Culture and Transfection-- HEK-293 cells and COS-7 cells were obtained from the American Type Culture Collection. HEK-293 cells stably expressing 1.5-2 pmol/mg of the HA-V2R or HA-V2beta 2R were generated by standard procedures using zeocin selection (0.4 mg/ml). HEK-293 cells were grown in Eagle's minimal essential medium with Earle's salt supplemented with 10% fetal bovine serum and 100 µg/ml gentamicin. COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 µg/ml gentamicin. Transient transfection of HEK-293 cells was performed using FuGENE 6 according to the manufacturers instructions. Transient transfection of COS-7 cells was performed using LipofectAMINE as previously described (12). Transfected cells were incubated overnight in serum-free growth medium supplemented with 10 mM HEPES, pH 7.4, 0.1% bovine serum albumin, and 100 µg/ml gentamicin prior to stimulation.

Confocal Microscopy-- HEK-293 cells were used for confocal microscopy because of their favorable morphology for comparing the cytoplasmic and plasma membrane distribution of proteins. Subconfluent monolayers in 100-mm dishes were transfected with expression plasmids encoding HA-AT1aR, HA-V2R, HA-alpha 1bAR, HA-beta 2AR, HA-V2beta 2R, or HA-beta 2V2R (8 µg/plate) along with GFP-beta -arrestin 2 (2 µg/plate). Twenty-four hours after transfection, cells were passed onto collagen-coated 35-mm glass bottom dishes and serum-starved overnight. For visualization of HA epitope-tagged receptors, cell surface receptors were stained using a 1:500 dilution of rhodamine-conjugated monoclonal anti-HA IgG in serum starving medium for 1 h at 37 °C. Cells were then washed with serum starving medium, stimulated as described in figure legends, washed with phosphate-buffered saline, fixed with 10% paraformaldehyde for 30 min at room temperature, and again washed prior to examination. Confocal microscopy was performed using a Zeiss LSM510 laser scanning microscope using a Zeiss 63 × 1.4 numerical aperture water immersion lens with dual line switching excitation (488 nm for GFP, 568 nm for rhodamine) and emission (515-540 nm for GFP, 590-610 nm for rhodamine) filter sets.

Immunoprecipitation and Immunoblotting-- Immunoprecipitation of FLAG epitope-tagged beta -arrestin 2 was performed following transient transfection of COS-7 cells in 100-mm dishes. Cells were transfected with expression plasmids encoding HA-AT1aR, HA-V2R, HA-alpha 1bAR, HA-beta 2AR, HA-V2beta 2AR, or HA-beta 2V2AR (2 µg/plate) and GFP-ERK2 (1 µg/plate) with or without plasmid encoding FLAG-beta -arrestin 2 (3 µg/plate), as indicated. Stimulation was performed as described in the figure legends. After stimulation, monolayers were washed with phosphate-buffered saline, solubilized in 0.8 ml of glycerol lysis buffer (50 mM Hepes, 50 mM NaCl, 10% (v/v) glycerol, 0.5% (v/v) Nonidet P-40, 2 mM EDTA, 100 µM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 4 µg/ml leupeptin, 2.5 µg/ml aprotinin), and clarified by centrifugation. For determination of protein expression and total cellular phospho-ERK1/2, a 50-µl aliquot of each clarified whole cell lysate was removed and mixed with an equal volume of 2× Laemmli sample buffer. Approximately 50 µg of protein from each lysate was resolved by SDS-PAGE and transferred to polyvinylidene difluoride membrane for immunoblotting. To isolate beta -arrestin-bound GFP-ERK2, bovine serum albumin was added to each lysate to a final concentration of 1%, and immunoprecipitation was performed using 20 µl of 50% slurry of monoclonal M2 anti-FLAG affinity agarose, with constant agitation overnight at 4 °C. Immune complexes were washed three times with glycerol lysis buffer and boiled in Laemmli sample buffer. Immunoprecipitated proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membrane for immunoblotting.

For experiments involving the visualization of covalently cross-linked receptor-beta -arrestin-ERK complexes, COS-7 cells in 100-mm dishes were transfected with expression plasmids encoding HA-V2R or HA-beta 2V2AR (6 µg/plate). Stimulations were performed at 37 °C in 4.6 ml of Dulbecco's phosphate-buffered saline. Incubations were terminated by the addition of 0.4 ml of 25 mM dithiobis(succinimidylpropionate) in dimethyl sulfoxide. Monolayers were agitated gently for 30 min at room temperature, washed with Dulbecco's phosphate-buffered saline containing 50 mM Tris-HCl, pH 7.4, to neutralize unreacted dithiobis(succinimidyl propionate), and lysed in 0.5 ml of glycerol lysis buffer. 25-µl aliquots of clarified cross-linked detergent lysates were mixed with an equal volume of 2× Laemmli sample buffer with or without beta -mercaptoethanol, for electrophoresis under reducing or nonreducing conditions, respectively. The remainder of each lysate was agitated overnight at 4 °C with 20 µl of 50% slurry of monoclonal anti-HA affinity agarose to immunoprecipitate HA-epitope-tagged receptor and any cross-linked proteins. After washing, immunoprecipitates were resolved by SDS-PAGE under reducing conditions and subjected to immunoblotting.

Immunoblotting of FLAG-beta -arrestin 2 was performed using rabbit polyclonal anti-FLAG IgG. Immunoblotting of endogenous beta -arrestins was performed using rabbit polyclonal anti-beta -arrestin. Endogenous ERK1/2 and phospho-ERK1/2, and GFP-ERK2 and phospho-GFP-ERK2 were detected using polyclonal anti-ERK1/2 and anti-phospho-ERK1/2 IgG. Horseradish peroxidase-conjugated polyclonal donkey anti-rabbit IgG was employed as secondary antibody. Immune complexes on polyvinylidene difluoride filters were visualized by enzyme-linked chemiluminescence using the SuperSignal chemiluminescence reagent and immunoblots were quantified using a Fluor-S MultiImager.

Quantitation of Nuclear Phospho-ERK1/2-- For the separation of nuclear and extranuclear pools of endogenous ERK1/2, COS-7 cells on 100-mm plates were transfected with plasmids encoding either the HA-V2R or HA-V2beta 2R (2 µg/plate) and FLAG-beta -arrestin 2 (0.5 µg/plate). Serum-starved cells were stimulated with vasopressin (1 µM) for 5 min. Monolayers were washed twice with ice-cold phosphate-buffered saline and collected in 2 ml of hypotonic lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl, 0.3% (v/v) Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin). Cells were incubated on ice for 3 min to allow lysis, and an aliquot of the lysed cell suspension was removed prior to centrifugation for measurement of total phospho-ERK1/2. Lysates were then centrifugation at 500 × g for 5 min to pellet nuclei. The supernatants, containing a mixture of plasma membrane, microsomal vesicles, cytoskeleton, and cytosol, represented the extranuclear fraction. Pellets containing cell nuclei were washed in lysis buffer without Nonidet P-40 and again pelleted at 500 × g. Both the extranuclear and nuclear fractions were solubilized in 2× Laemmli sample buffer and phospho-ERK1/2 was determined by protein immunoblotting. The purity of each fraction was verified by immunoblotting with antibodies to nuclear lamin B, retinoblastoma protein, and actin as described previously (12).

Elk-1 Luciferase Reporter Assays-- ERK1/2-dependent transcription was measured using an Elk-1 driven luciferase reporter system as described previously (12). Briefly, HEK-293 cells stably expressing either HA-V2R or HA-V2beta 2R receptors were plated in 100-mm dishes and transfected with GAL4-Elk-1 (1 ng/plate), pFR-luc (1 µg/plate), and pRL-tk-luc (20 ng/plate). The GAL4-Elk-1 plasmid encodes a fusion protein containing the GAL4 DNA binding domain and the transactivation domain of Elk-1, pFR-luc encodes the firefly luciferase gene under the control of the GAL4 DNA binding element, and pRL-tk-luc encodes Renilla luciferase under the control of the thymidine kinase promoter. One day following transfection, the cells were passed into 6-well dishes and serum-starved overnight. Stimulation with vasopressin (1 µM) was carried out for 6 h. Luciferase activities were determined using a dual luciferase assay kit (Promega). Cells were extracted and assayed sequentially for firefly and Renilla luciferase activities. 5-µl aliquots of cell lysate were incubated with 50 µl of luciferin reagent and luminescence was recorded for 5 s. Stop and GloTM reagent (50 µl) was added and the specific luminescence from Renilla luciferase was recorded for an additional 5 s. Firefly activities were normalized to Renilla luciferase activity.

[3H]Thymidine Incorporation-- HEK-293 cells stably expressing either V2R or V2beta 2R in 24-well plates were grown to subconfluence and serum-starved overnight. Subsequently, cells were incubated with either vasopressin (1 µM) or 2-10% fetal calf serum for 24 h at 37 °C. During last 4 h of incubation, [3H]thymidine (0.5 µCi/well) was added. Cells were washed, DNA was obtained by solubilization in 2 M guanidine in 50% phenol, and incorporated [3H]thymidine was measured by scintillation counting.

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The Stability of the GPCR-beta -Arrestin Complex Determines the Extent of Activation of a beta -Arrestin-bound Pool of ERK-- Oakley et al. (3) have shown that most GPCRs exhibit one of two patterns of receptor-beta -arrestin interaction. Fig. 1 illustrates each of these patterns, using anti-HA rhodamine staining and GFP-beta -arrestin 2 to track the cellular localization of an HA epitope-tagged receptor and beta -arrestin, respectively. As shown for the HA-beta 2AR in Fig. 1A, in the absence of agonist, rhodamine-stained receptors (left panel, red) were confined to the plasma membrane, whereas GFP-beta -arrestin 2 (middle panel, green) was diffusely cytosolic in distribution. An indistinguishable pattern of receptor and beta -arrestin distribution was observed in unstimulated cells expressing the alpha 1bAR, AT1aR, and V2R (data not shown). As shown in Fig. 1B, 15 min of agonist treatment led to the coalescence of both the beta 2AR and alpha 1bAR (left panels, red) and GFP-beta -arrestin 2 (middle panels, green) in small puncta along the plasma membrane. This redistribution reflects the recruitment of beta -arrestin to agonist-occupied GPCR and the accumulation of GPCR-beta -arrestin complexes in clathrin-coated pits or nascent endosomes. Internalized receptors, represented by the intracellular accumulation of HA-rhodamine, did not colocalize with GFP-beta -arrestin 2 (right panels), suggesting that the receptor-beta -arrestin complex dissociates at or near the plasma membrane following receptor internalization.


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Fig. 1.   The pattern of GFP-beta -arrestin 2 binding defines two classes of GPCR. A, distribution of HA-rhodamine-stained beta 2AR and GFP-beta -arrestin 2 in nonstimulated HEK-293 cells. HEK-293 cells were transiently transfected with plasmids encoding HA-beta 2AR and GFP-beta -arrestin 2 as described under "Experimental Procedures." The HA epitope on the cell surface was labeled using rhodamine-conjugated anti-HA monoclonal IgG, and unstimulated cells were fixed in paraformaldehyde and examined by confocal immunofluorescence microscopy. B, formation of transient membrane-associated GPCR-beta -arrestin complexes. Cells were transiently transfected with plasmids encoding GFP-beta -arrestin 2, and either HA-beta 2AR or HA-alpha 1bAR. Cells were prestained with anti-HA rhodamine, stimulated for 15 min with isoproterenol (1 µM) or phenylephrine (1 µM), respectively, fixed with paraformaldehyde, and examined by confocal microscopy. C, formation of stable GPCR-beta -arrestin complexes that colocalize in endosomes. Cells were transiently transfected with plasmids encoding GFP-beta -arrestin 2, and either HA-AT1aR or HA-V2R. Cells were prestained with anti-HA rhodamine, stimulated for 15 min with angiotensin II (1 µM) or arginine-vasopressin (1 µM), respectively, fixed with paraformaldehyde, and examined by confocal microscopy. In each image, the distribution of rhodamine-labeled HA receptors (red) and GFP-beta -arrestin 2 (green) are shown in the single channel images. Colocalization of HA receptors and GFP-beta -arrestin 2 is shown in the overlay images (yellow).

As shown in Fig. 1C, a markedly different pattern was observed for the AT1aR and V2R. With these receptors, 15 min of agonist exposure led to removal of most of the HA-rhodamine-labeled receptors from the plasma membrane and their accumulation in large endosomal vesicles (left panels, red). An overlapping pattern of redistribution of GFP-beta -arrestin 2 was observed (middle panels, green; right panels, yellow), indicative of the formation of stable receptor-beta -arrestin complexes that remain associated throughout receptor endocytosis and sorting.

In overexpression studies, we have previously demonstrated that beta -arrestins coprecipitate with cRaf-1, MEK1, and ERK2, and that overexpression of cRaf-1 or stimulation of AT1aRs increases the phosphorylation of a beta -arrestin-bound pool of ERK2 (10, 11), suggesting that beta -arrestins can function as scaffolds for the component kinases of the ERK1/2 cascade. We have also found that overexpression of beta -arrestin promotes the targeting of endogenous phospho-ERK1/2, along with the AT1aR, to endosomal vesicles and away from the nucleus, resulting in a smaller nuclear pool of activated ERK1/2 and a reduction in the Elk-1-driven transcriptional response to angiotensin II stimulation (12). Similar results have been reported for the PAR2 (9). However, both the AT1aR and PAR2 form stable receptor-beta -arrestin complexes that remain associated following internalization of the receptor. Because GPCRs differ in their ability to form stable complexes with beta -arrestins, we hypothesized that they might also differ in the extent to which they activated beta -arrestin-bound ERK.

To determine whether the stability of the receptor-beta -arrestin interaction affects the ability of GPCRs to activate a beta -arrestin-bound pool of ERK, COS-7 cells were transfected with HA epitope-tagged receptor, FLAG epitope-tagged beta -arrestin 2, and GFP-tagged ERK2, and the phosphorylation state of GFP-ERK2 in FLAG-beta -arrestin 2 immunoprecipitates was determined before and after agonist stimulation. GFP-ERK2 was employed in these studies to facilitate quantitation of ERK phosphorylation in the transfected cell pool, because its slower electrophoretic mobility allows it to be easily resolved from endogenous ERK1/2. GFP-ERK2 has previously been shown to undergo agonist-stimulated phosphorylation and nuclear translocation in a manner analogous to endogenous ERK (9).

As shown in Fig. 2A, 5 min stimulation of AT1aR, V2R, and alpha 1bAR expressed in COS-7 cells produced equivalent levels of whole cell GFP-ERK2 phosphorylation (top immunoblot). Under these conditions, FLAG-beta -arrestin 2 and GFP-ERK2 were constitutively associated, as shown by their coprecipitation in both the presence and absence of agonist treatment (second and third immunoblots). The beta -arrestin-bound pool of GFP-ERK2 was minimally phosphorylated in the absence of agonist. In response to stimulation of AT1aR and V2R, phosphorylation of beta -arrestin-bound GFP-ERK2 increased substantially. In contrast, stimulation of alpha 1bAR had little effect on the phosphorylation of beta -arrestin-bound GFP-ERK2, despite a robust increase in the level of phospho-GFP-ERK2 in the whole cell lysate (bottom immunoblot). Fig. 2B, provides a quantitative assessment of the relative extent of phosphorylation of beta -arrestin-bound GFP-ERK2 following stimulation of each of the three receptors. While no significant difference was observed between the AT1aR and V2R, the alpha 1bAR was 4-5-fold less efficient at activating the FLAG-beta -arrestin-bound pool of GFP-ERK2.


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Fig. 2.   GPCR-mediated phosphorylation of GFP-ERK2 in whole cell lysates and FLAG-beta -arrestin immunoprecipitates. A, COS-7 cells were transfected with plasmids encoding HA-AT1aR, HA-V2R, or HA-alpha 1bAR along with GFP-ERK2 and either empty vector or FLAG-beta -arrestin 2 as indicated. Serum-starved cells were incubated for 5 min in the presence or absence of angiotensin II (Ang II, 1 µM), arginine-vasopressin (Vaso, 1 µM), or phenylephrine (Phe, 1 µM). Phosphorylation of GFP-ERK2 in whole cell detergent lysates was determined by immunoblotting using phospho-ERK1/2-specific IgG as described (upper immunoblot). Anti-FLAG immunoprecipitates con- taining FLAG-beta -arrestin 2 were probed for FLAG-beta -arrestin 2 (second immunoblot), coprecipitated GFP-ERK2 (third immunoblot), and phosphorylated GFP-ERK2 (lower immunoblot). B, bar graph depicting the amount of phosphorylated GFP-ERK2 present in FLAG-beta -arrestin 2 immunoprecipitates following agonist treatment. In each experiment, the amount phospho-GFP-ERK2 in the FLAG-beta -arrestin 2 immunoprecipitate was normalized to the amount of GFP-ERK2 present. Data are expressed as a percentage of the beta -arrestin 2-associated phospho-ERK signal observed in angiotensin II-stimulated cells. Data shown represent the mean ± S.E. from four separate experiments. *, less than AT1aR, p < 0.05.

The AT1aR and alpha 1bAR couple primarily to Gq/11 family heterotrimeric G proteins to stimulate phosphatidylinositol hydrolysis (13), whereas the V2R couples primarily to the Gs-adenylyl cyclase pathway (4). As shown in Fig. 1, the AT1aR and V2R form stable receptor-beta -arrestin complexes, whereas the alpha 1bAR interacts with beta -arrestin transiently. Thus, the ability of these receptors to activate beta -arrestin-bound ERK2 correlated with their ability to form stable receptor-beta -arrestin complexes, rather than with the activation of a specific G protein pool.

The GPCR C-terminal Tail Regulates the Activation of beta -Arrestin-bound ERK-- The binding of beta -arrestin to an agonist-occupied GPCR is dependent upon GRK-mediated phosphorylation of serine and threonine residues located within the C-terminal tail of the receptor (1, 2). Previous studies have demonstrated that the structure of the C-terminal tail is also the principal determinant of the stability of the receptor-beta -arrestin interaction (3, 4, 14). As shown in Fig. 3A, a chimeric GPCR composed of the V2R, a stable beta -arrestin binder, substituted with the C terminus of the beta 2AR, a transient beta -arrestin binder (V2beta 2R), exhibits transient beta -arrestin binding like the beta 2AR. Instead of accumulating in endosomes along with the receptor, GFP-beta -arrestin 2 colocalizes with the V2beta 2R only at the plasma membrane. As shown in Fig. 3B, the converse is true of a chimeric beta 2AR receptor containing the V2R C terminus (beta 2V2R), where the characteristic beta 2AR pattern of receptor-beta -arrestin complex formation only at the plasma membrane is converted into one of colocalized receptor and beta -arrestin in endosomes.


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Fig. 3.   Chimeric V2beta 2 and beta 2V2 receptors exhibit a reversal of the pattern of receptor-beta -arrestin interaction. A, comparison of the pattern of receptor-beta -arrestin binding by wild type V2R (V2Rwt) and the chimeric V2beta 2R. HEK-293 cells were transiently transfected with plasmids encoding GFP-beta -arrestin 2, and either HA-V2R or HA-V2beta 2R. Cells were prestained with anti-HA rhodamine, stimulated for 15 min with arginine-vasopressin (1 µM), fixed with paraformaldehyde, and examined by confocal microscopy. Qualitatively similar redistribution of receptors and beta -arrestin 2 was observed in COS-7 cells transiently expressing HA-V2R and HA-V2beta 2R and GFP-beta -arrestin 2 (data not shown). B, comparison of the pattern of receptor-beta -arrestin binding by wild type beta 2AR and the chimeric beta 2V2R. HEK-293 cells were transiently transfected with plasmids encoding GFP-beta -arrestin 2, and either beta 2AR or beta 2V2R. Cells were prestained with anti-HA rhodamine, stimulated for 15 min with isoproterenol (1 µM), fixed with paraformaldehyde, and examined by confocal microscopy. In each image, the distribution of rhodamine-labeled HA receptors (red) and GFP-beta -arrestin 2 (green) are shown in the single channel images. Colocalization of HA receptors and GFP-beta -arrestin 2 is shown in the overlay images (yellow).

If the ability of a GPCR to activate beta -arrestin-bound ERK correlates with the formation of stable receptor-beta -arrestin complexes, then one might expect that exchanging the C terminus of receptors that differ in their pattern of beta -arrestin binding would reverse the pattern of ERK activation. To test this hypothesis, we compared the ability of the V2R and beta 2AR to activate beta -arrestin-bound GFP-ERK2, with that of the chimeric V2beta 2R and beta 2V2R. As shown in Fig. 4A, 5 min stimulation of COS-7 cells expressing V2R and V2beta 2R produced equivalent activation of GFP-ERK2 measured in the whole cell lysate. However, when the phosphorylation state of beta -arrestin-bound GFP-ERK2 was compared, the V2R and V2beta 2R differed significantly. As shown quantitatively in Fig. 4B, the V2R was about three times more effective than the V2beta 2R at mediating the activation of beta -arrestin-bound GFP-ERK.


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Fig. 4.   Effect of exchanging the C terminus of the V2R and beta 2AR on GPCR-mediated phosphorylation of GFP-ERK2 in whole cell lysates and FLAG-beta -arrestin immunoprecipitates. A, COS-7 cells were transfected with plasmids encoding either HA-V2R or HA-V2beta 2R along with GFP-ERK2 and either empty vector or FLAG-beta -arrestin 2 as indicated. Serum-starved cells were incubated for 5 min in the presence or absence of arginine-vasopressin (Vaso, 1 µM). Phosphorylation of GFP-ERK2 in whole cell detergent lysates was determined as described (upper immunoblot). Anti-FLAG immunoprecipitates containing FLAG-beta -arrestin 2 were probed for FLAG-beta -arrestin 2 (second immunoblot), coprecipitated GFP-ERK2 (third immunoblot), and phosphorylated GFP-ERK2 (lower immunoblot). B, bar graph depicting the amount of phosphorylated GFP-ERK2 present in FLAG-beta -arrestin 2 immunoprecipitates following stimulation of V2R and V2beta 2R. C, cells were transfected with plasmids encoding either HA-beta 2AR or HA-beta 2V2R along with GFP-ERK2 and either empty vector or FLAG-beta -arrestin 2 as indicated. Serum-starved cells were incubated for 5 min in the presence or absence of isoproterenol (Iso, 1 µM). Phosphorylation of GFP-ERK2 in whole cell detergent lysates was determined as described (upper immunoblot). Anti-FLAG immunoprecipitates containing FLAG-beta -arrestin 2 were probed for FLAG-beta -arrestin 2 (second immunoblot), coprecipitated GFP-ERK2 (third immunoblot), and phosphorylated GFP-ERK2 (lower immunoblot). D, bar graph depicting the amount of phosphorylated GFP-ERK2 present in FLAG-beta -arrestin 2 immunoprecipitates following stimulation of beta 2AR or beta 2V2R. For the experiments shown in panels B and D, the amount phospho-GFP-ERK2 in the FLAG-beta -arrestin 2 immunoprecipitate was normalized to the amount of GFP-ERK2 present. The response of the chimeric receptors is expressed as a percentage of the beta -arrestin 2-associated phospho-ERK signal observed in cells expressing the corresponding wild type receptor. Data shown represent the mean ± S.E. from four separate experiments. *, greater or less than wild type, p < 0.05.

Fig. 4C depicts the results of an analogous experiment performed with the beta 2AR and beta 2V2R. Unlike the V2R and V2beta 2R, the wild type beta 2AR was a relatively poor activator of ERK2 in COS-7 cells. Substitution of the V2R tail, which causes the beta 2V2R chimera to bind beta -arrestin 2 tightly, produced a receptor capable of eliciting a substantially greater increase in GFP-ERK2 phosphorylation, even when ERK phosphorylation was measured in the whole cell lysate. Associated with this increase in overall GFP-ERK2 phosphorylation, we observed a 2-fold increase in isoproterenol-stimulated phospho-ERK2 bound to beta -arrestin for the beta 2V2R. This result, shown quantitatively in Fig. 4D, suggests that the increase in whole cell ERK phosphorylation seen with the V2beta 2R reflects increased utilization of a beta -arrestin scaffold by the chimeric receptor.

beta -Arrestin Binding Affects the Cellular Distribution of Endogenous Phospho-ERK1/2 following Stimulation of the Wild Type V2R and Chimeric V2beta 2R-- One reported consequence of the formation of beta -arrestin-ERK complexes is the cytosolic retention of beta -arrestin-bound ERK (9, 11, 12). Whereas the preceding data suggest that some fraction of the ERK activated in response to GPCR stimulation is associated with beta -arrestin, and that the stability of the GPCR-beta -arrestin complex affects the efficiency with which beta -arrestin-bound ERK is activated, they do not address whether the beta -arrestin-bound ERK pool represents a large enough fraction of the total cellular pool of GPCR-activated ERK to affect its overall cellular distribution.

To address this question, we first analyzed the distribution of endogenous phospho-ERK1/2 in whole cell lysates and receptor immunoprecipitates following covalent cross-linking with the membrane-permeable reversible cross-linker, dithiobis(succinimidyl propionate). COS-7 cells transiently expressing HA-tagged V2 or V2beta 2 receptors were stimulated for varying times prior to cross-linking and detergent lysis. As shown in Fig. 5A, when these lysates were resolved by SDS-PAGE under nonreducing conditions, which preserve the covalent cross-links, agonist stimulation resulted in the appearance of a heterogenous population of high apparent molecular weight bands that immunoblotted for HA-tagged receptor, beta -arrestin, ERK1/2, and phospho-ERK1/2. When the same samples were resolved under reducing conditions, which break the cross-links, these high molecular weight complexes were absent. As shown in Fig. 5B, when receptor immunoprecipitates from cross-linked lysates were resolved under reducing conditions, we observed agonist-dependent coprecipitation of endogenous beta -arrestin and phospho-ERK with the epitope-tagged receptor. Thus, the migration of receptor, beta -arrestin, and ERK as high molecular weight species in cross-linked whole cell detergent lysates reflected, as least in part, the agonist-induced formation of receptor-beta -arrestin-ERK complexes. To directly compare the effect of beta -arrestin binding on the distribution of V2R-activated endogenous ERK1/2, we measured the fraction of the total phospho-ERK1/2 in cross-linked whole cell lysates that was present in complexes with apparent molecular weight >100,000 following stimulation of V2R and V2beta 2R. As shown in Fig. 5C (upper immunoblot and bar graph), ~75% of the phospho-ERK1/2 generated by the V2R was present in these complexes, compared with ~35% of that generated by the V2beta 2R. Conversely, about 20% of the phospho-ERK1/2 migrated at 42-44 kDa when the V2R was stimulated, compared with 55% for the V2beta 2R. In these experiments, the total amount of phospho-ERK1/2 generated by the two receptors was indistinguishable, as demonstrated when the cross-linked lysates were electrophoresed under reducing conditions (lower immunoblot). As the V2R and V2beta 2R differ only in the stability of beta -arrestin binding (3), these data suggest that stable beta -arrestin binding is associated with the enhanced formation of high molecular weight complexes containing the receptor, beta -arrestin, and ERK, and that these complexes contain a significant fraction of the phospho-ERK generated by V2R.


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Fig. 5.   Analysis of the distribution of endogenous beta -arrestin, ERK1/2, and phospho-ERK1/2 in covalently cross-linked cell lysates following stimulation of wild type V2R and chimeric V2beta 2R. COS-7 cells expressing either HA-V2R or HA-V2beta 2R were stimulated with vasopressin (1 µM) for the indicated times prior to covalent cross-linking with dithiobis(succinimidyl propionate) and preparation of detergent lysates. A, cross-linked detergent lysates from stimulated V2R-expressing cells were resolved by SDS-PAGE under reducing (beta -ME, mercaptoethanol) or nonreducing conditions. Identical immunoblots were probed for HA-epitope (left panel), beta -arrestin (second panel), ERK1/2 (third panel), and phospho-ERK1/2 (right panel) as described. The representative immunoblots shown are from one of six separate experiments. B, HA-V2R was immunoprecipitated from cross-linked detergent lysates and resolved by SDS-PAGE under reducing conditions. Identical immunoblots were probed for HA epitope (left panel), and coprecipitated endogenous beta -arrestin (center panel) and phospho-ERK1/2 (right panel). The representative immunoblots shown are from one of three separate experiments. C, cross-linked detergent lysates from stimulated V2R- and V2beta 2R-expressing cells were resolved by SDS-PAGE under reducing (lower immunoblot) or nonreducing (upper immunoblot) conditions. The percent of the total phospho-ERK1/2 signal present in each stimulated lane that migrated with an apparent molecular weight of 42,000-44,000 or >100,000 was quantified by scanning densitometry. The bar graph compares the distribution of phospho-ERK1/2 between pools of different apparent molecular weight, following V2R and V2beta 2R stimulation. Data shown represent the mean ± S.E. from three to six separate experiments.

To further examine the effect of beta -arrestin binding on the cellular distribution of active ERK1/2, we compared the fraction of total cellular phospho-ERK1/2 that translocated to the nucleus following stimulation of the V2R and V2beta 2R. In these experiments, COS-7 cells transiently expressing HA-tagged V2 or V2beta 2 receptors were stimulated for 5 min prior to isolation of cell nuclei by differential centrifugation. As shown in Fig. 6A, we found that with the V2R less than 10% of the total cellular phospho-ERK1/2 was present within the nucleus 5 min after stimulation, compared with about 25% with the V2beta 2 chimera. Conversely, as shown in Fig. 6B, the amount of phospho-ERK1/2 present in the extranuclear fraction, representing plasma membrane, microsomes, cytoskeleton, and cytosol, was proportionally greater for the V2R than the V2beta 2R. At this time point, the total amount of phospho-ERK1/2 in the V2 and V2beta 2 receptor-expressing cells was indistinguishable. Thus, the data obtained by measuring the fraction of active ERK1/2 in high molecular weight complexes (Fig. 5) and those obtained by measuring the nuclear fraction of phospho-ERK1/2 are complementary, and suggest that stimulation of the wild type V2R generates more phospho-ERK1/2 in large multiprotein complexes and less phospho-ERK1/2 in the nucleus than stimulation of the V2beta 2 receptor.


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Fig. 6.   Effect of exchanging the C terminus of the V2R and beta 2AR on nuclear translocation of activated endogenous ERK1/2 following agonist stimulation. COS-7 cells were transfected with plasmids for either HA-V2R or HA-V2beta 2R. Serum-starved cells were stimulated with vasopressin (1 µM) for 5 min, cell nuclei were isolated as described, and the phospho-ERK1/2 content of the nuclear and extranuclear fractions was determined by immunoblotting. A, phospho-ERK1/2 content of the whole cell lysate collected prior to fractionation (upper immunoblot) and the nuclear fraction (lower immunoblot) following stimulation of V2R and V2beta 2R. B, phospho-ERK1/2 content of whole cell lysate collected prior to fractionation (upper immunoblot) and the extranuclear fraction (lower immunoblot) following stimulation of V2R and V2beta 2R. In the bar graphs, data are expressed as the percentage of the total cellular phospho-ERK1/2 pool present in the nuclear or extranuclear fraction. Data shown represent the mean ± S.E. of four independent experiments. *, greater or less than V2R, p < 0.05.

These data are also consistent with comparisons of the fraction of beta -arrestin-associated phospho-ERK1/2 formed in response to stimulation of the wild type PAR2 receptor and a mutant receptor lacking GRK phosphorylation sites that does not bind beta -arrestin (9). Using a gel filtration approach, these authors found that greater than 80% of the phospho-ERK formed in response to wild type receptor activation coeluted with the receptor, Raf-1, and beta -arrestin, whereas less than 2% of the phospho-ERK coeluted with the mutant receptor. These authors also found that the wild type receptor activated a predominantly non-nuclear pool of ERK, whereas the mutant predominantly activated nuclear phospho-ERK. The presence of a nuclear export signal in beta -arrestin 2, which was recently shown to account for the nuclear exclusion of beta -arrestin 2-bound JNK3 in cells overexpressing beta -arrestin 2 (15), may account for the effect of beta -arrestin binding on ERK distribution.

The Transcriptional Activity of GPCR-activated ERK1/2 Is Regulated by the Stability of the Receptor-beta -Arrestin Interaction-- The preceding data demonstrate that the ability of a GPCR to bind stably to beta -arrestin correlates with increased activation of beta -arrestin-bound of ERK1/2, and decreased nuclear translocation of phospho-ERK1/2. To determine whether the functional consequences of ERK activation are affected by the stability of the receptor-beta -arrestin interaction at endogenous levels of beta -arrestin and ERK1/2 expression, we employed stably transfected HEK-293 cell lines expressing the V2R and V2beta 2R. These cell lines exhibit comparable levels of cAMP production in response to vasopressin stimulation, but differ in the stability of the receptor-beta -arrestin interaction and the pattern of receptor internalization, dephosphorylation, and recycling.

Fig. 7A depicts the time course of vasopressin-stimulated ERK1/2 phosphorylation over 4 h in the V2R- and V2beta 2R-expressing HEK-293 cells. At short time points, up to ~10 min, the responses were indistinguishable. Although the overall level of ERK1/2 phosphorylation declined dramatically after 30 min for both receptors, the level of persistent ERK1/2 phosphorylation was modestly, but significantly, greater for the V2beta 2R than the V2R. Previous work has shown replacement of the V2R tail with that of the beta 2AR increases the rate of receptor dephosphorylation and recycling after endocytosis (3). Thus, this persistent signal may represent the effect of beta -arrestin binding on the rate of receptor recycling and the steady state level of undesensitized receptors on the plasma membrane in the continuous presence of agonist. Fig. 7B compares the ability of V2R and V2beta 2R to induce transcription of an Elk1-driven luciferase reporter. This response is dependent on activation of endogenous ERK1/2, because it is completely eliminated by incubation with the MEK inhibitor, PD98059 (data not shown). As shown, the chimeric receptor produced a transcriptional response that was ~8-fold greater than the wild type receptor. The response to stimulation of endogenous epidermal growth factor (EGF) receptors was comparable between the two cell lines.


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Fig. 7.   Effect of exchanging the C terminus of the V2R and beta 2AR on the time course of vasopressin-induced phosphorylation of endogenous ERK1/2 and induction of an Elk1-luciferase reporter. A, HEK-293 cells stably expressing either V2R (upper immunoblot) or V2beta 2R (lower immunoblot) were stimulated for the indicated times with vasopressin (1 µM) and whole cell phospho-ERK1/2 was determined by immunoblotting. The graph represents the mean ± S.E. of five separate experiments. B, HEK-293 cells stably expressing either V2R or V2beta 2R were transfected with plasmids for pFR-luciferase, GAL-4-Elk-1, and TK-Renilla as described. Luciferase activity was determined after 4 h incubation of serum-starved cells in the presence or absence (NS) of vasopressin (1 µM) or EGF (10 ng/ml). Luciferase activity is expressed as the -fold increase relative to unstimulated V2R cells and represents mean ± S.E. from three separate experiments. *, greater than V2R, p < 0.05.

Compared with the V2R, activation of V2beta 2R resulted in greater nuclear translocation of activated ERK1/2 (Fig. 6), more persistent ERK1/2 activation, and enhanced ERK1/2-dependent transcription (Fig. 7). Because nuclear translocation of ERK1/2 is required for growth factor-stimulated mitogenesis (16), we compared the ability of the V2R and V2beta 2R to stimulate DNA synthesis in the two cell lines. As shown in Fig. 8, the V2R, despite its ability to mediate robust activation of ERK1/2, failed to elicit a detectable increase in [3H]thymidine incorporation into DNA. In contrast, the V2beta 2R was weakly, but significantly, mitogenic, eliciting a response comparable with refeeding with 2% fetal calf serum. Because these two receptors differ only in their C termini, these data suggest that the stability of the receptor-beta -arrestin interaction influences the mitogenic potential of the V2R. It is likely that this effect is because of both binding of ERK1/2 to beta -arrestin leading to its retention in the cytosol, and to beta -arrestin-dependent removal of receptors from the cell surface leading to a diminished steady state level of ERK activation.


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Fig. 8.   Effect of exchanging the C terminus of the V2R and beta 2AR on vasopressin-stimulated DNA synthesis. Serum-starved HEK-293 cells stably expressing either V2R or V2beta 2R were incubated for 24 h in the presence or absence (NS) of vasopressin (1 µM) or fetal bovine serum (FBS, 2% v/v) prior to the determination of [3H]thymidine incorporation into DNA. Results are expressed as the -fold increase relative to unstimulated cells. Experiments were performed in duplicate and the data shown represent mean ± S.E. from three independent experiments. For reference, refeeding with medium containing 10% fetal calf serum resulted in a 10-12-fold increase in [3H]thymidine incorporation. *, greater than NS, p < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The binding of beta -arrestins to agonist-occupied GPCRs serves two broad functions. By uncoupling the receptor from heterotrimeric G proteins and targeting it for endocytosis, beta -arrestin binding is central to the processes of GPCR desensitization and sequestration that lead to rapid termination of G protein-dependent signals. At the same time, beta -arrestins function as adapter proteins, bringing various signaling molecules into complex with the receptor and initiating additional beta -arrestin-dependent signaling events. The finding that beta -arrestins can interact directly with potential enzymatic effectors such as Src family tyrosine kinases (8, 17-19) and ubiquitin ligases (20), as well as components of the ERK1/2 (8, 9, 11, 12) and JNK3 (10, 15, 21) mitogen-activated protein kinase modules, suggests that beta -arrestins may serve in a variety of signaling roles.

Activation of the ERK cascade by heptahelical receptors is a complex process that can simultaneously involve multiple mechanisms, and in which the predominant mechanism varies depending on both the GPCR and the cellular context in which the receptor is expressed (22, 23). For example, in S49 lymphoma cells, beta 2AR-mediated ERK1/2 activation occurs via a Gs-, adenylyl cyclase-, and PKA-dependent pathway that causes activation of the small GTPase, Rap1 (24, 25), whereas in fibroblasts, cardiomyocytes, and pancreatic acinar cells ERK1/2 activation is achieved largely through a Ras-dependent mechanism involving activation of pertussis toxin-sensitive G proteins and "transactivation" of EGF receptors (26-29). Other receptors, notably the AT1aR, and the PAR2 and neurokinin NK-1 receptors, appear to utilize beta -arrestins as scaffolds to a significant extent (8, 9, 11, 12), although AT1aRs are also clearly capable of mediating ERK activation through cross-talk with EGF or platelet-derived growth factor receptors (30-32). We have previously shown that overexpression of beta -arrestins in COS-7 cells enhances AT1aR-mediated ERK1/2 activation, but leads to cytosolic retention of ERK and a diminished transcriptional response (11, 12).

Given their ability both to dampen receptor-G protein coupling and to act as scaffolds for ERK activation, beta -arrestins are uniquely positioned to influence the balance between these different mechanisms. Furthermore, because the mechanism used to activate ERK influences its spatial distribution and transcriptional activity, the binding of beta -arrestin and receptor might dictate the cellular response to ERK activation. In a number of systems, the proliferative response to GPCR stimulation involves cross-talk between GPCRs and EGF receptors (31, 33). In contrast, beta -arrestin-dependent ERK activation does not appear to lead to proliferative signaling. Wild type PAR2 receptors, which mediate beta -arrestin-dependent activation of a predominantly cytosolic pool of ERK1/2 in KNRK cells, do not stimulate [3H]thymidine incorporation or cell replication (9). As we have shown, altering the stability of the V2R receptor-beta -arrestin interaction by replacing the C-terminal tail of the V2R with that of the beta 2AR was sufficient to confer mitogenic potential on a receptor that, in this system, did not detectably stimulate [3H]thymidine incorporation (Fig. 8).

Fig. 9 depicts this conceptual role of beta -arrestins in regulating the mechanism and functional consequences of GPCR-stimulated ERK activation. Activation of ERK1/2 via classical G protein-dependent mechanisms, potentially including transactivation of receptor tyrosine kinases, leads to nuclear translocation of ERK1/2, activation of ERK1/2-dependent transcription and a mitogenic response. In contrast, activation of ERK1/2 in a beta -arrestin-bound pool leads to localized ERK1/2 activity, with less nuclear phospho-ERK1/2 and little or no mitogenic potential. Significantly, beta -arrestin binding to the receptor not only confers the ability to activate beta -arrestin-bound ERK1/2, it also attenuates signaling via G protein-dependent pathways and determines the rate of receptor recycling. As we have shown, altering the stability of the receptor-beta -arrestin interaction affects not only the efficiency of GPCR-mediated activation of a beta -arrestin-bound ERK pool, but also the balance between the nuclear and cytosolic phospho-ERK1/2 and the steady-state level of ERK1/2 activity during prolonged stimulation. This shift undoubtedly reflects the role of beta -arrestins in receptor desensitization, sequestration, and recycling as much as it does the enhanced utilization of beta -arrestin scaffolds by receptors that bind stably to beta -arrestin.


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Fig. 9.   Proposed model of the effect of beta -arrestin binding on ERK1/2 activation and function. The binding of agonist to a GPCR initially leads to ERK activation via G protein-dependent pathways. The binding of beta -arrestin simultaneously inhibits G protein-dependent ERK1/2 activation, by inducing homologous receptor desensitization and sequestration, and initiates the activation of a beta -arrestin-bound pool of phospho-ERK1/2. For GPCRs that form stable receptor-beta -arrestin complexes, activation of the beta -arrestin-dependent pathway is more pronounced, leading to the formation of a functionally distinct pool of phospho-ERK1/2.

The extent to which "G protein-dependent" and "beta -arrestin-dependent" ERK activation are genuinely independent processes is unclear. In murine fibroblasts derived from beta -arrestin 1/beta -arrestin 2 homozygous null embryos, lysophosphatidic acid-stimulated ERK1/2 activation is sensitive to inhibitors of the EGF receptor tyrosine kinase, suggesting that GPCR-stimulated EGF receptor transactivation can occur in a beta -arrestin null background.2 On the other hand, beta -arrestin recruitment requires GRK-mediated receptor phosphorylation, and receptor phosphorylation by GRK2 or GRK3 requires Gbeta gamma subunit-mediated membrane translocation of the kinase. Thus, one might expect that beta -arrestin-dependent ERK activation would not be G protein-independent. Rather, it would represent a shift in the mechanism of ERK activation coincident with homologous desensitization that leads to the localized activation of a functionally distinct pool of ERK1/2. Interestingly, however, Seta et al. (34) have recently reported that in Chinese hamster ovary cells, a mutant AT1aR that is markedly impaired in G protein coupling is still fully competent to induce ERK1/2 activation, but that ERK activated by the mutant receptor is retained in the cytosol and is transcriptionally inactive (34). While these authors do not implicate beta -arrestins in the putatively "G protein-independent" activation of ERK, their data do suggest the existence of one or more mechanisms of ERK activation that do not require G protein activation, and that, like the beta -arrestin-dependent activation of ERK, leads to localized ERK activation outside the of the cell nucleus.

Our data demonstrate the significant impact of the receptor-beta -arrestin interaction on the mechanism and functional consequences of ERK activation. The activation of ERK bound to beta -arrestin, which is favored in the setting of a stable receptor-beta -arrestin interaction, limits nuclear translocation of ERK and attenuates Elk-1-driven transcription. However, any additional functions of beta -arrestin-bound ERK1/2 remain to be discovered. ERK1/2 are known to phosphorylate multiple plasma membrane, cytoplasmic, and cytoskeletal substrates (16), including several proteins involved in heptahelical receptor signaling, such as beta -arrestin 1 (35), GRK2 (36, 37), and the Galpha -interacting protein GAIP (38). Thus, one role of beta -arrestin-ERK complexes could be to target ERK1/2 to substrates involved in the regulation of GPCR signaling or intracellular trafficking. Alternatively, beta -arrestin-bound ERK1/2 may phosphorylate other cytosolic kinases involved in transcriptional regulation, such as p90 RSK (39), which in turn relay signals to the nucleus. In such a model, transcriptional events mediated directly by the nuclear pool of ERK1/2 would be attenuated, whereas alternate pathways of ERK-dependent transcription would persist, resulting in an altered pattern of GPCR-stimulated transcription.

    ACKNOWLEDGEMENTS

We thank Donna Addison and Julie Turnbough for excellent secretarial assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DK55524 (to L. M. L.), HL16037 (to R. J. L.), and NS19576 (to M. G. C.).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.

** Investigators with the Howard Hughes Medical Institute.

§§  To whom correspondence should be addressed: N3019 GRECC, Durham Veterans Affairs Medical Center, 508 Fulton St., Durham, NC 27705. Tel.: 919-286-0411 (ext. 7196); Fax: 919-416-5823; E-mail: luttrell@receptor-biol.duke.edu.

Published, JBC Papers in Press, December 6, 2002, DOI 10.1074/jbc.M212231200

2 T. Kohout and R. J. Lefkowitz, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; AR, adrenergic receptor; AT1aR, angiotensin II type 1a receptor; ERK1/2, extracellular signal-regulated kinases 1 and 2; G protein, heterotrimeric guanine nucleotide-binding protein; GFP, green fluorescent protein; GRK, G protein-coupled receptor kinase; HA, influenza virus hemagglutinin; JNK3, c-Jun N-terminal kinase 3; PAR2, protease-activated receptor type 2; V2R, vasopressin receptor type 2; EGF, epidermal growth factor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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