Cellular Trafficking of G Protein-coupled Receptor/beta -Arrestin Endocytic Complexes*

Jie ZhangDagger , Larry S. BarakDagger , Pieter H. Anborgh§, Stephane A. LaporteDagger parallel , Marc G. CaronDagger **, and Stephen S. G. Ferguson§Dagger Dagger

From the Dagger  Howard Hughes Medical Institute Laboratories, Departments of Cell Biology and Medicine, Duke University Medical Center, Durham, North Carolina 27710 and the § John P. Robarts Research Institute, Departments of Physiology and Pharmacology and Toxicology, University of Western Ontario, P.O. Box 5015, 100 Perth Drive, London, Ontario N6A 5K8, Canada

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

beta -Arrestins are multifunctional proteins identified on the basis of their ability to bind and uncouple G protein-coupled receptors (GPCR) from heterotrimeric G proteins. In addition, beta -arrestins play a central role in mediating GPCR endocytosis, a key regulatory step in receptor resensitization. In this study, we visualize the intracellular trafficking of beta -arrestin2 in response to activation of several distinct GPCRs including the beta 2-adrenergic receptor (beta 2AR), angiotensin II type 1A receptor (AT1AR), dopamine D1A receptor (D1AR), endothelin type A receptor (ETAR), and neurotensin receptor (NTR). Our results reveal that in response to beta 2AR activation, beta -arrestin2 translocation to the plasma membrane shares the same pharmacological profile as described for receptor activation and sequestration, consistent with a role for beta -arrestin as the agonist-driven switch initiating receptor endocytosis. Whereas redistributed beta -arrestins are confined to the periphery of cells and do not traffic along with activated beta 2AR, D1AR, and ETAR in endocytic vesicles, activation of AT1AR and NTR triggers a clear time-dependent redistribution of beta -arrestins to intracellular vesicular compartments where they colocalize with internalized receptors. Activation of a chimeric AT1AR with the beta 2AR carboxyl-terminal tail results in a beta -arrestin membrane localization pattern similar to that observed in response to beta 2AR activation. In contrast, the corresponding chimeric beta 2AR with the AT1AR carboxyl-terminal tail gains the ability to translocate beta -arrestin to intracellular vesicles. These results demonstrate that the cellular trafficking of beta -arrestin proteins is differentially regulated by the activation of distinct GPCRs. Furthermore, they suggest that the carboxyl-tail of the receptors might be involved in determining the stability of receptor/beta arrestin complexes and cellular distribution of beta -arrestins.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Signal transduction via G protein-coupled receptors (GPCRs)1 is intimately associated with a wide variety of biological processes including neurotransmission, chemoattraction, cardiac function, olfaction, and vision. beta -Arrestin proteins play an important role in regulating the responsiveness of GPCRs by contributing to mechanisms involved in both GPCR desensitization and resensitization (1-5). beta -Arrestins regulate GPCR desensitization by binding and uncoupling the receptors from heterotrimeric G proteins once they have been phosphorylated by G protein-coupled receptor kinases (GRKs) (1, 3). In addition, they are also required for the sequestration (endocytosis) of a growing number of GPCRs, including the CCR-5, follitropin receptor, lutropin/choriogonadotropin receptor, m2 muscarinic acetylcholine receptor, mu opioid receptor, substance P receptor, and the beta 2-adrenergic receptor (beta 2AR) (4, 6-11). At least in the case of the beta 2AR, the agonist-dependent sequestration of the receptor to an endosomal compartment not only promotes receptor dephosphorylation but is essential for the re-establishment of normal receptor responsiveness (5, 12-15).

Recent studies suggest that beta -arrestins participate in GPCR sequestration by directing receptors to clathrin-coated vesicles (4, 16, 17). beta -Arrestins have been shown to undergo redistribution in response to receptor activation both in live cells and following the fixation of cells, and to co-localize with clathrin (7, 11, 18, 19). However, while the phenomenon of beta -arrestin cellular trafficking is potentially important for understanding mechanisms underlying GPCR internalization and resensitization, the detailed pharmacology of the receptor-mediated beta -arrestin redistribution has never been characterized. As a consequence, it is not clear whether the pharmacological profile of beta -arrestin translocation recapitulates the pharmacology described for GPCR endocytosis. Moreover, the cellular fate of beta -arrestin proteins following association with various GPCRs also remains unknown, as well as where and when beta -arrestins dissociate from each receptor. In the case of rhodopsin, it was demonstrated that the interaction of visual arrestin with rhodopsin prevented the dephosphorylation and resensitization of the receptor (20, 21). Therefore, it is likely that the dissociation of the beta -arrestin/receptor complex contributes to the regulation of responsiveness for other GPCRs.

In the present study, we used a green fluorescent protein conjugate of beta -arrestin2 (beta arr2GFP) (18) to examine the cellular trafficking of beta -arrestin upon stimulation of several distinct GPCRs. Our data demonstrate that the pharmacology of beta -arrestin2 translocation in living cells could account for the agonist dependence of beta 2AR sequestration. Moreover, beta -arrestin2 was observed to redistribute to distinct subcellular locations in response to activation of different GPCRs. This differential redistribution of beta -arrestins likely involves the function of the carboxyl-terminal region of the receptors.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- Human embryonic kidney (HEK 293) cells were provided by the American Type Culture Collection (ATCC). Tissue culture media and fetal bovine serum were obtained from Life Technologies, Inc. Isoproterenol and dopamine were purchased from Research Biochemicals International. Endothelin and neurotensin were from Peninsula Laboratories, and angiotensin II was from Sigma. Rabbit anti-HA polyclonal antibody and mouse anti-HA 12CA5 monoclonal antibody were obtained from Babco and Boehringer Mannheim, respectively. Fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody was purchased from Sigma, and rhodamine-conjugated goat anti-rabbit Fabs was obtained commercially from Organon Teknika. [125I]Cyanopindolol was purchased from NEN Life Science Products.

DNA Construction-- All recombinant DNA procedures were carried out following standard protocols. beta -Arrestin1 with GFP conjugated to its COOH terminus (beta arr1GFP) was constructed in a manner similar to beta arr2GFP (18) by replacing the terminal stop codon of beta -arrestin1 with a SalI restriction site and inserting the modified cDNA in frame into the polylinker of p(S65T)GFP-N3 (CLONTECH) (18). pcDNA3-AT1A R-beta 2AR-CT and pcDNA1-Amp-beta 2AR-AT1AR-CT were constructed by polymerase chain reaction. The chimeric AT1AR with the beta 2AR carboxyl-terminal tail (AT1AR-beta 2AR-CT) contains the first 302 amino acids (Met1-Tyr302) of the AT1AR fused to the last 87 amino acids (Cys327-Leu413) of the beta 2AR. The chimeric beta 2AR with the AT1AR carboxyl-terminal tail (beta 2AR-AT1AR-CT) includes the first 348 amino acids (Met1-Lys348) of the beta 2AR fused to the last 38 amino acids (Ala324-Glu359) of the AT1AR. The sequences of the DNA constructs were confirmed by DNA sequencing.

Cell Culture and Transfection-- HEK 293 cells were grown in Eagle's minimal essential medium with Earle's salt (MEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum and gentamicin (100 µg/ml). The cells were seeded at a density of 2.5 × 106 cells/100-mm dish and transiently transfected with the cDNAs described in the figure legends by a modified calcium phosphate method (22). Following transfection (~18 h), the cells were incubated with fresh medium and allowed to recover 7-9 h before being reseeded into 35-mm glass-bottomed culture dishes (MatTek) or into six-well dishes (Falcon) containing or not containing 22-mm square glass coverslips coated with collagen (Sigma). The use of HEK 293 cells expressing beta 2AR-GFP was described previously (23).

Receptor Expression-- beta 2AR expression was measured by saturating [125I]cyanopindolol binding done at 30 °C for 60 min (24). The expression of other receptors was measured by flow cytometry and normalized according to the beta 2AR expression measured at the same time by both flow cytometry and saturating binding (16). The receptor expression levels was between 2000 and 4000 fmol/mg of whole cell protein for the experiments assessing beta -arrestin translocation with confocal microscope (see below) and between 1000 and 2000 fmol/mg of whole cell protein for all other experiments.

Confocal Microscopy-- Confocal microscopy was performed on a Zeiss LSM-410 laser scanning microscope using either Zeiss 40 × 1.3 or Zeiss 63 × 1.4 numerical aperture oil immersion lenses. For characterizing the pharmacology of beta -arrestin translocation in living cells, HEK 293 cells expressing beta 2AR and low levels of beta arr2GFP were plated on 35-mm glass-bottomed culture dishes and kept warm at 30 °C in serum-free MEM on a heated microscope stage. beta arr2GFP fluorescent signals were collected sequentially using the Zeiss LSM software time scan function in the photon counting mode using single line excitation (488 nm). Drugs were applied to the cells either prior to or during the scanning of beta arr2GFP labeled cells. For studying beta -arrestin trafficking in response to activation of other receptors, HEK 293 cells expressing the receptor of interest and low levels of beta arr2GFP were seeded on 22-mm square glass coverslips, stimulated with saturating concentrations of drugs at 30 °C for 1 h or as indicated, and then fixed with 3.7% paraformaldehyde in phosphate-buffered saline. beta arr2GFP fluorescent signals were collected using single line excitation (488 nm). Colocalization studies of beta arr2GFP and rhodamine-labeled receptor fluorescence were performed using dual excitation (488, 568 nm) and emission (515-540 nm, GFP; 590-610 nm, rhodamine) filter sets. Specificity of labeling and absence of signal cross-over were established by examination of single-labeled samples.

Immunofluorescent Labeling-- For performing colocalization studies of beta arr2GFP and rhodamine-labeled beta 2AR in live cells, HEK 293 cells expressing HA epitope-tagged beta 2AR and beta arr2GFP grown on 35-mm glass-bottomed culture dishes were incubated in serum-free MEM containing anti-HA polyclonal antibody at 37 °C for 30 min. Cells were washed three times with ice-cold MEM and incubated for 30 min on ice in the presence of rhodamine-conjugated goat anti-rabbit Fabs. Cells were washed an additional three times with serum-free MEM at 30 °C and imaged by confocal microscopy as described above. For studying colocalization of beta arr2GFP and AT1AR, HEK 293 cells expressing HA epitope-tagged AT1AR were grown on 22-mm square glass coverslips and incubated in serum-free MEM containing rhodamine-conjugated anti-HA 12CA5 monoclonal antibody on ice for 45 min. Cells were then washed, stimulated with a saturating concentration (500 nM) of angiotensin II for 1 h, washed again, fixed with 3.7% paraformaldehyde in phosphate-buffered saline, and imaged by confocal microscopy as described above.

Data Analysis-- The changes in beta arr2GFP distribution to the plasma membrane were analyzed with IP Labs software. The magnitude of beta arr2GFP fluorescence at the plasma membrane were determined by integration of the fluorescence signal along the cell perimeter. The relative magnitude of beta arr2GFP distribution along a linear slice of the cell was quantitated by the line scan function provided with the Zeiss LSM 410 image analysis software. beta arr2GFP translocation time course and dose-response curves were analyzed using GraphPad Prism. All data points represent the mean ± S.D.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

As described previously (18), in the absence of receptor activation beta arr2GFP fluorescence was evenly distributed throughout the cytoplasm and exhibited no apparent enhanced plasma membrane localization (Fig. 1, A and C; control). However, upon agonist-activation of the beta 2AR, a time-dependent rapid redistribution of beta arr2GFP to the plasma membrane occurred (Fig. 1, A and B). The time course of beta -arrestin translocation determined here, t1/2 = 2.3 min (Fig. 1B), followed beta 2AR phosphorylation (t1/2 = 15-40 s) (24, 25) and preceded beta 2AR internalization (t1/2 = 10 min) (4). At first beta arr2GFP appeared diffusely at the plasma membrane, but with time a punctate pattern became apparent. Moreover, the redistribution of beta arr2GFP from the cytosol to the plasma membrane was agonist dose-dependent (Fig. 1, C and D). The half-maximal effective concentration (EC50) of agonist was calculated to be 6 nM (Fig. 1D), a value comparable to that reported for beta 2AR sequestration in HEK 293 cells (11 nM) (24). No significant beta arr2GFP translocation in response to agonist exposure was observed in cells lacking overexpressed beta 2AR (data not shown). To further test the agonist specificity of beta arr2GFP translocation, cells were treated 5 min with 1 µM isoproterenol to induce beta arr2GFP translocation and then exposed to a saturating concentration of the antagonist propranolol in the presence of the agonist (Fig. 1E). Following the treatment of cells with the antagonist, the distribution of beta arr2GFP fluorescence reversed, with beta arr2GFP redistributing over time from the plasma membrane back to the cytoplasm. However, the redistribution of beta arr2GFP back into the cytoplasm was not immediate, but proceeded over a time course of 5-10 min consistent with previous reports describing agonist-dependent and independent steps of receptor internalization (26). Since the pharmacology of beta arr2GFP translocation accurately reflected the agonist dependence of beta 2AR sequestration, these results suggest that beta -arrestin translocation and receptor binding serve as the agonist-dependent switch triggering endocytosis of the beta 2AR.


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Fig. 1.   Pharmacology and agonist dependence of beta arr2GFP translocation in response to beta 2AR activation in HEK 293 cells. Visualization (A) and quantitation (B) of the time course for beta arr2GFP membrane translocation in HEK 293 cells expressing beta 2AR and beta arr2GFP in response to stimulation with 25 µM isoproterenol for 0-15 min. Shown are representative confocal microscopic images of beta arr2GFP fluorescence obtained prior to (control) and 90 s, 3 min, and 10 min following the addition of agonist to the medium. Visualization (C) and quantitation (D) of the agonist dose-dependent membrane translocation of beta arr2GFP in HEK 293 cells in response to 5-min exposures to increasing concentration of isoproterenol 10-10 to 10-5 M. Shown are representative confocal microscopic images of beta arr2GFP fluorescence in HEK 293 cells obtained prior to (control) and following the addition of 10-10 M, 10-8 M, and 10-6 M isoproterenol. (E) Effect of treating cells with the antagonist propranolol on the localization of beta arr2GFP redistributed to the plasma membrane in response to receptor activation. Shown are representative confocal microscopic images of beta arr2GFP fluorescence in HEK 293 cells prior to (control) treatment for 5 min with 1 µM isoproterenol (agonist), following which antagonist (300 µM propranolol) was added to the agonist containing medium to compete for receptor binding sites containing medium and time scanned for an additional 10 min (agonist + antagonist). All cells were transfected with 1 µg of pGFP-N3/beta arr2 and 5 µg of 12CA5 epitope-tagged beta 2AR in pcDNA1-Amp, and experiments were performed independently on 4-8 different cells. Experiments were performed on a heated microscope stage set at 30 °C. Data points represent the mean ± S.D. of 8 (B) and 5 (D) different cells from separate transfections. Increased membrane localized fluorescence was quantitated using IPLab spectrum image analysis software (Signal Analytics Corp.). The inset bars represent 10 µm.

While beta arr2GFP was observed to translocate to the plasma membrane and cluster at coated pits (18), beta arr2GFP labeling of intracellular endocytic vesicles following beta 2AR activation was never observed. The overall distribution pattern of beta arr2GFP appeared different from that of a GFP-conjugated beta 2AR (23) (Fig. 2, A and B). In response to agonist stimulation, the beta 2AR-GFP redistributed from a diffuse plasma membrane localization to a membrane-associated vesiculated pattern, followed by the appearance of beta 2AR-GFP in endocytic vesicles randomly distributed throughout the cytosol of the cell (Fig. 2B). Therefore, we examined the agonist-induced intracellular trafficking of both the beta 2AR and beta arr2GFP in the same living cells. To do this, beta 2ARs engineered with an amino-terminal HA epitope tag were expressed in HEK 293 cells with beta arr2GFP and labeled with 12CA5 monoclonal antibodies, which were themselves labeled with rhodamine-conjugated anti- Fabs. beta 2ARs labeled in this manner were still able to respond normally to agonist activation (Fig. 2C). In the absence of agonist, beta 2AR immunofluorescence (red) was localized solely to the plasma membrane, whereas beta arr2GFP fluorescence (green) was limited to the cytoplasm (Fig. 2C). In response to agonist activation of beta 2ARs, beta arr2GFP translocated to the receptors at the plasma membrane. This was followed by the redistribution of both the receptors and beta -arrestin to clathrin-coated pits, as denoted by the appearance of yellow hot spots (Fig. 2C). However, while yellow vesicles could be observed close to the membrane surface, no colocalization of beta arr2GFP with beta 2AR-bearing vesicles was ever observed in the cytoplasm of the cell (Fig. 2C). A similar agonist-mediated redistribution of beta arr1GFP to plasma membrane-localized beta 2AR, but not beta 2AR localized in endocytic vesicles, was also observed (data not shown). These results demonstrate that beta 2AR/beta -arrestin complex dissociates at or close to the plasma membrane, and therefore beta -arrestins are excluded from endocytic vesicles shortly following their formation.


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Fig. 2.   Redistribution and colocalization of the beta 2AR and beta arr2GFP following agonist stimulation. A, visualization of the redistribution of beta arr2GFP membrane fluorescence with time in response to the activation of the beta 2AR with 25 µM isoproterenol. HEK 293 cells were transfected with 1 µg of pGFP-N3/beta arr2 and 5 µg of 12CA5 epitope-tagged beta 2AR in pcDNA1-Amp. Shown are representative confocal microscopic images of beta arr2GFP fluorescence in the same HEK 293 cells exposed to agonist for the times indicated. B, visualization of the redistribution of beta 2AR-GFP membrane fluorescence with time in response to the activation of the beta 2AR. HEK 293 cells were permanently transfected to express the 12CA5 epitope-tagged beta 2AR-GFP construct (1.6 pmol/mg of protein) (23). Shown are representative confocal microscopic images of beta 2AR-GFP fluorescence obtained prior (control) to the exposure of the same field of cells to 10 µM isoproterenol for 5 min and 20 min. C, confocal visualization of the intracellular distribution and colocalization (yellow) of beta arr2GFP (green) with 12CA5 epitope-tagged beta 2ARs (red) labeled at 37 °C with 12CA5 monoclonal antibody followed by labeling with rhodamine-conjugated anti-rabbit Fabs at 4 °C in HEK 293 cells. Shown are representative confocal microscopic images of beta 2AR and beta arr2GFP distribution prior to (control) and following the beta 2AR activation with 25 µM isoproterenol at 30 °C for 5 and 20 min. Cells were transfected with 1 µg of pGFP-N3/beta arr2 and 5 µg of 12CA5 epitope-tagged beta 2AR in pcDNA1-Amp. All experiments were performed at 30 °C on three to six different occasions. The inset bars represent 10 µm.

In a previous study, we have demonstrated that although the AT1AR can utilize a distinct endocytic mechanism, overexpression of exogenous beta -arrestins mobilizes the receptor for internalization via clathrin-mediated endocytosis similar to that utilized by the beta 2AR (16). To further characterize the interaction of beta -arrestin with the AT1AR, receptor-mediated beta arr2GFP trafficking was examined in HEK 293 cells co-expressing the AT1AR and beta arr2GFP. The cells were stimulated with angiotensin II for various periods of time at 30 °C and observed under confocal microscope. Similar to that observed with the beta 2AR, beta arr2GFP was evenly distributed throughout the cytoplasm in the absence of agonist, but underwent a rapid translocation to the plasma membrane in response to AT1AR activation (Fig. 3A). However, activation of the AT1AR for a longer period of time (>4 min) resulted in a clear redistribution of beta arr2GFP to intracellular endocytic vesicles. beta arr1GFP was also observed to undergo a similar redistribution to AT1AR-containing endocytic vesicles.2 With time, the beta arr2GFP-containing vesicular structures grew in size and were mobilized to cluster at the perinuclear region of the cells (Fig. 3A). In contrast, under parallel conditions, beta arr2GFP remained confined to the plasma membrane even when the beta 2AR was activated by isoproterenol for 1 h (Fig. 3B). To further confirm that the redistribution of beta -arrestins is receptor-driven, we examined the localization of agonist-activated AT1AR and beta arr2GFP in the same HEK 293 cells. To do this, AT1ARs engineered with an amino-terminal HA epitope tag were expressed in HEK 293 cells with beta arr2GFP and labeled with rhodamine-conjugated anti-HA 12CA5 monoclonal antibodies. When the cells were activated by angiotensin II, an agonist-dependent colocalization of AT1AR red immunofluorescence and beta arr2GFP green fluorescence was observed and persisted for up to 1 h, as reflected by the predominant intracellular yellow vesicular structures located at the perinuclear region (Fig. 4). These data demonstrate a functional interaction between the AT1AR and beta -arrestin proteins. In addition, our results also indicate that, unlike the beta 2AR/beta -arrestin complex, the AT1AR/beta -arrestin complex remains intact and is mobilized to the interior of the cell driven by receptor internalization.


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Fig. 3.   Differential cellular trafficking of beta arr2GFP in response to activation of the AT1AR and beta 2AR. Visualization of the redistribution of beta arr2GFP fluorescence with time in response to the activation of the AT1AR with 500 nM angiotensin II (A) or the beta 2AR with 25 µM isoproterenol (B) for 0-60 min. HEK 293 cells were transfected with 1 µg of pGFP-N3/beta arr2 and 5 µg of 12CA5 epitope-tagged AT1AR or beta 2AR in pcDNA1-Amp. Shown are representative confocal microscopic images of beta arr2GFP fluorescence in fixed HEK 293 cells exposed to agonist for the times indicated. All experiments were performed at 30 °C on three to five different occasions.


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Fig. 4.   Colocalization of the AT1AR and beta arr2GFP following stimulation by angiotensin II. Confocal visualization of the intracellular distribution and colocalization (overlay) of beta arr2GFP (green) with 12CA5 epitope-tagged AT1ARs (red) labeled at 4 °C with rhodamine-conjugated 12CA5 monoclonal antibody in HEK 293 cells. Cells were transfected with 1 µg of pGFP-N3/beta arr2 and 5 µg of 12CA5 epitope-tagged AT1AR in pcDNA1-Amp. Shown are representative confocal microscopic images of AT1AR and beta arr2GFP distribution following AT1AR activation with 500 nM angiotensin II at 30 °C for 1 h. The experiment was performed on two different occasions.

Since the activation of beta 2AR and AT1AR promoted beta -arrestin trafficking to distinct subcellular locations, we examined the redistribution of beta arr2GFP in response to activation of several other GPCRs to address the generality of the two different beta -arrestin translocation patterns. To do this, beta arr2GFP was co-expressed in HEK 293 cells with different GPCRs including dopamine D1A receptor (D1AR), endothelin type A receptor (ETAR) and neurotensin receptor (NTR). As shown in Fig. 5, in response to activation of the D1AR and the ETAR, beta arr2GFP underwent a rapid membrane translocation and remained in a punctate pattern at the periphery of the cells for as long as 1 h, similar to that observed following beta 2AR stimulation. In contrast, in HEK 293 cells overexpressing the NTR, neurotensin stimulation resulted in the redistribution of beta arr2GFP fluorescence to intracellular vesicular structures with a pattern reminiscent of that observed following AT1AR activation. Therefore, different GPCRs either separate from beta -arrestins at the level of plasma membrane or internalize with beta -arrestin in intracellular vesicles. This property appears independent of both the types of G protein-coupling and agonists.


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Fig. 5.   Translocation of beta arr2GFP in response to activation of the D1AR, ETAR, and NTR. Visualization of the redistribution of beta arr2GFP fluorescence in response to activation of D1AR with 10 µM dopamine, ETAR with 100 nM endothelin, or NTR with 1 µM neurotensin. HEK 293 cells were transfected with 1 µg of pGFP-N3/beta arr2 and 5 µg of 12CA5 epitope-tagged D1AR in pcDNA1-Amp, ETAR in pcDNA1, or NTR in pcDNA3. Shown are representative confocal microscopic images of beta arr2GFP fluorescence in fixed HEK 293 cells exposed to agonist for 60 min. All experiments were performed at 30 °C on three different occasions.

Previous studies of GPCR internalization have suggested that the carboxyl-terminal region is important for receptor interaction with beta -arrestins (4). Therefore, to examine whether the carboxyl-terminal tail of the receptors contributes to the differential trafficking of beta -arrestin, we engineered chimeric mutants of the AT1AR and beta 2AR with their carboxyl-terminal region exchanged for that of the other. Like the wild-type AT1AR and beta 2AR, the chimeric AT1AR and beta 2AR mutants (namely AT1AR-beta 2AR-CT and beta 2AR-AT1AR-CT) underwent rapid internalization in response to agonist stimulation (Fig. 6B). However, when co-expressed with beta arr2GFP, angiotensin II activation of the AT1AR-beta 2AR-CT did not result in beta -arrestin trafficking to intracellular vesicles, but rather resulted in a beta -arrestin membrane localization pattern similar to that observed in response to beta 2AR activation (Fig. 6A). In fact, beta arr2GFP fluorescence was retained at the plasma membrane for up to 1 h and was never localized to intracellular vesicular structures in response to activation of the chimeric AT1AR mutant. In contrast, the corresponding beta 2AR chimeric mutant with AT1AR carboxyl-tail displayed "AT1AR-like" phenotype and acquired the ability to mediate beta -arrestin translocation to intracellular vesicles (Fig. 6A). In addition, a beta 2AR-beta arr2GFP fusion protein with beta arr2GFP attached to the carboxyl terminus of the beta 2AR was also localized to large intracellular vesicular structures similar to the vesicular population containing beta arr2GFP following AT1AR stimulation (Fig. 6C). These results demonstrate that the carboxyl-terminal region of the receptors is involved in determining the mode of interaction of the receptors with beta -arrestin proteins, and subsequently the agonist-dependent redistribution of beta -arrestins.


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Fig. 6.   Effect of the carboxyl-terminal regions of the AT1AR and beta 2AR on beta arr2GFP localization. A, visualization of the redistribution of beta arr2GFP fluorescence with time in response to stimulation of the AT1AR-beta 2AR-CT with 500 nM angiotensin II or the beta 2AR-AT1AR-CT with 10 µM isoproterenol. HEK 293 cells were transfected with 1 µg of pGFP-N3/beta arr2 and 5 µg of AT1AR-beta 2AR-CT or beta 2AR-AT1AR-CT in pcDNA3. Shown are representative confocal microscopic images of beta arr2GFP fluorescence in HEK 293 cells exposed to agonist for the times indicated. B, sequestration of the wild-type AT1AR, beta 2AR and the chimeric mutants AT1AR-beta 2AR-CT and beta 2AR-AT1AR-CT. In these experiments, HEK 293 cells were also transiently transfected with 1 µg of pGFP-N3/beta arr2 and with plasmids containing cDNAs for HA epitope-tagged AT1AR-beta 2AR-CT and Flag epitope-tagged beta 2AR-AT1AR-CT. Receptor sequestration was assessed by flow cytometry as described previously (16). The data represent the mean ± S.E. of six experiments. C, cellular localization of a beta 2AR-beta arr2GFP fusion protein. Cells were transfected with 5 µg of pGFP-beta 2AR-beta arr2GFP. Shown are representative confocal microscopic images of beta 2AR-beta arr2GFP distribution in HEK 293 cells.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present work, we use green fluorescent protein-conjugated beta -arrestin2 to study the cellular trafficking of beta -arrestin proteins in response to several different GPCRs in both living and fixed cells. By doing so, we show that the pharmacology of beta -arrestin translocation underlies the inherent agonist dependence of beta 2AR endocytosis. More interestingly, our results demonstrate that the cellular fate of beta -arrestin2 (or beta -arrestin1) is differentially regulated following activation of distinct GPCRs. A popular assumption in the field has been that beta -arrestins are associated with internalized receptors in intracellular vesicles (reviewed in Refs. 21, 27, and 28). While this appears to be true for some receptors such as the AT1AR and NTR, we found that in the case of the beta 2AR, D1AR, and ETAR, beta -arrestins do not traffic with the receptors to endosomes and appear to dissociate from receptor-bearing vesicles shortly following their formation. In addition, swapping of the carboxyl-terminal regions between the AT1AR and beta 2AR switches the phenotype of both receptors in terms of their ability to mobilize plasma membrane-associated beta -arrestin to cytosolic vesicular structures. This indicates that the carboxyl-terminal region of the receptors is important in determining receptor/beta -arrestin association and receptor-mediated beta -arrestin cellular distribution to the plasma membrane and/or endosomes.

Considerable effort has been expended to uncover receptor endocytic motifs underlying the agonist-dependent endocytosis of GPCRs. The expectation was that discrete amino acid motifs on the GPCRs, similar in function to those utilized by single transmembrane spanning receptors, would be identified (21, 29, 30). However, the matching pharmacology for beta -arrestin translocation and beta 2AR sequestration described here strongly indicates that beta -arrestin binding replaces the exposure of discrete amino acid motifs as the agonist-driven switch regulating receptor endocytosis. In contrast, the agonist dependence of the endocytosis of receptor tyrosine kinases is thought to involve the exposure of tyrosine-containing motifs on the receptors (30-34).

It is likely that the effect of agonist binding to the receptor is to induce an intramolecular rearrangement of multiple intracellular GPCR domains. This results in a generalized receptor conformation that is necessary to promote GRK phosphorylation and beta -arrestin binding. Interestingly, this conformational requirement may account for the failure of some opioid agonists to induce the sequestration of the mu opioid receptor and the sequestration defect described for the beta 2AR-Y326A mutant (11, 24, 35). Furthermore, experiments using beta 2AR/beta 3AR chimeric receptors showed that normal sequestration of the resulting chimeras required the swapping of several intracellular domains, including the first and second intracellular loops and the carboxyl tail between the two receptor proteins (36). Thus, it would appear that the endocytic switching function of beta -arrestins is related to their role in receptor desensitization, i.e. the binding to and uncoupling of the receptor from its G protein. Once receptors are bound to beta -arrestin, they then gain the ability to traffic to clathrin-coated pits. Indeed, photobleaching experiments of beta 2AR-GFP in HEK 293 cells suggest that desensitized receptors (i.e. complexed with beta -arrestins) are free moving in the plasma membrane and that their movement to coated pits is not rate-limiting for beta 2AR endocytosis (23). This suggests that the interaction of beta -arrestin with the beta 2AR represents the initiating event for receptor endocytosis.

Whereas there is growing evidence supporting beta -arrestins as a general endocytic intermediate for many GPCRs, it is somewhat surprising that beta -arrestins do not traffic to the same cellular compartments upon activation of distinct receptors. The internalization of beta -arrestin with some GPCRs but not with others suggests that the properties of beta -arrestin/receptor interactions differ for different GPCRs. For example, in the case of the alpha 2-adrenergic receptor, beta -arrestins were demonstrated to bind to the third intracellular loop, whereas beta -arrestin interactions with the beta 2AR appear to involve multiple receptor domains including the receptor carboxyl terminus (36, 37). In addition, peptide inhibition studies suggest that the third and, to a lesser extent, the first intracellular loops of rhodopsin may play an important role in arrestin binding to light-activated forms of rhodopsin (38). In the present study, these differences are highlighted by the ability of beta -arrestin2 to internalize with the AT1AR and NTR, but not the beta 2AR, D1AR, and ETAR. In the case of the AT1AR, it appears that the carboxyl-terminal tail contributes directly to beta -arrestin interactions. While it is plausible that these differences are the consequence of the high affinity and slow off-rate of peptidic ligands that might trap receptors in a conformation favoring stable beta -arrestin binding, the observation that the ETAR do not internalize with beta -arrestin bound does not support this apparently simple explanation. Rather, the present experiments with an AT1AR-beta 2AR carboxyl-terminal tail chimera as well as a beta 2AR-AT1AR carboxyl-terminal tail chimera suggest that these differences appear to be regulated by differences in either the tertiary structure or beta -arrestin-interacting sequences in the carboxyl-terminal domains of these receptors. Presumably, the carboxyl-terminal domain in conjunction with other intracellular receptor domains determines the relative stability of receptor/beta -arrestin complexes.

In a previous study, we have reported that, although the AT1AR is capable of utilizing a dynamin- and beta -arrestin-independent endocytic pathway, co-expression of beta -arrestins significantly increases the level of dynamin-dependent AT1AR internalization (16). This suggests that the AT1AR has the ability to directly interact with beta -arrestins. In this study, using the GFP-conjugated beta -arrestin2, we were able to visualize an agonist-dependent co-trafficking of the receptor with beta -arrestins to endocytic vesicles. This represents the first direct demonstration of AT1AR association with beta -arrestins. As GRKs were shown to phosphorylate and desensitize the AT1AR (39), it is probable that, similar to their role in beta 2AR function, beta -arrestins also play an important role in AT1AR regulation by binding to the GRK-phosphorylated form of the receptor. In addition to the AT1AR and beta 2AR, we also visualized the trafficking of several other receptors with beta -arrestins, including the D1R, ETAR, and NTR. The activation of the NTR resulted in an "AT1AR-like" beta -arrestin distribution pattern, whereas the activation of the other two receptors triggered a membrane localization of beta -arrestins similar to that observed for the beta 2AR. These observations indicate that, while GPCR/arrestin interactions represent a general GPCR regulatory mechanism, the stability of receptor/beta -arrestin complexes differs from receptor to receptor.

The binding of arrestin proteins to GRK-phosphorylated GPCRs serves to desensitize various GPCRs, following which, the agonist-promoted receptor internalization is proposed to contribute to receptor dephosphorylation and resensitization (21, 40). A critical step leading to effective GPCR dephosphorylation and resensitization is the dissociation of the GPCR/arrestin complex, since dephosphorylation of rhodopsin was demonstrated to be blocked when the receptor is arrestin-bound (20). Our results indicate that beta -arrestins dissociate from the beta 2AR shortly following the redistribution of beta -arrestins to coated pits. This early dissociation of GPCR/beta -arrestin complexes is presumably appropriate to allow the beta 2AR to associate with receptor phosphatase and dephosphorylate in early endosomes (15). On the other hand, for GPCRs that internalize with beta -arrestins bound, it might be expected that the kinetics of dephosphorylation of these receptors would be slower. While beta -arrestin binding to receptors may be a general feature of GPCR regulation, our results suggest that the nature of this association or the stability of receptor/beta -arrestin complex differs depending upon the receptor studied. Therefore, studies on the dissociation of receptor/beta -arrestin complex should be valuable for understanding the mechanisms by which receptor desensitization and resensitization are achieved. The development of mutant or chimeric receptors with altered ability to interact with beta -arrestins should greatly facilitate this goal.

    ACKNOWLEDGEMENT

We thank Sobha Budduluri for expert technical assistance.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant NS 19576, an unrestricted Neuroscience Award from Bristol-Myers Squibb, and an unrestricted grant from Zeneca Pharmaceutical Co. (to M. G. C.), Heart and Stroke Foundation of Ontario Grant NA-3349 (to S. S. G. F.), and National Institutes of Health Grant HL 03422 (to L. S. B.).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.

Recipient of a Merck Frosst Canada postdoctoral fellowship.

parallel Recipient of a fellowship award from the Heart and Stroke Foundation of Canada.

** Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, Box 3287, Duke University Medical Center, Durham NC 27710. Tel.: 919-684-5433; Fax: 919-681-8641.

Dagger Dagger Recipient of a McDonald scholarship from the Heart and Stroke Foundation of Canada.

2 R. Oakley, personal communication.

    ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; AT1AR, angiotensin II type 1A receptor; AT1AR-beta 2AR-CT, chimeric AT1AR mutant with the beta 2AR carboxyl-terminal tail; beta 2AR, beta 2-adrenergic receptor; beta 2AR-AT1AR-CT, chimeric beta 2AR mutant with the AT1AR carboxyl-terminal tail; beta arr2GFP, green fluorescent protein conjugate of beta -arrestin2; beta arr1GFP, green fluorescent protein conjugate of beta -arrestin1; D1AR, dopamine D1A receptor; ETAR, endothelin type A receptor; GFP, green fluorescent protein; GRK, G protein-coupled receptor kinase; HA, hemagglutinin; MEM, minimal essential medium; NTR, neurotensin receptor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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