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J Biol Chem, Vol. 274, Issue 45, 32248-32257, November 5, 1999

Association of -Arrestin with G Protein-coupled Receptors during Clathrin-mediated Endocytosis Dictates the Profile of Receptor Resensitization^*

Robert H. Oakley, Stéphane A. Laporte, Jason A. Holt, Larry S. Barak, and Marc G. Caron§

From the Howard Hughes Medical Institute Laboratories, Departments of Cell Biology and Medicine, Duke University Medical Center, Durham, North Carolina 27710

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Resensitization of G protein-coupled receptors (GPCRs) following agonist-mediated desensitization is a necessary step for maintaining physiological responsiveness. However, the molecular mechanisms governing the nature of GPCR resensitization are poorly understood. Here, we examine the role of -arrestin in the resensitization of the ₂ adrenergic receptor (₂AR), known to recycle and resensitize rapidly, and the vasopressin V2 receptor (V2R), known to recycle and resensitize slowly. Upon agonist activation, both receptors recruit -arrestin to the plasma membrane and internalize in a -arrestin- and clathrin-dependent manner. However, whereas -arrestin dissociates from the ₂AR at the plasma membrane, it internalizes with the V2R into endosomes. The differential trafficking of -arrestin and the ability of these two receptors to dephosphorylate, recycle, and resensitize is completely reversed when the carboxyl-terminal tails of these two receptors are switched. Moreover, the ability of -arrestin to remain associated with desensitized GPCRs during clathrin-mediated endocytosis is mediated by a specific cluster of phosphorylated serine residues in the receptor carboxyl-terminal tail. These results demonstrate that the interaction of -arrestin with a specific motif in the GPCR carboxyl-terminal tail dictates the rate of receptor dephosphorylation, recycling, and resensitization, and thus provide direct evidence for a novel mechanism by which -arrestins regulate the reestablishment of GPCR responsiveness.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

G protein-coupled receptor (GPCR)¹ signaling plays a pivotal role in regulating various physiological functions including vision, olfaction, neurotransmission, cardiac output, and fluid and electrolyte balance. The magnitudes of these physiological responses are linked intimately to the delicate balance between GPCR signal generation and signal termination. The termination of GPCR signaling is tightly regulated by a family of intracellular proteins termed -arrestins (1-5). -Arrestins bind with high affinity to agonist-activated GPCRs that have been phosphorylated by G protein-coupled receptor kinases (GRKs). The interaction of -arrestins with a phosphorylated receptor uncouples the receptor from heterotrimeric G proteins, producing a nonsignaling, desensitized receptor. For many GPCRs, like the ₂-adrenergic receptor (₂AR), -arrestins target the desensitized receptor to clathrin-coated pits for endocytosis (4, 6-10). In this process, -arrestins function as docking proteins that link receptors to components of the endocytic machinery such as AP-2 and clathrin (11, 12). Intracellular trafficking of GPCRs following -arrestin-mediated desensitization is necessary for receptor resensitization (5, 13, 14). Therefore, -arrestins are involved not only in terminating receptor G protein coupling but also in initiating processes that regulate re-establishment of receptor responsiveness.

While it is evident that responsiveness to most GPCR-activating ligands can be regained, the biochemical and kinetic properties of the cellular processes mediating resensitization may differ considerably among receptors. Some internalized GPCRs recycle rapidly back to the plasma membrane fully resensitized, while others are retained inside the cell and recycle slowly or not at all (5, 14-22). The molecular mechanisms governing the rate at which receptors recycle and re-establish agonist responsiveness are poorly understood; however, the dephosphorylation of GRK-phosphorylated receptors in early endosomes appears to be a critical step in the resensitization pathway (14, 17, 23-25). An event presumably necessary for the dephosphorylation of GPCRs is their dissociation from -arrestin, as suggested by in vitro evidence that the binding of visual arrestin to GRK-phosphorylated rhodopsin prevents rhodopsin dephosphorylation (26). A common assumption is that -arrestins do not dissociate from desensitized receptors at the plasma membrane but traffic with them into early endosomes (27, 28). However, recent observations have demonstrated that the fate of the GPCR--arrestin complex can differ among receptors (29). For some receptors, the receptor--arrestin complex dissociates at or near the plasma membrane shortly after the formation of clathrin-coated pits, and -arrestin is excluded from receptor-containing endocytic vesicles. For other receptors, the receptor--arrestin complex remains intact and is internalized into endosomes. The ability of -arrestin to remain associated with some receptors but not others suggests that -arrestin may regulate the cellular trafficking and dephosphorylation of receptors and ultimately their kinetics of resensitization.

In order to investigate the role of -arrestin in the regulation of GPCR resensitization, two GPCRs, the ₂AR and the vasopressin V2 receptor (V2R), which share many of the same signaling properties but differ markedly in their ability to recycle and resensitize (5, 14, 20), were chosen. We demonstrate using mutagenesis and chimeric receptors that the ability of -arrestin to remain associated with desensitized GPCRs and internalize with them into endosomes dictates the properties of GPCR resensitization. These observations, therefore, provide direct evidence for an important mechanism by which -arrestins can regulate the physiological responsiveness of GPCRs.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Isoproterenol was purchased from Research Biochemicals Inc., and arginine vasopressin (AVP) was obtained from Sigma. The anti-HA 12CA5 mouse monoclonal antibody and the rhodamine-conjugated anti-HA 12CA5 mouse monoclonal antibody were purchased from Roche Molecular Biochemicals. [¹²⁵I]Cyanopindolol, [³H]AVP, [³H]adenine, [¹⁴C]cAMP, [³²P]ATP, [³H]ATP, [³H]cAMP, and [³²P]orthophosphate were purchased from NEN Life Science Products.

Plasmid DNA-- Construction of plasmids containing the hemagglutinin epitope (HA)-tagged ₂AR, arr2-GFP, arr1-GFP, -arrestin1, -arrestin2, -arrestin1 dominant negative mutant V53D, and dynaminI dominant negative mutant K44A have been described previously (4, 29-31). The HA-tagged V2R cDNA was kindly provided by Dr. Jurgen Wess (National Institutes of Health, Bethesda, MD). All other constructs were generated by polymerase chain reaction following standard protocols and contain the HA epitope. The ₂AR-V2R chimera contains the first 341 amino acids of the ₂AR (Met-1 to Cys-341) fused to the last 29 amino acids of the V2R (Ala-343 to Ser-371). The V2R-₂AR chimera contains the first 342 amino acids of the V2R (Met-1 to Cys-342) fused to the last 72 amino acids of the ₂AR (Leu-342 to Leu-413). The V2R-S362X truncation mutant was generated by replacing nucleotides CCG encoding Ser-362 of the V2R with nucleotides TAA encoding a stop codon. The V2R-SSSTSS/AAAAAA mutant was generated by replacing Ser-362, Ser-363, Ser-364, Thr-369, Ser-370, and Ser-371 of the V2R with alanine residues. The V2R-TSS/AAA mutant was generated by replacing Thr-369, Ser-370, and Ser-371 of the V2R with alanine residues. The V2R-SSS/AAA and ₂AR-V2R-SSS/AAA mutants were generated by replacing Ser-362, Ser-363, and Ser-364 of the V2R with alanine residues. The ₂AR413-V2R10 chimera contains the full-length ₂AR (Met-1 to Leu-413) fused to the last 10 amino acids of the V2R (Ser-362 to Ser-371). The ₂AR360-V2R10 chimera contains the first 360 amino acids of the ₂AR (Met-1 to Thr-360) fused to the last 10 amino acids of the V2R (Ser-362 to Ser-371). Sequences of the DNA constructs were confirmed by DNA sequencing.

Cell Culture and Transfection-- HEK-293 and COS-7 cells, grown as described previously (32), were seeded at a density of 2 × 10⁶ cells/100-mm dish and 5 × 10⁵ cells/100-mm dish, respectively. Transient transfections were performed using a modified calcium phosphate coprecipitation method as described previously (32).

Receptor Binding-- Membrane binding assays were performed on transfected HEK-293 cells as described previously (33). Briefly, membrane proteins (2 µg) from cells expressing the ₂AR and ₂AR-V2R chimera were incubated in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) at room temperature in the presence of 15 pM [¹²⁵I]cyanopindolol and increasing concentrations of isoproterenol (10 pM to 30 µM). Membrane proteins (10 µg) from cells expressing the V2R and V2R-₂AR chimera were incubated in PBS containing 2% BSA at room temperature with increasing concentrations of [³H]AVP (0.5 nM to 16.0 nM). Binding was terminated by rapid filtration and consecutive washes with ice-cold wash buffer (120 mM NaCl, 50 mM Tris-HCl, pH = 7.2). Wild-type and chimeric receptor expression levels were measured on whole cells as described previously (32). Transfected HEK-293 cells expressing the V2R and V2R-₂AR chimera were incubated 2 h on ice in PBS containing 2% BSA with a saturating concentration of [³H]AVP, and bound radioactivity was extracted with 0.1 M NaOH. Nonspecific binding was determined under each respective condition in the presence of 10 µM propranolol or 10 µM unlabeled AVP. Receptor expression levels varied between 2000 and 4000 fmol/mg of whole cell protein for experiments with arr2-GFP and between 500 and 1500 fmol/mg of whole cell protein for all other experiments.

Receptor Sequestration and Recycling-- Receptor sequestration was assessed by flow cytometry as described previously (30). Sequestration was defined as the fraction of total cell surface receptors that, after exposure to agonist, were removed from the plasma membrane and thus not accessible to antibodies from outside the cell. For recycling experiments, isoproterenol was removed by three rapid washes with serum-free medium at 37 °C and AVP was removed by successive washes with ice-cold PBS, acid (150 mM NaCl/5 mM acetic acid), PBS, and serum-free medium.

Confocal Microscopy-- arr2-GFP trafficking was visualized in transfected HEK-293 cells on a heated (37 °C) microscope stage as described previously (31). Images were collected sequentially using single line excitation (488 nm) with a Zeiss laser scanning confocal microscope (LSM-510). For experiments assessing arr2-GFP trafficking after agonist removal, cells were washed as described above to remove agonist and returned to a 37 °C incubator for 60 min. Colocalization of arr2-GFP with rhodamine-labeled receptors was performed on transfected cells pre-incubated in serum-free medium containing a rhodamine-conjugated anti-HA 12CA5 mouse monoclonal antibody (1:100) for 45 min at 37 °C. Cells were then washed three times with serum-free medium, treated with the appropriate agonist at 37 °C for 30 min, and imaged by confocal microscopy. arr2-GFP 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.

Adenylyl Cyclase Assays-- Whole cell cyclase assays were performed on transfected HEK-293 cells using varying concentrations of isoproterenol (1 × 10¹² M to 1 × 10⁵ M) or AVP (1 × 10¹² M to 1 × 10⁵ M) as described previously (5). For membrane adenylyl cyclase assays, transfected HEK-293 cells were harvested by scraping in ice-cold lysis buffer (10 mM Tris-HCl, 5 mM EDTA, pH = 7.4) and membranes were prepared by disruption with a Polytron homogenizer for 20 s at 20,000 rpm followed by centrifugation at 40,000 × g. The cell membrane was resuspended in lysis buffer by Polytron homogenization for 15 s at 20,000 rpm, centrifuged, and resuspended in ice-cold assay buffer (75 mM Tris-HCl, 2 mM EDTA, 15 mM MgCl₂, pH = 7.4) to a final concentration of 1-2 µg/µl membrane protein. Equivalent amounts of membrane protein in 20-µl aliquots were assayed for agonist-stimulated adenylyl cyclase activity in a final volume of 50 µl as described previously (5).

Whole Cell Phosphorylation-- Receptor phosphorylation was performed as described previously (5). In brief, transfected HEK-293 cells were labeled for 1 h at 37 °C with [³²P]orthophosphate (100 µCi/ml) in phosphate-free medium. Cells were stimulated with agonist for 10 min at 37 °C and then washed three times on ice with ice-cold PBS. For resensitization experiments, cells were washed to remove agonist as described above and either maintained on ice or allowed to recover at 37 °C. All cells were scraped in radioimmune precipitation buffer (150 mM NaCl, 50 mM Tris, 5 mM EDTA, 10 mM NaF, 10 mM disodium pyrophosphate, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) containing protease inhibitors and solubilized for 1 h at 4 °C. After centrifugation, supernatants were collected and assayed for protein concentration (Bio-Rad DC protein assay kit). HA-tagged receptors were immunoprecipitated at 4 °C using the anti-HA 12CA5 mouse monoclonal antibody. Equivalent amounts of receptor, as determined by receptor expression and the amount of solubilized protein in each sample, were subjected to SDS-polyacrylamide gel electrophoresis and processed for autoradiography. Receptor phosphorylation was quantitated using a Molecular Dynamics PhosphorImager and ImageQuant software.

Data Analysis-- The mean and standard error of the mean are expressed for values obtained from the number of independent experiments indicated. Statistical significance was determined using a two-tailed t test. Binding and dose-response data were analyzed using GraphPad Prism software.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Construction and Characterization of the ₂AR-V2R and V2R-₂AR Chimeras-- The ₂AR and V2R share many of the same signaling properties: both agonist-activated receptors couple to G_s and stimulate adenylyl cyclase, desensitize in a GRK-dependent fashion, and internalize into early endosomes (5, 15, 34-37). However, whereas the ₂AR recycles rapidly back to the plasma membrane fully resensitized, the V2R is retained inside the cell (5, 14, 20). -Arrestin binding to many GPCRs is mediated by GRK-phosphorylated serine and threonine residues located in the receptor carboxyl-terminal tails. In addition, residues in the carboxyl terminus of the V2R have been shown to play a critical, but undefined, role in the intracellular retention of this sequestered receptor (20). Therefore, to gain insight into the mechanisms regulating the ability of these two receptors to recycle and resensitize, chimeric receptors were constructed in which the carboxyl-terminal tails of the ₂AR and V2R were exchanged, one for the other, after the putative sites of palmitoylation. The ₂AR-V2R chimera contains the first 341 amino acids of the ₂AR (Met-1 to Cys-341) fused to the last 29 amino acids of the V2R (Ala-343 to Ser-371). The V2R- ₂AR chimera contains the first 342 amino acids of the V2R (Met-1 to Cys-342) fused to the last 72 amino acids of the ₂AR (Leu-342 to Leu-413). The chimeric receptors were essentially indistinguishable from their wild-type counterparts with respect to their affinity for agonist, level of expression, and half-maximal effective concentration (EC₅₀) for adenylyl cyclase activation (Table I).

View this table: [in this window] [in a new window]   Table I Binding, expression, and adenylyl cyclase activation parameters for the wild-type and chimeric receptors The ligand binding properties, expression, and half-maximal effective concentration (EC50) for the activation of adenylyl cyclase were determined for the wild-type and chimeric receptors in transfected HEK-293 cells. Isoproterenol competition binding assays were performed on membranes to determine the equilibrium dissociation binding constants (Kd) for the 2AR and 2AR-V2R chimera. Kh and Kl indicate the Kd values for the high and low affinity states, respectively. [3H]AVP saturation binding assays were performed on membranes to determine the affinity of AVP for the V2R and V2R-2AR chimera. Saturation binding assays were performed on whole cells using [125I]cyanopindolol or [3H]AVP to determine the Bmax of the wild-type and chimeric receptors. The EC50 for activation of adenylyl cyclase was determined on whole cells using varying concentrations of isoproterenol or AVP. All values are expressed as the mean ± S.E. (n = 3).

Sequestration of the ₂AR, V2R, and ₂AR-V2R and V2R-₂AR Chimeras-- Differences in the ability of the ₂AR and V2R to recycle and resensitize might be due to differences in their sequestration pathways. The ₂AR internalizes in a -arrestin- and clathrin-dependent manner (34), but little is known about the sequestration pathway of the V2R other than it is blocked nonspecifically by sucrose (36). Therefore, we evaluated the -arrestin and clathrin dependence of the V2R sequestration pathway. HEK-293 cells were transfected with each receptor alone or each receptor together with the -arrestin1 dominant negative mutant V53D (V53D), which blocks ₂AR sequestration (34), or the dynaminI dominant negative mutant K44A (K44A), which blocks clathrin-mediated endocytosis (34). Agonist-induced sequestration of the ₂AR, V2R, and their chimeras was blocked by overexpression of V53D or K44A (Fig. 1A). V53D was less effective inhibiting sequestration of the V2R (30 ± 4% reduction) compared with the ₂AR (62 ± 1% reduction). The differential sensitivity to V53D was reversed in the chimeric receptors (31 ± 4% and 58 ± 1% reduction for the ₂AR-V2R and V2R-₂AR chimeras, respectively) and presumably reflects a tighter interaction of the endogenous -arrestins with the V2R tail. COS-7 cells express lower levels of endogenous -arrestins than many other cell types and therefore provide an alternative system for assessing the role -arrestin plays in ₂AR and V2R sequestration (38). COS-7 cells were transfected with each receptor alone or each receptor together with -arrestin1 or -arrestin2 (Fig. 1B). In the absence of overexpressed -arrestin, only a small amount of sequestration (compared with HEK-293 cells) was observed for the ₂AR, V2R, and their chimeras following a 30-min exposure to agonist. In contrast, overexpression of -arrestin1 or -arrestin2 enhanced the ability of each receptor to sequester to levels similar to that observed in HEK-293 cells. These sequestration studies demonstrate that the ₂AR and V2R both internalize in a -arrestin- and clathrin-mediated pathway.

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Sequestration of the 2AR, V2R, and 2AR-V2R and V2R-2AR chimeras. A, wild-type and chimeric receptors were transiently expressed in HEK-293 cells together with empty vector (CON), the -arrestin1 dominant negative mutant V53D, or the dynaminI dominant negative mutant K44A. After treating the cells for 30 min at 37 °C with or without 10 µM isoproterenol (2AR and 2AR-V2R chimera) or 100 nM AVP (V2R and V2R-2AR chimera), cell surface receptors were assessed by flow cytometry. Receptor sequestration is expressed as a loss of cell surface immunofluorescence. B, wild-type and chimeric receptors were transiently expressed in COS-7 cells together with empty vector (CON), -arrestin1, or -arrestin2. Cells were treated and processed as described above. The data represent the mean ± S.E. of three independent experiments (*, p < 0.05; **, p < 0.01; and ***, p < 0.001 compared with control values; t test).

Cellular Trafficking of -Arrestin with the ₂AR, V2R, and ₂AR-V2R and V2R-₂AR Chimeras-- We employed a functional arr2-GFP to visualize whether differences exist in -arrestin trafficking with the ₂AR and V2R in transfected HEK-293 cells (31). In the absence of agonist, arr2-GFP was evenly distributed throughout the cytoplasm of cells expressing either the ₂AR or the V2R as indicated by the homogenous arr2-GFP fluorescence (Fig. 2, A and C, 0 min). Addition of isoproterenol promoted the rapid redistribution of arr2-GFP from the cytosol to the ₂AR at the plasma membrane (Fig. 2A, 2 min) (28, 30). The punctate pattern of arr2-GFP fluorescence at the plasma membrane reflects its localization with the receptor in clathrin-coated pits (11, 29, 31). Activation of the V2R with AVP also promoted the rapid redistribution of arr2-GFP from the cytoplasm to the receptor at the plasma membrane in the same time frame and with the same punctate pattern as that observed for the ₂AR (Fig. 2C, 2 min). A more prolonged exposure to agonist produced a striking difference in the trafficking of -arrestin. arr2-GFP remained at the plasma membrane in cells expressing the ₂AR even after 1 h of agonist treatment (Fig. 2A, 15 min and data not shown). In contrast, arr2-GFP redistributed within 2 to 3 min of agonist stimulation to endocytic vesicles in cells expressing the V2R and remained in these vesicles even after 1 h of agonist treatment (Fig. 2C, 15 min and data not shown). The differential trafficking of arr2-GFP with the ₂AR and V2R was completely reversed when the carboxyl-terminal tails of these two receptors were switched. arr2-GFP redistributed to endocytic vesicles in cells expressing the ₂AR-V2R chimera but remained at the plasma membrane in cells expressing the V2R-₂AR chimera (Fig. 2, B and D, compare 15 min images). Similar results were found using a functional arr1-GFP (data not shown).

View larger version (88K): [in this window] [in a new window]   Fig. 2.   Cellular trafficking of arr2-GFP with the 2AR, V2R, and 2AR-V2R and V2R-2AR chimeras. HEK-293 cells were co-transfected with plasmids containing the cDNA for arr2-GFP and the cDNA for the 2AR (A), 2AR-V2R chimera (B), V2R (C), or V2R-2AR chimera (D). The distribution of arr2-GFP fluorescence was visualized in cells before and after treatment with 10 µM isoproterenol (2AR and 2AR-V2R chimera) or 100 nM AVP (V2R and V2R-2AR chimera). Shown are confocal microscopic images of arr2-GFP fluorescence in the same HEK-293 cells treated with agonist for 0, 2, and 15 min at 37 °C.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Colocalization of arr2-GFP with the internalized 2AR, V2R, and 2AR-V2R and V2R-2AR chimeras. HEK-293 cells were co-transfected with plasmids containing the cDNA for arr2-GFP and the cDNA for the 2AR (A), 2AR-V2R chimera (B), V2R (C), or V2R-2AR chimera (D). Cells pre-labeled with rhodamine-conjugated anti-HA mouse monoclonal antibody were treated for 15 min at 37 °C with 10 µM isoproterenol (2AR and 2AR-V2R chimera) or 100 nM AVP (V2R and V2R-2AR chimera). Shown are the confocal visualizations of the receptor immunofluorescence (red) and the arr2-GFP fluorescence (green). Colocalization (yellow) of the receptor with arr2-GFP is indicated in the overlay.

To determine whether -arrestin colocalized with the V2R in endocytic vesicles, we examined the agonist-induced redistribution of the receptor and arr2-GFP in the same living HEK-293 cell. Cell surface receptors were pre-labeled with fluorescent antibodies prior to agonist stimulation. Under these conditions, V2R immunofluorescence was localized at the plasma membrane and arr2-GFP fluorescence was evenly distributed in the cytoplasm (data not shown). Following a 15-min stimulation with AVP, an extensive colocalization (yellow) of the V2R immunofluorescence (red) and the arr2-GFP fluorescence (green) was observed in endocytic vesicles (Fig. 3C). In contrast, arr2-GFP fluorescence (green) did not colocalize with ₂AR immunofluorescence (red) emanating from endocytic vesicles (Fig. 3A). Switching the carboxyl-terminal tails of these two receptors completely reversed these phenotypes. arr2-GFP colocalized with the ₂AR-V2R chimera in endocytic vesicles but did not colocalize with vesicles containing the V2R-₂AR chimera (Fig. 3, B and D). These results demonstrate that the endocytic pathways of the ₂AR and V2R share a common recruitment of -arrestin to the receptor at the plasma membrane during the initial stages of clathrin-mediated endocytosis but then diverge. The ₂AR--arrestin complex dissociates at or close to the plasma membrane, and -arrestin is excluded from receptor-bearing endocytic vesicles. In contrast, the V2R--arrestin complex remains intact and is internalized into endocytic vesicles. Moreover, these results demonstrate that the differential trafficking of -arrestin to endosomes is mediated by the carboxyl-terminal tails of these two receptors.

Recycling and Resensitization of the ₂AR, V2R, and ₂AR-V2R and V2R-₂AR Chimeras-- The ability of internalized -arrestin-free and -arrestin-complexed receptors to recycle back to the plasma membrane was examined by flow cytometry in transfected HEK-293 cells. Cells were treated with agonist for a period of 30 min to promote receptor sequestration. Agonist was then removed, and the return of sequestered receptors to the cell surface was monitored over time. Sixty minutes after agonist removal, 65 ± 6% of the internalized ₂AR but only 11 ± 4% of the internalized ₂AR-V2R chimera recycled back to the plasma membrane (Fig. 4A). Similarly, only 23 ± 5% of the internalized V2R but 89 ± 7% of the internalized V2R-₂AR chimera recycled back to the plasma membrane 60 min after agonist removal (Fig. 4B). Thus, by switching the carboxyl-terminal tails of the recycling ₂AR and nonrecycling V2R and altering their ability to recruit -arrestin into endocytic vesicles, the ability of these two receptors to recycle was completely reversed.

View larger version (62K): [in this window] [in a new window]   Fig. 4.   Receptor recycling and the redistribution of arr2-GFP following agonist removal. A and B, time course of receptor recycling. HEK-293 cells were transfected with plasmids containing the cDNA for the 2AR, 2AR-V2R chimera, V2R, or V2R-2AR chimera. Cells were treated for 30 min at 37 °C with or without 10 µM isoproterenol (2AR and 2AR-V2R chimera, A) or 100 nM AVP (V2R and V2R-2AR chimera, B). The cells were then washed to remove agonist and maintained on ice or incubated at 37 °C for 0, 15, 30, or 60 min. Cell surface receptors were assessed by flow cytometry and are plotted as percentage of total cell surface immunofluorescence measured in control cells not treated with agonist. Data represent the mean ± S.E. of 4-5 independent experiments. C and D, visualization of arr2-GFP fluorescence after agonist removal. HEK-293 cells were co-transfected with plasmids containing the cDNA for arr2-GFP and the cDNA for the 2AR, 2AR-V2R chimera, V2R, or V2R-2AR chimera. Cells were treated with agonist as described above, washed to remove agonist, and incubated at 37 °C for 60 min. The distribution of arr2-GFP fluorescence was visualized in cells immediately before agonist removal (0 min) and immediately after the 60-min recovery period (60 min). Shown are confocal microscopic images of arr2-GFP fluorescence in representative cells.

The fate of arr2-GFP following agonist removal was also examined in this "recycling" paradigm for each of the wild-type and chimeric receptors. After a 30-min treatment with agonist, arr2-GFP fluorescence was observed in a punctate pattern at the plasma membrane of cells expressing the recycling ₂AR and V2R-₂AR chimera and localized to endocytic vesicles in cells expressing the nonrecycling V2R and ₂AR-V2R chimera (Fig. 4, C and D, 0 min). The cells were then washed to remove agonist, and arr2-GFP fluorescence was re-evaluated after a 60-min recovery period. In cells expressing the recycling ₂AR and V2R-₂AR chimera, arr2-GFP redistributed from the plasma membrane back to the cytoplasm as reflected by the homogeneous arr2-GFP fluorescence (Fig. 4, C and D, 60 min). In contrast, in cells expressing the nonrecycling V2R and ₂AR-V2R chimera, arr2-GFP remained localized with the receptor in endocytic vesicles (Fig. 4, C and D, 60 min). These results demonstrate that the interaction of -arrestin with the nonrecycling V2R and ₂AR-V2R chimera is a stable interaction, as it is preserved in endocytic vesicles even 1 h after agonist removal.

We next tested whether the differences in the ability of the wild-type and chimeric receptors to recycle lead to corresponding differences in the ability of these receptors to resensitize. Receptor-expressing HEK-293 cells were treated with vehicle for 15 min (Naive), with agonist for 15 min (Desensitized), or with agonist for 15 min and allowed to recover for 60 min in agonist-free medium (Resensitized) (Fig. 5). Cell membranes were then prepared and agonist-mediated adenylyl cyclase activity was measured for each condition. For both the ₂AR and ₂AR-V2R chimera, desensitization was characterized by a decrease in the maximal velocity (V_max) of adenylyl cyclase activity (Fig. 5, A and B). One hour after agonist removal, the recycling ₂AR had fully resensitized as indicated by the complete recovery in V_max (100 ± 3% of V_max measured under Naive conditions) (Fig. 5A). In contrast, recovery of the V_max for the nonrecycling ₂AR-V2R chimera was impaired by 66 ± 3% (Fig. 5B). Similar results were obtained for the V2R and V2R-₂AR chimera. Desensitization was characterized for both receptors by a decrease in V_max and a rightward shift in the EC₅₀ (Fig. 5, C and D). One hour after agonist removal, the recycling V2R-₂AR chimera fully resensitized as indicated by the complete recovery in V_max (102 ± 2% of V_max measured under Naive conditions) (Fig. 5D). In contrast, recovery of the V_max for the nonrecycling V2R was impaired by 54 ± 1% (Fig. 5C). Thus, differences in the ability of the wild-type and chimeric receptors to recycle lead to corresponding differences in the ability of these receptors to resensitize and re-establish agonist responsiveness.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Resensitization of the 2AR, V2R, and 2AR-V2R and V2R-2AR chimeras following agonist removal. HEK-293 cells were transfected with plasmids containing the cDNA for the 2AR (A), 2AR-V2R chimera (B), V2R (C), or V2R-2AR chimera (D). Cells were incubated for 15 min at 37 °C in the absence (Naive) or presence (Desensitized, Resensitized) of 10 µM isoproterenol (2AR and 2AR-V2R chimera) or 100 nM AVP (V2R and V2R-2AR chimera). The cells were then washed to remove agonist and maintained on ice (Naive, Desensitized) or incubated for 1 h at 37 °C (Resensitized). Cell membranes were prepared and adenylyl cyclase activity measured under basal conditions and in the presence of increasing concentrations of agonist and 10 µM forskolin. Adenylyl cyclase activity was normalized to the forskolin-stimulated cyclase response and is expressed as the percentage of the maximal response in Naive cells. The EC50 values for 2AR- and 2AR-V2R-mediated activation of adenylyl cyclase were 91 and 61 nM, respectively. The EC50 values for V2R- and V2R-2AR-mediated activation of adenylyl cyclase were 4.7 and 4.0 nM, respectively. The data represent the mean ± S.E. of four independent experiments performed in duplicate.

Phosphorylation and Dephosphorylation of the ₂AR, V2R, and ₂AR-V2R and V2R-₂AR Chimeras-- Dissociation of -arrestin from desensitized receptors may be necessary for their dephosphorylation and resensitization (26). As shown in Fig. 6, each of the wild-type and chimeric receptors expressed in HEK-293 cells are phosphorylated after 10 min of agonist treatment. To assess the rate of receptor dephosphorylation, receptor-expressing cells were treated for 10 min with agonist, washed to remove agonist, and either maintained on ice (Desensitized) or returned to a 37 °C incubator for 30 or 60 min (Resensitized) (Fig. 7, A and B). A 48 ± 5% reduction in the phosphorylation of the recycling ₂AR and a 67 ± 7% reduction in the phosphorylation of the recycling V2R-₂AR chimera were observed 60 min after agonist removal. In contrast, very little dephosphorylation was observed for the nonrecycling V2R (3 ± 6% decrease) and no dephosphorylation was observed for the nonrecycling ₂AR-V2R chimera (12 ± 13% increase) after the 60-min recovery period. These data suggest that the stability of the -arrestin interaction with the carboxyl-terminal tail of GPCRs dictates the rate of receptor dephosphorylation.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Phosphorylation of the agonist-activated 2AR, V2R, and 2AR-V2R and V2R-2AR chimeras. HEK-293 cells were transfected with empty vector (mock) or with plasmids containing the cDNA for the 2AR, 2AR-V2R chimera, V2R, or V2R-2AR chimera. Cells were treated for 10 min at 37 °C with or without 10 µM isoproterenol (2AR and 2AR-V2R chimera, A) or 100 nM AVP (V2R and V2R-2AR chimera, B). Receptors were then immunoprecipitated and assayed for phosphorylation. Upper panel shows a representative autoradiograph, and lower panel shows the mean ± S.E. of three to four independent experiments quantified by PhosphorImager analysis.

View larger version (39K): [in this window] [in a new window]   Fig. 7.   Dephosphorylation of the 2AR, V2R, and 2AR-V2R and V2R-2AR chimeras following agonist removal. HEK-293 cells were transfected with plasmids containing the cDNA for the 2AR, 2AR-V2R chimera, V2R, or V2R-2AR chimera. Cells were treated for 10 min at 37 °C with or without 10 µM isoproterenol (2AR and 2AR-V2R chimera, A) or 100 nM AVP (V2R and V2R-2AR chimera, B). The cells were then washed to remove agonist and either maintained on ice (Desensitized, D) or incubated at 37 °C for 30 min (Resensitized, R30) or 60 min (Resensitized, R60). Receptors were then immunoprecipitated and assayed for phosphorylation. Upper panel shows a representative autoradiograph, and lower panel shows the mean ± S.E. of three independent experiments quantified by PhosphorImager analysis. Values are expressed as the percentage of phosphorylation measured for the desensitized receptor.

Identification of Residues within the V2R Carboxyl Terminus That Allow -Arrestin to Remain Associated with Receptors in Endocytic Vesicles-- To identify residues in the V2R tail that stabilize the receptor's interaction with -arrestin, mutations were made in the putative phosphate acceptor sites (Fig. 8A). These mutant receptors expressed in HEK-293 cells sequestered to levels similar to that observed for the wild-type V2R and induced translocation of arr2-GFP to the plasma membrane upon agonist activation (data not shown). Removal of the two clusters of serine/threonine residues contained within the last 10 amino acids of the V2R tail, either by truncation or alanine substitution, produced mutant receptors (V2R-S362X and V2R-SSSTSS/AAAAAA) that did not recruit -arrestin into endocytic vesicles (Fig. 8A, images 2 and 3, respectively). Removal of the cluster of serine/threonine residues at the end of the V2R tail by alanine substitution produced a mutant receptor (V2R-TSS/AAA) that still recruited -arrestin into endocytic vesicles (Fig. 8A, image 4). However, removal of the more proximal cluster of serine residues (Ser-362, Ser-363, and Ser-364) by alanine substitution produced a mutant receptor (V2R-SSS/AAA) that failed to recruit -arrestin into endocytic vesicles (Fig. 8A, image 5). Similar results were found when this serine cluster was mutated to alanines in the ₂AR-V2R chimera (₂AR-V2R-SSS/AAA) (Fig. 8A, image 6). The importance of this serine cluster for recruiting -arrestin into endocytic vesicles was further tested by adding only the last 10 amino acids of the V2R to the end of the full-length ₂AR. arr2-GFP translocated to this mutant receptor (₂AR413-V2R10) at the plasma membrane upon agonist activation but did not internalize with the receptor into endocytic vesicles (Fig. 8A, image 8). Similar results were found when the last 29 amino acids of the V2R were added to the end of the full-length ₂AR (data not shown). However, when the last 10 amino acids of the V2R were positioned closer to the putative palmitoylated cysteine of the ₂AR, the mutant receptor (₂AR360-V2R10) gained the ability to recruit -arrestin into endocytic vesicles (Fig. 8A, image 9). These findings identify a cluster of three serine residues located in the V2R carboxyl terminus that mediate the trafficking of -arrestin with the V2R into endocytic vesicles. Moreover, they suggest that the position of the serine cluster within the receptor carboxyl-terminal tail is critical for the formation of a stable receptor--arrestin complex that internalizes into endocytic vesicles.

View larger version (63K): [in this window] [in a new window]   Fig. 8.   Cellular trafficking of arr2-GFP with the V2R into endocytic vesicles is mediated by a cluster of three serine residues at the V2R carboxyl terminus. A, upper panel shows the amino acid composition of the carboxyl-terminal tails of the V2R, 2AR, and various mutant receptors beginning with the putative sites of palmitoylation in bold (Cys-342 for the V2R constructs 1-5, and Cys-341 for the 2AR constructs 6-9). Underlined are the mutations made by alanine substitution and the last 10 amino acids of the V2R tail when added to the 2AR. Lower panel shows the distribution of arr2-GFP fluorescence in HEK-293 cells expressing wild-type or mutant receptors following a 30-min treatment at 37 °C with 100 nM AVP (images 1-5) or 10 µM isoproterenol (images 6-9). The number on each image corresponds to the number of the transfected receptor construct shown in the upper panel. B, amino acid composition of the carboxyl-terminal tails of the V2R, NTR1, and AT1AR beginning with the conserved NPXXY motif in bold. Clusters of serine/threonine residues are underlined, and putative sites of palmitoylation are indicated with an asterisk (*).

Whole cell phosphorylation assays were performed on HEK-293 cells expressing the wild-type or mutant V2Rs in order to assess whether the proximal cluster of three serine residues is actually phosphorylated. As shown in Fig. 9, agonist-induced phosphorylation of the V2R-SSSTSS/AAAAAA mutant, in which both the proximal and distal clusters of serine/threonine residues are mutated, was reduced 86.3 ± 1.4% compared with the wild-type V2R. Agonist-induced phosphorylation of the V2R-TSS/AAA mutant, in which only the distal cluster of serine/threonine residues is mutated, was reduced 4.7 ± 6.9%. However, agonist-induced phosphorylation of the V2R-SSS/AAA mutant, in which only the proximal cluster of three serine residues is mutated, was reduced 84.2 ± 0.6%. Therefore, the proximal cluster of three serine residues, which mediates the formation of stable V2R--arrestin complexes that internalize into endocytic vesicles, is the principal site of V2R phosphorylation.

View larger version (36K): [in this window] [in a new window]   Fig. 9.   The proximal cluster of three serine residues in the V2R carboxyl terminus is the principal site of V2R phosphorylation. HEK-293 cells were transfected with plasmids containing the cDNA for the V2R, V2R-SSSTSS/AAAAAA, V2R-TSS/AAA, or V2R-SSS/AAA. Cells were treated for 10 min at 37 °C with or without 100 nM AVP. Receptors were then immunoprecipitated and equivalent amounts assayed for phosphorylation. No significant differences were observed among the receptors in the level of basal phosphorylation, and the average -fold induction for each receptor after agonist treatment was approximately 11.0 (V2R), 1.5 (V2R-SSSTSS/AAAAAA), 10.7 (V2R-TSS/AAA), and 1.7 (V2R-SSS/AAA). Upper panel shows a representative autoradiograph. In the lower panel, data are expressed as the percentage of the -fold induction measured for the V2R and represent the mean ± S.E. of two to four independent experiments quantified by PhosphorImager analysis.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present study, we evaluated the role of -arrestin in the resensitization of the ₂AR and V2R. Both these receptors, upon agonist activation, recruit -arrestin to the plasma membrane and internalize in a -arrestin- and clathrin-dependent fashion. The ₂AR--arrestin complex dissociates at or near the plasma membrane, presumably allowing the receptor to associate rapidly with receptor phosphatases and be dephosphorylated in the endosomal compartment. The dephosphorylated ₂AR is then recycled rapidly back to the plasma membrane, and normal receptor responsiveness is re-established. The more stable V2R--arrestin complex does not dissociate at the plasma membrane and is internalized into endosomes. Preservation of this complex presumably prevents the association of this receptor with receptor phosphatases. Consequently, the V2R is not dephosphorylated efficiently in endosomes and recycles only slowly back to the plasma membrane. The molecular determinants that stabilize the receptor--arrestin complex appear to reside in the receptor carboxyl terminus since switching the tails of the ₂AR and V2R completely reverses their dephosphorylation, recycling, and resensitization kinetics. For the V2R, a cluster of three serine residues located in the carboxyl terminus and serving as the principal site for GRK-mediated phosphorylation determines whether a stable V2R--arrestin complex forms. Moreover, this same serine cluster plays a critical role in preventing V2R recycling (20). Therefore, the interaction of -arrestin with phosphorylated serine/threonine clusters in the carboxyl-terminal tail of GPCRs regulates the rate of GPCR dephosphorylation, recycling, and resensitization by presumably impeding the interaction between GPCRs and receptor phosphatases.

-Arrestins bind with high affinity to agonist-activated, GRK-phosphorylated GPCRs in what is thought to involve the simultaneous engagement of two regions of the -arrestin molecule with two corresponding regions of the receptor (28, 39, 40). A large region within the amino-terminal half of -arrestin (termed the activation-recognition domain) recognizes the agonist-activated state of GPCRs. This domain of -arrestin appears to bind the third cytoplasmic loop of a variety of GPCRs, including the m2 and m3 muscarinic acetylcholine receptors and the 2A adrenergic receptor (41). The amino-terminal half of -arrestin also contains a positively charged, smaller domain of approximately 20 amino acids (termed the phosphorylation-recognition domain; net charge +7), which appears to interact with the GRK-phosphorylated carboxyl termini of GPCRs (42). GRK-mediated phosphorylation of the serine cluster in the tail of the V2R would produce a localized concentration of negative charges. Therefore, the strong ionic interaction between the cluster of negative charges in the V2R tail and the cluster of positive charges in the phosphorylation-recognition domain of -arrestin might be responsible for promoting the formation of a stable receptor--arrestin complex that internalizes into endocytic vesicles. Consistent with this hypothesis, mutation of the serine cluster in the V2R-SSS/AAA and V2R-SSSTSS/AAAAAA mutants results in a marked reduction (approximately 85%) in the level of agonist-induced phosphorylation of these receptors as well as the inability of these receptors to recruit -arrestin into endocytic vesicles. Nevertheless, these mutant receptors still exhibit a small amount of agonist-induced phosphorylation, a robust recruitment of -arrestin to the plasma membrane, and normal levels of sequestration. This suggests that other serine/threonine residues in the V2R may also serve as substrates for GRK-mediated phosphorylation, thus enabling -arrestin to interact, albeit more weakly, with other intracellular regions of the agonist-occupied V2R.

We have recently demonstrated that -arrestins dissociate from the dopamine D1A receptor (D1AR) and the endothelin type A receptor (ETAR) at the plasma membrane but traffic with the neurotensin receptor 1 (NTR1) and the angiotensin II type 1A receptor (AT1AR) into endocytic vesicles (29). A comparison of the carboxyl-terminal tails of these receptors with the ₂AR and V2R reveals striking similarities with respect to the absence or presence of clusters of putative phosphate acceptor sites (defined as serine/threonine residues occupying three consecutive positions or three out of four positions). Clusters of putative phosphorylation sites are noticeably absent from the carboxyl-terminal tails of the ₂AR, D1AR, and ETAR even though potential phosphate acceptor sites are abundant. In contrast, clusters of putative phosphorylation sites are not only present in the V2R, NTR1, and AT1AR carboxyl-terminal tails but also occupy similar positions with respect to the conserved NPXXY motif (marking the end of the seventh transmembrane domain) and the end of the receptor (Fig. 8B). Evidence that the location of the cluster of putative phosphate acceptor sites is critical for the formation of a stable receptor--arrestin complex comes from our finding that the ₂AR gains the ability to recruit -arrestin into endocytic vesicles when the V2R serine cluster is added to the ₂AR truncated at position 360 but not when added to the full-length ₂AR. Clearly, an important goal of future studies will be to test whether the clusters of putative phosphate acceptor sites found in the tails of other GPCRs are critical to the recruitment of -arrestin with these receptors into endocytic vesicles and to test whether these complexes can interact with other accessory proteins or signaling molecules.

In summary, -arrestins are multifunctional proteins that play a pleiotropic role in regulation of GPCR responsiveness. They regulate GPCR desensitization by uncoupling receptors from their cognate G proteins and they regulate GPCR sequestration by targeting desensitized receptors to clathrin-coated pits for endocytosis. We now show that -arrestins, by interacting with a specific motif in the carboxyl-terminal tails of GPCRs, dictate the rate of receptor dephosphorylation, recycling, and resensitization. Thus, the stability with which -arrestins associate with GPCRs controls the intracellular trafficking of receptors and consequently the re-establishment of physiological responsiveness following desensitization.

	ACKNOWLEDGEMENT

We thank Brian Curtin for technical assistance.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grant NS 19576 (to M. G. C.), an unrestricted Neuroscience Award from Bristol-Myers Squibb (to M. G. C.), and National Institutes of Health Grant HL 61365 (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 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 Inst., Box 3287, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-5433; Fax: 919-681-8641; E-mail: caron002@mc.duke.edu.

	ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; ₂AR, ₂-adrenergic receptor; V2R, vasopressin V2 receptor; AVP, arginine vasopressin; arr2-GFP, -arrestin2-green fluorescent protein conjugate; arr1-GFP, -arrestin1-green fluorescent protein conjugate; D1AR, dopamine D1A receptor; ETAR, endothelin type A receptor; NTR1, neurotensin receptor 1; AT1AR, angiotensin II type 1A receptor; HA, hemagglutinin; BSA, bovine serum albumin; PBS, phosphate-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES