Heterologous Inhibition of G Protein-coupled Receptor Endocytosis Mediated by Receptor-specific Trafficking of b -Arrestins*

We have observed an unexpected type of nonreciprocal “cross-regulation” of the agonist-induced endocytosis of G protein-coupled receptors by clathrin-coated pits. Isoproterenol-dependent internalization of b 2 -ad- renergic receptors in stably transfected HEK293 cells was specifically blocked ( > 65% inhibition) by vasopressin-induced activation of V2 vasopressin receptors co-expressed at similar levels. In contrast, activation of b 2 receptors caused no detectable effect on V2 receptor internalization in the same cells. Several pieces of evidence suggest that this nonreciprocal inhibition of endocytosis is mediated by receptor-spec-ific intracellular trafficking of b -arrestins. First, previous studies showed that the activation of V2 but not b 2 receptors caused pronounced recruitment of b -a-rrestins to endocytic membranes (Oakley, R. H., Laporte, S. A., J. A., L. S., and Caron, M. G. (1999) J. Biol. Chem. 274, 32248–32257). Second, overexpression of arrestin 2 or 3 ( b -arrestin 1 or 2) abolished the V2 receptor-mediated inhibition of b 2 receptor int- ernalization. Third, mutations of the V2 receptor that block endomembrane recruitment of b -arrestins eliminated the V2 receptor-dependent blockade of b 2 receptor internalization. These results identify a novel type of heterologous regulation of G protein-coupled receptors, define a new functional role of receptor-spec-ific intracellular trafficking of b -arrestins, and suggest an experimental method to rapidly modulate the functional activity of b -arrestins in intact cells.

G protein-coupled receptors (GPCRs) 1 are regulated by a set of highly conserved molecular mechanisms (1)(2)(3). Because most cells express multiple types of GPCR that serve distinct functions, the specificity of these regulatory mechanisms is of great physiological interest. Extensive studies have led to the classification of distinct "heterologous" and "homologous" mechanisms of GPCR regulation. An example of a heterologous regulatory mechanism is rapid desensitization of the ␤ 2 -adrenergic receptor (␤ 2 AR) mediated by the cyclic AMPdependent protein kinase. Activated cAMP-dependent protein kinase can phosphorylate both the ␤ 2 AR as well as a number of other GPCRs irrespective of whether or not these receptors have bound agonist. A well characterized mechanism of homologous regulation is desensitization of the ␤ 2 AR mediated by G protein-coupled receptor kinases. In general G protein-coupled receptor kinases preferentially phosphorylate ligand-activated receptors without affecting other GPCRs present in the same cells that are not activated by their respective agonist (4 -6).
Many GPCRs, such as the prototypic ␤ 2 AR, are regulated by agonist-induced endocytosis via clathrin-coated pits (7)(8)(9)(10). This process is promoted by G protein-coupled receptor kinasemediated phosphorylation of receptors followed by membrane recruitment of nonvisual arrestins (or ␤-arrestins), which link receptors to the clathrin/AP-2 endocytic coat (9,11,12). Because high affinity interaction of GPCRs with arrestins is promoted by both G protein-coupled receptor kinase-mediated phosphorylation and an agonist-induced conformation of the receptor protein (13,14), endocytosis by clathrin-coated pits is thought to represent a highly homologous mechanism of GPCR regulation (8,11). Consistent with this, distinct GPCRs, even when co-expressed at high levels, are endocytosed by coated pits in a highly selective manner after activation by their respective agonist (15).
Arrestins play a highly conserved role in promoting endocytosis of various GPCRs by clathrin-coated pits (11). Recent studies indicate that particular GPCRs differ substantially in their effects on the intracellular trafficking of arrestins. Many G protein-coupled receptors (including the ␤ 2 AR) recruit arrestins to the plasma membrane but do not remain associated with arrestins after endocytosis (16,17). In contrast, the V2 vasopressin receptor (V2R) has been shown to mediate recruitment of arrestins both to the plasma membrane and to V2Rcontaining endocytic vesicles (17). This receptor-specific difference in the endomembrane recruitment of arrestins is mediated by a persistent phosphorylation of internalized V2Rs, which inhibits recycling of receptors to the plasma membrane and causes a prolonged state of receptor desensitization (17,18). Endosome-associated arrestins have been proposed to play an important role in determining the specificity of downstream signal transduction by endocytosed GPCRs (19,20). However, to our knowledge no previous studies have examined the possibility that arrestin trafficking may modulate the specificity of GPCR endocytosis itself. In the present study we have observed an unexpected type of heterologous and nonreciprocal inhibition of ␤ 2 AR endocytosis mediated by agonist-induced activation of the V2R. This heterologous inhibition is dependent on endomembrane recruitment of ␤-arrestins, suggesting a novel role of receptor-specific trafficking of arrestins in modulating endocytosis of certain GPCRs. * These studies were supported by research grants from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

EXPERIMENTAL PROCEDURES
cDNA Constructs and Mutagenesis-The coding sequence of the human V2R was amplified by PCR and inserted into a tagging vector containing the influenza hemagglutinin signal sequence and the FLAG epitope (21). The N-terminal epitope-tagged receptor sequence was subcloned into the mammalian expression vector pcDNA3.1 (Invitrogen). The human ␤ 2 AR and the murine ␦-opioid receptor were tagged in the N-terminal extracellular domains with the HA epitope (YPYDVP-DYA) and subcloned into pcDNA3.1, as previously described (22). Two truncated forms of the V2R were obtained through oligonucleotidedirected mutagenesis using the polymerase chain reaction with 3Ј antisense primers incorporating a stop codon followed by a suitable restriction site (345T and 362T: stop codons substituted for residues at positions 345 and 362 of the human V2R amino acid sequence, respectively) (18). PCR products were subcloned into pcDNA3.1. GFP-tagged arrestin 3 (␤-arrestin 2) was a generous gift of Dr. Marc Caron. A C-terminally EE epitope-tagged version of arrestin 2 (␤-arrestin-1, a generous gift of Dr. Jeffrey Benovic) was constructed by ligating a synthetic adaptor oligonucleotide encoding the epitope tag sequence and cloning into pcDNA3. All constructs were verified by DNA sequencing (University of California San Francisco Biomolecular Resource Center).
Cell Lines and Transfection-HEK293 cells (ATCC) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin (University of California San Francisco Cell Culture Facility) in a humidified incubator with 7% CO 2 at 37°C. Cells were transiently transfected using calcium phosphate co-precipitation (23). For generation of stable transformants, cells after transfection were selected for resistance to 0.5 mg/ml G418 (Geneticin, Life Technologies, Inc.). Single clones were isolated after 2-3 weeks of culture and screened for expression by immunofluorescence microscopy and radioligand binding assays.
Immunofluorescence Microscopy-Cells were grown on glass coverslips in 6-well tissue culture plates. For co-localization of FLAG-tagged V2R and HA-tagged ␤ 2 AR or HA-tagged ␦-opioid receptor, cells were incubated with the monoclonal anti-epitope antibodies anti-FLAG M1 (Sigma) and anti-HA HA.11 (Covance), respectively, both at a 1:500 dilution for 30 min at 37°C. Cells were then treated under the respective conditions with agonists, fixed using 4% formaldehyde in phosphate-buffered saline, and permeabilized with blocking buffer (3% nonfat dry milk, 0.1% Triton X-100, 50 mM Tris/HCl, pH 7.5, 1 mM CaCl 2 ). Bound M1 antibody was detected using incubations with a rabbit antimouse IgG 2b subtype-specific linker antibody (1:800 dilution, 45 min at room temperature) followed by a Texas Red-conjugated donkey antirabbit antibody (1:1000 dilution, 20 min at room temperature). Bound HA.11 antibody was detected using fluorescein isothiocyanate-conjugated goat anti-mouse IgG 1 subtype-specific antibody (1:800 dilution, 20 min at room temperature). Immuno-stained coverslips were mounted on microscopy slides and examined by epifluorescence or confocal microscopy (see below).
Membrane Preparation-Individual cell clones stably expressing the ␤ 2 AR and/or the V2R were grown on 10-cm dishes to confluency. Cells were lifted in phosphate-buffered saline containing 2 mM EDTA and centrifuged at 500 ϫ g for 10 min. Pellets were resuspended in 1 ml of lysis buffer (5 mM Hepes/NaOH, pH 7.4, 5 mM EDTA, 5 mM EGTA, 0.5 mM Pefabloc SC) and homogenized using a glass Dounce homogenizer.
The suspension was centrifuged for 15 min at 20,000 ϫ g, and the pellets were resuspended and homogenized as above. The final pellet was resuspended in 200 l of membrane buffer (20 mM Hepes/NaOH, pH 7.4, 1 mM EDTA, 5 mM MgCl 2 , 0.5 mM Pefabloc SC). Protein concentration was estimated by the method of Bradford (25). Membranes were stored frozen at Ϫ80°C.
Scoring of Receptor Localization by Fluorescence Microscopy-Coverslips were processed for indirect immunofluorescence staining, as above, and coded such that the identity and treatment conditions of the specimen were not specified. Examination of coded specimens by epifluorescence microscopy was performed by a second individual not familiar with the details of the experiment. The localization of fluorochromes representing individual receptors were classified in multiple cells examined at random, positive for expression of both receptors. Cells with detectable expression of only one of the two receptors were excluded from the scoring. Immunostaining was classified according to the following criteria: non-internalized localization (bright staining around the cell periphery with Ͻ10 immunoreactive puncta visualized within the cytoplasm); intermediate appearance (10 -20 internal puncta); internalized (Ͼ20 immunoreactive puncta resolved within the cytoplasm). At least 25 cells per coverslip were scored by these criteria, and all specimens from an individual experiment were scored in a single session before the code was broken and results were tabulated. For studies employing GFP-tagged arrestins, receptor localization for GFP positive and negative cells were scored in the same sitting, and the specificity of observed effects was confirmed by transfection of GFP not fused to ␤-arrestin. Numbers in figures represent the mean Ϯ S.D. for results from a representative experiment, and all reported experiments were performed independently at least three times with similar results.
Estimation of Receptor Internalization by Fluorescence Flow Cytometry-A previously established (26) flow cytometric assay was used to quantitate immunoreactive receptors present on the surface of intact cells after dissociation from the cell culture dish. All data points represent quantitation of 20,000 cells using a FACScan cytometer (Becton Dickenson) performed in triplicate (representing three independently treated dishes) for each experiment. The extent of receptor internalization was calculated according to the agonist-induced reduction in mean surface immunoreactivity (26). Figures indicate mean Ϯ S.D. for results compiled from three separate experiments.

RESULTS AND DISCUSSION
We chose to examine membrane trafficking of the ␤ 2 AR and V2R in HEK293 cells because both receptors stimulate adenylyl cyclase activity via the G s -signaling pathway and undergo agonist-induced endocytosis by an arrestin-dependent mechanism mediated by clathrin-coated pits, yet these receptors are well established to differ substantially in their effects on the intracellular trafficking of ␤-arrestins in this cell type (17). A functional HA-tagged ␤ 2 AR and FLAG-tagged V2R were coexpressed in HEK293 cells by stable transfection. Because a single selection marker (neomycin) was used to isolate stably transfected cells after transfection of separate plasmids encoding the ␤ 2 AR and V2R, many cell populations obtained by this method expressed either the ␤ 2 AR or V2R but not both. Dual label fluorescence microscopy was used to identify stably trans-fected cell populations that expressed both the ␤ 2 AR and V2R in the majority (ϳ70%) of cells. A cell clone expressing both receptors at moderate levels (3.8 Ϯ 0.3 pmol/mg for the V2R and 4.7 Ϯ 0.2 pmol/mg for the ␤ 2 AR, respectively) was selected from this group for further analysis because these levels of expression are comparable to those used previously for studies of GPCR endocytosis and arrestin trafficking in HEK293 cells (16,17,22).
Both the ␤ 2 AR and V2R were visualized by fluorescence microscopy in the plasma membrane of cells incubated in the absence of agonist (Fig. 1, panels a and b). In the presence of saturating concentrations (10 M) of the ␤ 2 AR agonist isoproterenol (ISO), ␤ 2 AR redistributed from the plasma membrane to endocytic vesicles within 15 min, whereas co-expressed V2Rs remained in the plasma membrane and did not exhibit any detectable internalization (Fig. 1, panels c and d). Conversely, in the presence of saturating concentrations (10 M) of the V2R agonist AVP, V2Rs were selectively endocytosed without any detectable internalization of co-expressed ␤ 2 ARs (Fig. 1, panels e and f). Agonist-induced endocytosis of the ␤ 2 AR in HEK293 cells is mediated by clathrin-coated pits, as indicated by morphological studies and the effects of biochemical and genetic inhibitors of clathrin-coated pit function (7)(8)(9)22). Recent studies indicate that agonist-induced endocytosis of the V2R is also mediated by clathrin-coated pits (17,27). Consistent with this, we confirmed that AVP-induced internalization of the FLAGtagged V2R was blocked by mild hypertonicity (0.45 M sucrose) and was also specifically inhibited by overexpression of K44E (dominant-negative) mutant dynamin (not shown).
In contrast to the ability of the ␤ 2 AR or V2R to endocytose selectively when activated separately by ISO or AVP, respectively, surprising results were observed in cells exposed to a saturating concentration (10 M) of both ISO and AVP. Under these conditions, pronounced internalization of the V2R was still observed, but co-expressed ␤ 2 ARs remained in the plasma membrane and appeared to be completely resistant to agonistinduced internalization (Fig. 1, panels g and h, the black arrow indicates an example of a cell co-expressing both receptors). Indeed, the only cells in which ␤ 2 AR internalization was observed under these conditions were the minority of cells in the transfected population (ϳ10 -20% of total, depending on the cell line) that expressed only ␤ 2 AR without any detectable V2R (white arrow in Fig. 1, panels g and h, indicates an example of such a cell). Quantitation of multiple cells (selected at random in coded specimens, see "Experimental Procedures") confirmed these observations ( Fig. 2A). Similar results were also observed in transiently transfected cells that vary more widely in ␤ 2 AR and V2R expression levels (see below). Transient transfection of Chinese hamster ovary cells yielded comparable results, demonstrating that the inhibition can be observed in more than one cell type (not shown).
The heterologous inhibition of ␤ 2 AR internalization caused by activation of co-expressed V2Rs was further confirmed using an established flow cytometric assay (26) applied to stably transfected cells expressing both receptors at closely similar levels (ϳ4 pmol/mg), as assessed by radioligand binding assays (see "Experimental Procedures"). Consistent with observations made by fluorescence microscopy, AVP and ISO specifically promoted internalization of the V2R and ␤ 2 AR, respectively, in a strictly homologous manner when added separately to the culture medium (Fig. 2B, first, second, fourth, and fifth bars). In contrast, in cells exposed to both AVP and ISO, ISO-induced activation of the ␤ 2 AR was markedly and specifically inhibited (Fig. 2B, third and sixth bars). The magnitude of AVP-induced inhibition of ␤ 2 AR internalization measured by this assay was ϳ65% and was statistically highly significant (Fig. 2B legend), whereas ISO-induced activation of the ␤ 2 AR caused no detect-  (g and h). The distribution of both receptors after treatment was visualized by immunofluorescence microscopy. The upper panels show the localization of the V2R, and the lower panels show the localization of the ␤ 2 AR in the same field. Black arrows in panels g and h indicate a cell co-expressing both receptors, and white arrows indicate a cell expressing only the ␤ 2 AR without any detectable V2R.

FIG. 2. Quantitation of agonist-induced internalization of the V2R and ␤ 2 AR.
HEK293 cells stably expressing both FLAG epitopetagged V2R and HA epitope-tagged ␤ 2 AR at equal levels were treated for 20 min at 37°C either with no agonist or 10 M ISO alone, AVP alone, or both ISO and AVP. A, internalization for both receptors was estimated by scoring receptor localization after visualization by immunofluorescence microscopy (see "Experimental Procedures"). Only cells with detectable expression of both receptors were included in the scoring. B, quantitation of receptor internalization by fluorescence flow cytometry. In the presence of ISO alone, cells show 60 Ϯ 1% loss of cell surface immunoreactivity (internalization) for the ␤ 2 AR. In the presence of AVP alone, the V2R shows 29 Ϯ 3% internalization. Co-stimulation with both AVP and ISO reduces the extent of ␤ 2 AR internalization by ϳ65% to 21 Ϯ 3%, whereas V2R internalization remains unaffected (33 Ϯ 3%). The statistical significance of the AVP-induced inhibition of ␤ 2 AR internalization was confirmed using Student's t test (p Ͻ 0.001).
able effect on AVP-induced internalization of the V2R. Furthermore, the extent of V2R-mediated inhibition of ␤ 2 AR internalization measured in these experiments was underestimated because of the presence of a subpopulation of stably transfected cells expressing ␤ 2 AR without detectable V2R (ϳ15% of total), in which no AVP-induced inhibition of ␤ 2 AR internalization is observed (Fig. 1, panels g and h). A similar extent of inhibition of ␤ 2 AR internalization was observed 60 min after incubation of co-transfected cells with ISO and AVP (not shown). When stimulated separately, the extent of internalization of both receptors reached steady state within 15 min after the addition of agonist (AVP or ISO, respectively). Taken together, these observations suggest that AVP-induced activation of the V2R in these cells causes a nearly complete blockade of the internalization of co-expressed ␤ 2 ARs.
The nonreciprocal nature of the cross-inhibition of ␤ 2 AR internalization induced by V2R activation was particularly remarkable because both receptors were expressed at closely similar levels (see above), and the flow cytometric analysis indicated that the ␤ 2 AR (when activated individually in the absence of V2R activation) can internalize in these cells to a significantly greater extent at steady state than the V2R (Fig.  2B). To begin to examine the mechanism of the selective V2Rmediated endocytic inhibition, we examined its generality to several other receptors that endocytose via clathrin-coated pits. AVP-induced activation of the V2R caused no detectable inhibition of constitutive endocytosis of endogenously expressed transferrin receptors, as visualized by fluorescence microscopy using Texas Red-conjugated transferrin (not shown). However, V2R activation did cause a nearly complete blockade of etorphine-stimulated internalization of co-expressed HA-tagged ␦-opioid receptor, which was similar to that observed for the ␤ 2 AR and was also nonreciprocal (Fig. 3). Together, these observations indicate that the V2R-mediated inhibition is specific to the mechanism mediating agonist-induced endocytosis of certain GPCRs and does not reflect a more general inhibition or saturation of the clathrin-mediated endocytic pathway.
Opioid receptors, like the ␤ 2 AR, recruit ␤-arrestins to the plasma membrane of transfected HEK293 cells but fail to mediate detectable endomembrane recruitment of arrestins (28 -31). In contrast, the V2R recruits ␤-arrestins both to the plasma membrane and to V2R-containing endocytic vesicles (17). These observations suggest that the nonreciprocal inhibition of ␤ 2 AR internalization by V2R activation might be mediated by receptor-specific trafficking of arrestins to endomembranes. To begin to test this hypothesis, we examined the effect of overexpressing ␤-arrestins on the V2R-mediated blockade of ␤ 2 AR internalization. Cells co-expressing FLAG-tagged V2R and HA-tagged ␤ 2 AR were transiently transfected with a plasmid encoding a GFP-tagged version of arrestin 3 (␤-arrestin 2) (24). Then cells were incubated as above in the presence of both AVP and ISO, and triple-color fluorescence microscopy was used to visualize the subcellular distribution of V2R, ␤ 2 AR, and GFP-tagged arrestin 3 in the same cells. In contrast to the complete lack of detectable ␤ 2 AR internalization in cells expressing ␤-arrestins at endogenous levels ( Figs. 1 and 2), internalization of the ␤ 2 AR was readily observed under the same conditions in cells overexpressing GFP-tagged arrestin 3 (Fig.  4A). Many of the punctate structures containing ␤ 2 ARs observed in cells overexpressing ␤-arrestins could be resolved from the plasma membrane by confocal optical sectioning and were inaccessible to antibody in nonpermeabilized cells (not shown), demonstrating that these structures represent bona fide ␤ 2 AR-containing endocytic vesicles (rather than clusters of receptors present in the plasma membrane). These results were confirmed in multiple cells examined in coded specimens (Fig. 4B). Similar results were observed when transiently transfecting both receptors into cells stably overexpressing an EE epitope-tagged arrestin 2 (␤-arrestin 1, not shown). Significantly, overexpression of ␤-arrestins has little effect on ISOinduced internalization of the ␤ 2 AR in HEK293 cells not expressing the V2R (32). Together these results suggest that overexpression of ␤-arrestins is sufficient to specifically "rescue" the V2R-mediated inhibition of ␤ 2 AR internalization. Furthermore we observed that ␤ 2 ARs and V2Rs visualized in arrestin-transfected cells were observed in an overlapping population of endocytic vesicles that colocalized extensively with GFP-tagged arrestin 3 (Fig. 4A, corresponding arrows in each panel indicate examples of such colocalized vesicles in a representative high-power field including several arrestintransfected cells). These results demonstrate that once the V2R-mediated "sequestration" of arrestin activity is overcome, the co-expressed ␤ 2 AR and V2R can enter a similar endocytic pathway.
To examine whether endomembrane trafficking of ␤-arrestins is actually necessary for the V2R-mediated inhibition of ␤ 2 AR internalization, we used a mutational strategy to disrupt V2R-mediated recruitment of ␤-arrestins to endocytic vesicles. The wild type V2R is highly resistant to dephosphorylation in endocytic vesicles, leading to endomembrane recruitment of arrestins and inhibited recycling of receptors (17,18). Truncation mutations (362T and 345T) of the cytoplasmic tail create functional receptors that undergo agonist-induced endocytosis with similarly rapid kinetics as the wild type receptor (18) but exhibit distinct defects in agonist-dependent phosphorylation/ dephosphorylation. The 345T mutant receptor does not exhibit detectable phosphorylation after exposure to agonist; the 362T mutant receptor undergoes agonist-induced phosphorylation but is rapidly dephosphorylated after endocytosis (17,18). Significantly, neither mutant receptor mediates detectable endomembrane recruitment of ␤-arrestins (17). We confirmed this result (not shown) and then compared the ability of FLAGtagged versions of full-length, 362T and 345T mutant V2Rs to mediate AVP-induced inhibition of the ␤ 2 AR (co-expressed in HEK293 cells without overexpression of ␤-arrestins). Consistent with observations in stably transfected cells (Figs. 1 and 2), activation of the full-length V2R strongly inhibited agonistinduced internalization of co-expressed ␤ 2 ARs in transiently transfected HEK293 cells (Fig. 5A, panels a and b). In contrast, neither the 345T (Fig. 5A, panels c and d) nor the 362T (Fig. 5A, panels e and f) mutant receptor mediated detectable inhibition of ␤ 2 AR internalization when examined under the same conditions. The ability of these mutations to abrogate the AVPinduced inhibition of ␤ 2 AR internalization was not a result of differences in expression levels of the mutant receptors, as immunofluorescence staining intensity present in individual transfected cells (estimated using an electronic camera connected to the fluorescence microscope, not shown) confirmed that 345T, 362T, and full-length V2Rs were expressed over a similar range of expression levels in individual transfected cells. The differential effects of mutant V2Rs on internalization of the co-expressed ␤ 2 AR were confirmed by examining multiple cells at random in coded specimens (Fig. 5B). We also observed extensive colocalization of the ␤ 2 AR with both the 362T and 345T mutant receptors in endocytic membranes visualized in the same cells by dual label fluorescence microscopy (arrows in Fig. 5A indicate examples of such colocalized endocytic vesicles). These results further confirm that, once the ␤ 2 AR endocytic inhibition is rescued either by overexpression of ␤-arrestins (Fig. 4) or by mutations of the V2R that block endomembrane recruitment of ␤-arrestins (Fig. 5), the ␤ 2 AR can enter a similar endocytic pathway as the V2R.
We conclude that AVP-induced activation of the V2R mediates a pronounced heterologous inhibition of agonist-induced endocytosis of the ␤ 2 AR as well as certain other GPCRs that endocytose via clathrin-coated pits. This inhibition is clearly not reciprocal, because activation of the ␤ 2 AR with saturating concentrations of agonist does not detectably inhibit agonist-induced endocytosis of the V2R, even in cells in which both receptors are expressed at closely similar levels and in which steady state internalization of the ␤ 2 AR (induced by ISO in the absence of AVP) is significantly greater than that of the V2R. The nonreciprocal nature of this inhibition is correlated with a previously described difference in the effects of the ␤ 2 AR and V2R on intracellular trafficking of ␤-arrestins (17). We established that overexpression of ␤-arrestins is sufficient to rescue the heterologous inhibition of ␤ 2 AR internalization mediated by the wild type V2R. Moreover, mutations of the V2R that prevent endomembrane recruitment of ␤-arrestins abrogate the AVP-induced inhibition of ␤ 2 AR internalization in cells expressing ␤-arrestins at endogenous levels. Thus we propose that receptor-specific intracellular trafficking of ␤-arrestins depletes the functional pool of cytoplasmic ␤-arrestins below that required to promote ligand-induced endocytosis of certain GPCRs.
Whereas receptor-specific differences in the intracellular trafficking of various GPCRs have been examined in considerable detail, relatively little is known about the functional consequences of receptor-specific differences in the intracellular trafficking of ␤-arrestins. Recent studies suggest that ␤-arrestins associated with endocytic membranes may play an important role in organizing or modulating downstream signal transduction via endocytosed GPCRs (19,20,33). However, to our knowledge the present results provide the first direct evidence for a specific functional consequence of ␤-arrestin trafficking in regulating GPCR endocytosis itself. Furthermore, we believe these results provide the first data implicating ␤-arrestins in a heterologous form of GPCR regulation, which may FIG. 5. Trafficking of ␤ 2 AR with co-expressed full-length or truncated forms of the V2R. HEK293 cells were transiently transfected with the ␤ 2 AR and either full-length (wt) V2R or one of the two truncated mutant V2 receptors, 345T or 362T (see "Experimental Procedures"). Cells were treated for 20 min at 37°C with both 10 M ISO and AVP. A, the distribution of both receptors after treatment was visualized by immunofluorescence microscopy. The upper panels show the localization of the different forms of V2R, and the lower panels show the localization of the ␤ 2 AR. The white arrows indicate examples of intracellular vesicles in which both receptors are co-localized. B, receptor internalization for both receptors was estimated by scoring of receptor localization after visualization by immunofluorescence microscopy (see "Experimental Procedures"). Only cells with detectable expression of both receptors were included in the scoring. complement previous studies suggesting heterologous regulation of certain G protein-coupled receptor kinases (34,35).
In this study we have focused primarily on V2R-mediated effects on regulated internalization of a subset of GPCRs, which have been established most definitively to endocytose in HEK293 cells by clathrin-coated pits. Therefore further studies will be necessary to examine the generality of our observations to other cell types and other GPCRs, particularly receptors that are endocytosed by "alternative" mechanisms (8,36) or further differ in their effects on intracellular trafficking of ␤-arrestins (16,37). It will also be important to determine whether the mechanism characterized in the present study is capable of mediating cross-inhibition of other important arrestin-dependent functions such as functional uncoupling of receptors from heterotrimeric G proteins or receptor-mediated signaling via mitogenic kinase cascades. The levels of receptor expression examined in the present studies, although moderate compared with other studies of transfected cells, may still be considerably higher than observed in native tissues. Thus the relevance of the present observations to endogenously expressed GPCRs remains to be determined. Nevertheless, as the process of endomembrane recruitment of ␤-arrestins can be observed in natively expressing neurons (37), we anticipate that the heterologous regulatory mechanism described in the present study may be of considerable physiological importance. For example it is tempting to speculate that this mechanism may be relevant to the recently reported ability of the V2R to bypass desensitization of myocardial adrenergic receptors in an experimental model of congestive heart failure (38). The potential physiological relevance of this regulatory mechanism notwithstanding, the present results also establish a novel experimental method by which an important functional activity of cytoplasmic ␤-arrestins can be modulated acutely in living cells. We anticipate that this approach may be useful as an adjunct to previously described methods (e.g. dominant loss-of-function mutations (39,40), antisense knockdown (41,42), and gene knockout approaches (43,44)) that typically allow cellular ␤-arrestins to be manipulated only over a much longer time scale.