Mutation of Three Residues in the Third Intracellular Loop of the Dopamine D2 Receptor Creates an Internalization-defective Receptor*

Background: Arrestin mediates G protein-independent signaling and internalization of the D2 receptor. Results: A D2 receptor mutant with modestly diminished ability to recruit arrestin and β2-adaptin did not internalize in response to agonists. Conclusion: Arrestin-mediated recruitment of receptor to AP2 is not sufficient for internalization. Significance: Receptor mutants lacking specific functions are tools for analysis of signaling mechanisms. Arrestins mediate desensitization and internalization of G protein-coupled receptors and also direct receptor signaling toward heterotrimeric G protein-independent signaling pathways. We previously identified a four-residue segment (residues 212–215) of the dopamine D2 receptor that is necessary for arrestin binding in an in vitro heterologous expression system but that also impairs receptor expression. We now describe the characterization of additional mutations at that arrestin binding site in the third intracellular loop. Mutating two (residues 214 and 215) or three (residues 213–215) of the four residues to alanine partially decreased agonist-induced recruitment of arrestin3 without altering activation of a G protein. Arrestin-dependent receptor internalization, which requires arrestin binding to β2-adaptin (the β2 subunit of the clathrin-associated adaptor protein AP2) and clathrin, was disproportionately affected by the three-residue mutation, with no agonist-induced internalization observed even in the presence of overexpressed arrestin or G protein-coupled receptor kinase 2. The disjunction between arrestin recruitment and internalization could not be explained by alterations in the time course of the receptor-arrestin interaction, the recruitment of G protein-coupled receptor kinase 2, or the receptor-induced interaction between arrestin and β2-adaptin, suggesting that the mutation impairs a property of the internalization complex that has not yet been identified.

Arrestins mediate desensitization and internalization of G protein-coupled receptors and also direct receptor signaling toward heterotrimeric G protein-independent signaling pathways. We previously identified a four-residue segment (residues 212-215) of the dopamine D2 receptor that is necessary for arrestin binding in an in vitro heterologous expression system but that also impairs receptor expression. We now describe the characterization of additional mutations at that arrestin binding site in the third intracellular loop. Mutating two (residues 214 and 215) or three (residues 213-215) of the four residues to alanine partially decreased agonist-induced recruitment of arrestin3 without altering activation of a G protein. Arrestin-dependent receptor internalization, which requires arrestin binding to ␤2-adaptin (the ␤2 subunit of the clathrin-associated adaptor protein AP2) and clathrin, was disproportionately affected by the three-residue mutation, with no agonist-induced internalization observed even in the presence of overexpressed arrestin or G protein-coupled receptor kinase 2. The disjunction between arrestin recruitment and internalization could not be explained by alterations in the time course of the receptor-arrestin interaction, the recruitment of G protein-coupled receptor kinase 2, or the receptor-induced interaction between arrestin and ␤2-adaptin, suggesting that the mutation impairs a property of the internalization complex that has not yet been identified.
Dopamine receptors belong to the large superfamily of G protein-coupled receptors (GPCRs) 4 and consist of five receptor subtypes (D [1][2][3][4][5] ) that are categorized into two subgroups (D1-like and D2-like) defined by several criteria, including primary structure, signaling properties, and pharmacological profile. Dopaminergic neurotransmission via the D 2 receptor contributes to the rewarding properties of drugs of abuse (1) and is the target of therapeutics used to treat disorders such as Parkinson's disease (2) and schizophrenia (3). The dopamine D 2 receptor activates heterotrimeric G i/o/z proteins that decrease the production of cyclic AMP (4 -7). Activated D 2 receptors are then phosphorylated by G protein receptor kinase 2/3 (GRK2/3) on serine and threonine residues in IL3 of the receptor, and subsequent receptor internalization is mediated through recruitment of arrestin3 (8 -11).
As well as being crucial for termination of G protein-dependent signaling and receptor internalization, arrestins act as scaffolds for signaling proteins (12). Agonist-induced recruitment of arrestin by a GPCR may activate a unique set of signaling pathways that have distinctive behavioral consequences. For example, the D 2 receptor mediates arrestin-dependent dephosphorylation of the protein kinase Akt by protein phosphatase 2A and a net increase in glycogen synthase kinase 3␤ activity, which contributes to amphetamine-induced locomotor activation (13,14). This signaling pathway may be involved in the pathophysiology of schizophrenia (15), suggesting that it would be valuable to develop tools, such as biased ligands (16,17) or a D 2 receptor that preferentially activates either G protein-or arrestin-dependent pathways, to investigate pathwayspecific behaviors.
In the standard model of arrestin-mediated desensitization, GPCR phosphorylation by GRK increases the affinity of arrestin for the receptor, leading to subsequent receptor desensitization and internalization (18,19). However, although IL3 of the D 2 receptor is the site of GRK2 phosphorylation and directly interacts with arrestin3, receptor phosphorylation by GRK2 may not be required for either arrestin3 recruitment or receptor internalization (20).
We have shown previously that arrestin3 binding to the D 2 receptor is greatly reduced when amino acids 212-215 (IYIV), located within IL3 of the D 2 receptor, were mutated to alanine residues. This mutation abolishes agonist-induced receptor internalization, with less effect on G protein-mediated signaling (9). This mutation also greatly impairs trafficking of the receptor to the cell membrane, thereby reducing its value as a tool to study the relationships between specific signaling pathways and dopamine-dependent behaviors. The purpose of this study was to develop a D 2 receptor mutant that is significantly impaired in arrestin3 recruitment yet maintains G protein signaling and receptor expression.
Herein we describe the creation and characterization of a mutant D 2 dopamine receptor that has modestly diminished agonist-induced recruitment of GRK2 and arrestin3 yet maintains G protein-dependent signaling and normal expression. Surprisingly, despite retaining substantial ability to recruit arrestin3 and to promote arrestin3-dependent interaction with ␤2-adaptin (the ␤2 subunit of the clathrin-associated adaptor protein AP2), this mutant D 2 receptor failed to undergo agonist-induced receptor internalization.
Cell Culture and Transfections-HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% bovine calf serum (Thermo Scientific, Logan, UT), 5% fetal clone serum (Thermo Scientific), and 1% penicillin-streptomycin solution (Thermo Scientific) at 37°C with 10% CO 2 . HEK293T cells were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin (Corning Inc.) at 37°C with 10% CO 2 . For the internalization and Western blot studies, stable expression of either wild type or mutant D 2L receptors in HEK293 cells was obtained by transfecting 6 g of D2 receptor cDNA using Lipofectamine 2000 (Invitrogen) per the manufacturer's recommended protocol. Pools of stably transfected cells were then maintained under selective pressure using 600 g/ml G418 for 3 weeks, after which the amount of G418 in the growth medium was decreased to 300 g/ml. Transient expression of arrestin3, GRK2, and/or pcDNA3.1 into HEK293 cells stably expressing wild type or mutant D 2L receptors was obtained by transfecting 3 g of cDNA using Lipofectamine 2000 (Invitrogen). After overnight incubation at 37°C, transfection complexes were rinsed off, and the cells were split into new plates and incubated for an additional 48 h before conducting experiments. Protein expression was confirmed by immunoblotting.
For all BRET studies, constructs were transiently transfected into either HEK293 or HEK293T cells using polyethyleneimine (PEI; Polysciences, Inc., Warrington, PA), as described previously (22). The quantity of transfected DNA was adjusted so that the levels of membrane expression of wild type and mutant D 2 receptors were similar as determined by radioligand binding assay or FACS, as described below.
For the arrestin3 mutant internalization studies, 2 g of GRK2 were contransfected with 2 g of pcDNA3.1, Arr3-WT, Arr3-⌬LIEFD, Arr3-R395E, or Arr3-⌬LIEFD/R395E into HEK293 cells stably expressing wild-type D 2L receptors using PEI at a ratio of 2 l of PEI (1 g/l) per 1 g of cDNA . After  overnight incubation at 37°C, cells were washed with PBS, split  into new plates, and incubated for an additional 48 h before  conducting experiments. BRET-HEK293 or HEK293T cells were transiently co-transfected with plasmids as described above. For mVenus-Arr3 and ␤2-AP-EYFP recruitment to the receptor, cells were harvested and split into two plates, one for radioligand binding assays and one for BRET assays. Forty-eight hours after transfection, cells were harvested, resuspended in assay buffer (PBS with Ca 2ϩ , MgCl 2 , and 0.2 M ascorbic acid), and plated at ϳ150,000 cells/ well in 96-well OptiPlates (PerkinElmer Life Sciences). Vehicle or quinpirole was added with 2 M coelenterazine h and incubated at 25°C. Emission of the donor (460 nm) and the acceptor (535 nm) was then measured once at 10 min or every 5 min for 30 min using a Victor TM X Light luminescence reader (PerkinElmer Life Sciences), and the BRET ratio was calculated as described previously (26). Cells used in the G protein activation, GRK2 recruitment to receptor, and arrestin3 recruitment to ␤2-AP-EYFP BRET assays were prepared and assayed as described previously (22) and were measured 2, 2, and 20 min, respectively, after the addition of quinpirole.
For the arrestin translocation assay, pcDNA3.1 encoding wild type or mutant D 2 receptors was cotransfected with plasmids containing GRK2, Rluc8-arrestin3-Sp1, and a membrane marker mem-linker-citrine-SH3. The Sp1 and SH3 helper peptides, adapted from the helper interaction FRET system (27), were used to enhance the interaction between arrestin recruited to the plasma membrane by receptor and the membrane marker, thus increasing the dynamic range of the assay. Dopamine-and quinpirole-induced arrestin recruitment to the membrane was measured after 10 min of stimulation. D 2 -mediated inhibition of cAMP was measured using a BRET-based cAMP sensor, CAMYEL. Cells transiently transfected with D 2 -WT or A4 receptor and CAMYEL were preincubated for 10 min with 10 M forskolin (Sigma) to stimulate cAMP production, followed by the addition of quinpirole. Quinpirole-mediated inhibition of forskolin-induced cAMP was measured after 10 min.
For all BRET studies, the vehicle BRET ratio was subtracted from the quinpirole BRET ratio and presented as a percentage of the E max fit of quinpirole at the wild type receptor. Data were analyzed by nonlinear regression using Prism (GraphPad, San Diego, CA). To depict the variability in the activity of D 2 -WT and mutant receptors across replicate experiments, we calculated the mean of the E max values for D 2 -WT, converted the E max value for each replicate to a percentage of that value, and then determined the mean Ϯ S.E. for all replicates.
D2 L Receptor Radioligand Binding-To compare membrane expression of D 2 receptors, cells were lysed in ice-cold hypotonic buffer (1 mM HEPES, 2 mM EDTA, pH 7.4) for 20 min at 4°C, scraped from the plate, and centrifuged at 17,000 ϫ g at 4°C for 20 min. The resulting pellet was resuspended in TBS (50 mM Tris, 120 mM NaCl, pH 7.4) and homogenized for 10 s using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY). Protein concentration of the membranes was determined using a BCA protein assay kit (Thermo Scientific) and 5 g of protein was incubated in TBS containing 0.02% BSA (Fisher) at a volume of 1 ml with [ 3 H]YM-09151-2 for 1 h at 25°C. Nonspecific binding was assessed using (ϩ)-butaclamol (2 M). Membranes were then harvested, and radioactivity was measured as described previously (9). Data were analyzed by nonlinear regression using GraphPad Prism to determine K d and B max values.
FACS-Transfected cells expressing N-terminal FLAG-or c-Myc-tagged D 2 receptor were dissociated, and surface receptors were labeled using mouse anti-FLAG or anti-c-Myc antibodies and goat anti-mouse-Alexa647 antibodies diluted 1:400 in PBS (with 0.1% BSA and 0.1% NaN3) and quantitated using a C6 Flow Cytometer (Accuri). This FACS assay was used to equalize receptor expression in the assays for G protein activation ( Figs. 1 and 3, A and B), inhibition of cAMP (Fig. 1D), GRK2-Venus recruitment (Fig. 6), and the interaction between arrestin and ␤ 2 -adaptin ( Fig. 9).
Internalization Assay-D 2 receptor internalization was measured by using a whole-cell [ 3 H]sulpiride binding assay described previously (28). HEK293 cells stably expressing wildtype or mutant D 2 receptors were transiently transfected with pcDNA3.1, wild type or mutant arrestin3, and/or GRK2 as described above. After overnight incubation with the transfection complexes, cells were harvested and split into three plates, one for confirmation of arrestin3 and/or GRK2 protein expression (described below) and two for the internalization assay. Forty-eight hours after transfection, cells were incubated with either a vehicle control, quinpirole (10 M), or dopamine (10 M) for 30 min at 37°C, washed three times with ice-cold PBS, and scraped into 2 ml of cold assay buffer (PBS containing 2 mM EDTA and 0.01% BSA). The total number of cells in each condition was determined using a NucleoCounter (Chemometec, Lillerød, Denmark), and 300,000 cells were incubated in assay buffer at a volume of 1 ml with the membrane-impermeant antagonist [ 3 H](Ϫ)-sulpiride (final concentration, 5 nM) for 3 h at 4°C with or without unlabeled haloperidol (10 M). Membranes were harvested, and radioactivity was measured as described previously (9).
Immunoblot-To confirm expression of arrestin3 and/or GRK2, cells were washed twice with cold PBS and lysed in icecold radioimmune precipitation assay lysis buffer (Millipore, Billerica, MA) containing phosphatase inhibitor mixture set II (1:100) and protease inhibitor mixture set III (1:200) for 20 min with gentle agitation at 4°C. Samples were then centrifuged for 15 min (14,000 ϫ g, 4°C), and the protein concentration of the supernatant was determined using a BCA protein assay. Proteins (30 -40 g total) were separated by SDS-PAGE on Criterion TM 10% Tris-HCl precast gels (Bio-Rad) and transferred onto PVDF membranes (Millipore). Membranes were washed once in TBS, blocked in blocking buffer (TBS containing 5% BSA) for 1 h at room temperature, and incubated in mouse anti-␤-arrestin2 (1:300) and rabbit anti-GRK2 (1:1000) antibodies overnight at 4°C. After primary antibody incubation, membranes were washed three times for 5 min in TBS containing 0.1% Tween 20 (TBST; Sigma-Aldrich), incubated with Alexa Fluor 488 goat anti-mouse (1:500) and Alexa Fluor 568 goat anti-rabbit (1:500) secondary antibodies diluted in blocking buffer for 2 h at room temperature, and then washed three times for 5 min in TBST and once for 5 min in TBS. Fluorescence was detected using a Typhoon 9410 variable mode imager (GE Healthcare). Antibody was then stripped off the membrane using Restore TM Western blot stripping buffer (Thermo Scientific) per the manufacturer's protocol and vigorously washed four times for 5 min in TBS. The membrane was then blocked in blocking buffer for 1 h at room temperature and incubated with rabbit anti-actin (1:500) diluted in blocking buffer overnight at 4°C. Membranes were then washed, incubated in Alexa Fluor 568 goat anti-rabbit, and washed again, and fluorescence was detected as described above.

D 2 -A4 Is Not Suitable for in Vivo Analyses of D 2 -mediated
Pathways-Mutation of amino acid residues 212-215 in IL3 of the D 2 receptor to alanines (D 2 -A4) creates a receptor that does not recruit arrestin3 and retains some G protein signaling but is poorly expressed at the cell membrane (9). To confirm these results, radioligand binding was used to determine D 2 -A4 membrane expression, and BRET-based assays were used to monitor receptor-mediated G protein activation and arrestin3 recruitment. When a constant amount of plasmid DNA encoding wild type D 2 receptor (D 2 -WT) or D 2 -A4 was transiently transfected into HEK293 cells, D 2 -A4 was expressed at ϳ10% of the density of D 2 -WT (data not shown). For the experiments in Fig. 1, D 2 -A4 DNA was adjusted to obtain expression equivalent to D 2 -WT.
The ability of D 2 -WT and D 2 -A4 to inhibit forskolin-mediated activation of adenylyl cyclase was measured to determine the capacity of each receptor to signal through G proteins. Quinpirole stimulation of D 2 -WT produced a dose-dependent increase in the inhibition of cyclic AMP accumulation (Fig. 1A), whereas activation of D 2 -A4 with quinpirole resulted in less inhibition of adenylyl cyclase activity (Fig. 1, A and B). This suggests that D 2 -A4 is a poor activator of G proteins. To confirm this hypothesis, D 2 -A4-mediated G protein activation was measured using a BRET assay in which agonist-bound receptor induces a separation of the G␣ energy donor (G␣ i1 -91-Rluc8) from the complemented G␤␥ acceptor (mVenus-G␤ 1 ␥ 2 ), thus decreasing the BRET signal ( Fig. 1C) (22). Activation of D 2 -WT produced a dose-dependent increase in G protein activation (Fig. 1D), whereas activation of D 2 -A4 with either quinpirole or dopamine resulted in significantly less G protein activation than seen at the wild-type receptor (Fig. 1, D and E). In combination, the cyclic AMP and BRET data suggest that D 2 -A4 is a poor G protein activator relative to D 2 -WT, which appears to differ from our previous results using cells stably expressing D 2 -A4 and D 2 -WT; under those conditions, agonist stimulation of D 2 -A4 resulted in a decrease in cAMP that was similar to that observed for the wild type receptor, but a decrease in the potency of dopamine in one experiment in that earlier work suggests that a receptor reserve might have obscured a partial loss of coupling efficiency (9). We used a BRET assay (24) to monitor the recruitment of mVenus-arrestin3 to Rluc8-tagged-D 2 -WT and D 2 -A4 receptors ( Fig. 2A). Activation of the wild-type receptor by quinpirole produced a dose-dependent increase in the BRET ratio ( Fig. 2B), which is indicative of the interaction between the receptor and arrestin3 (26). As we reported previously, quinpirole activation of D 2 -A4 produced essentially no increase in the BRET ratio (Fig. 2, B and C) even when membrane expression of D 2 -A4 was similar to the wild type receptor.
Taken together, these results suggest that despite its inability to recruit arrestin, the D 2 -A4 receptor would be a poor candidate for studying pathway-specific D 2 receptor-mediated behavior, given the impairment of its expression and G protein signaling. Based on these results, we sought to create a receptor that has an impaired interaction with arrestin3 but unimpaired ability to activate G proteins and to be expressed at the cell membrane.
D 2 -A3 and D 2 -A2 Receptor G Protein Activation and Arres-tin3 Recruitment-Residues 213-215 or residues 214 and 215 within IL3 of the D 2 receptor were mutated to alanines (D 2 -A3 and D 2 -A2, respectively), and receptor activation of G protein and interaction with arrestin3 were evaluated using the techniques described above. Agonist stimulation of D 2 -A3 and D 2 -A2 produced a dose-dependent increase (Fig. 3A) in G protein activation that was similar to that of D 2 -WT (Fig. 3B). In contrast, the extent of quinpirole-induced recruitment of arres-tin3 to the receptor was significantly less for the D 2 -A3 receptor than for D 2 -WT, with a trend for reduced recruitment of arrestin by the D 2 -A2 receptor mutant (59 and 76% of the wild type receptor, respectively; Fig. 3, C and D). Dopamine stimulation also produced modestly reduced arrestin3 recruitment to the receptor by the D 2 -A3 and D 2 -A2 receptor mutants that was not statistically different from recruitment by D 2 -WT (Fig. 3D). To monitor receptor-induced translocation of arrestin to the plasma membrane in a manner that does not require Rluc8 to be attached to the C terminus of the receptor, matching the internalization assay used below, we developed an assay in which untagged receptors were cotransfected with BRET sensors Rluc8-arrestin3-Sp1 and a plasma membrane marker, mem-linker-citrine-SH3 (see "Experimental Procedures"). Relative arrestin translocation to the membrane induced by the wild type D 2 receptor, D 2 -A3, and D 2 -A2 closely matched the recruitment to the receptor observed using Rluc8-tagged receptors, except that none of the reductions in agonist-induced translocation of arrestin to the membrane was significant ( Fig. 3, E and F). Sulpiride (10 M) completely blocked arrestin translocation in response to activation of all three receptors, demonstrating the specificity of the response in our novel BRET arrestin translocation assay (data not shown). Importantly, despite modest reductions in arrestin membrane translocation and recruitment to the receptor, both D 2 -A3 and D 2 -A2 receptors still robustly recruited arrestin3 in response to agonist stimulation.
Receptor Internalization-One of the functional consequences of arrestin3 interaction with the D 2 receptor is receptor internalization. Agonist-induced receptor internalization was measured using a whole-cell binding assay in HEK293 cells that stably express D 2 -WT, D 2 -A4, D 2 -A3, or D 2 -A2. We hypothesized that the amount of agonist-induced receptor endocytosis would be positively correlated with agonist-induced recruitment of arrestin3. Indeed, after treatment with quinpirole for 30 min, cell surface expression of the D 2 -WT receptor was significantly decreased (17 and 30% in the absence and presence of overexpressed arrestin3, respectively; Fig. 4A), whereas D 2 -A4 did not internalize at all in response to agonist stimulation, even when arrestin3 was overexpressed (Fig. 4, A and B). Quinpirole stimulation of D 2 -A2 receptors produced receptor internalization that was 44% of internalization of the wild-type receptor (Fig. 4A). Overexpression of arrestin3 with the D 2 -A2 receptor increased agonist-induced receptor internalization, consistent with the ability of this receptor to recruit arrestin3, although even in the presence of overexpressed arrestin, quinpirole-induced internalization of D 2 -A2 was less than that of D 2 -WT (Fig. 4A). Surprisingly, although D 2 -A3 also partially recruited arrestin, the D 2 -A3 mutant did not undergo either dopamine or quinpirole-induced internalization, even in cells that overexpressed arrestin3 (Fig. 4, A and B).
Kinetics of Arrestin3 Recruitment to D 2 Receptor-The discrepancy between the amount of arrestin3 recruited to the D 2 -A3 receptor and amount of receptor internalized could be due to a difference in the time of measurement between the assays (10 min of agonist stimulation in the arrestin recruitment BRET assay and 30 min in the internalization assay) (i.e. the interaction of arrestin3 and D 2 -A3 might be

Creating an Internalization-defective D 2 Receptor
NOVEMBER 28, 2014 • VOLUME 289 • NUMBER 48 relatively transient and absent after 30 min of agonist stimulation). To address this possibility, the time course of the BRET response to quinpirole or dopamine was determined for the D 2 -WT and D 2 -A3 receptors. The onset and gradual decay of the response appeared similar for the two receptors, with the D 2 -A3 BRET response being significantly less than the response to the wild type receptor after 10 -30 min of quinpirole stimulation (Fig. 5A). Notably, the ratio of the BRET response for D 2 -A3 to the response for D 2 -WT was stable from 10 min (0.69) to 30 min (0.66) (Fig. 5A). Similar results were observed for the time course of the response of the two receptors to dopamine (data not shown). Thus, the failure of D 2 -A3 to internalize does not reflect a more transient association with arrestin3. D 2 -A3 Interaction with Arrestin3 Occurs at the Cell Membrane-Because the BRET response being measured is from populations of whole cells rather than at the subcellular level (29), the interaction between D 2 -A3 and Arr3 may be occurring at a location other than the cell membrane (i.e. within intracellular compartments, from which, by definition, internalization cannot occur). To confirm that agonist-induced BRET takes place at the cell membrane, we pretreated cells expressing D 2 -WT or D 2 -A3 with the membrane-impermeable D 2 receptor antagonist, (S)-(Ϫ)-sulpiride (30), before treatment with quinpirole (1 M). When the cells were pretreated with increasing concentrations of sulpiride, there was a dose-dependent decrease in quinpirole-induced arrestin3 interactions with both receptors (Fig. 5B). As would be expected, D 2 -A3 recruited significantly less arrestin3 than D 2 -WT in response to quinpirole (72 Ϯ 3%, p ϭ 0.02, Student's t test). The potency of sulpiride, however, was similar (Table 1), and the BRET signal was completely abolished in both the D 2 -WT and D 2 -A3 receptor-expressing cells at concentrations of sulpiride above 1 M. These results suggest that quinpirole-induced recruitment of arrestin3 is due to activation of receptors on the cell membrane and not receptors expressed in intracellular compartments that are not subject to internalization.

GRK2 Recruitment to the D 2 -WT and D 2 -A3 Receptors-Typ-
ically, phosphorylation of GPCRs by GRKs produces an increased affinity of arrestin for the receptor, resulting in endocytosis (31). We hypothesized that the A3 mutation within the D 2 receptor may impair the ability of the receptor to interact with GRK2, subsequently disrupting the ability of the receptor to recruit arrestin3 and undergo receptor internalization. To test this hypothesis, we first measured the amount of BRET between GRK2-Venus and D 2 -WT or D 2 -A3 receptors fused to Rluc8 (Fig. 6A). Quinpirole produced a dose-dependent increase in recruitment of GRK2 to D 2 -WT and, to a lesser extent, to D 2 -A3 (62% of WT) (Fig. 6, B and C). Similarly, dopamine-induced recruitment of GRK2 to D 2 -A3 was significantly reduced when compared with recruitment to D 2 -WT (Fig. 6C).
The functional consequences of GRK2 interactions with D 2 -WT and D 2 -A3 were then assessed by overexpressing GRK2 and measuring recruitment of arrestin3 and agonist-induced receptor internalization. GRK2 was overexpressed in cells expressing D 2 -WT-Rluc8 or D 2 -A3-Rluc8 and mVenus-Arr3 ( Fig. 2A). Similar to previous experiments, quinpirole stimula-   NOVEMBER 28, 2014 • VOLUME 289 • NUMBER 48 tion induced less recruitment of Arr3 to D 2 -A3 than to D 2 -WT (Fig. 7A). Interestingly, when GRK2 was overexpressed with D 2 -WT and D 2 -A3, at both receptors there was an almost 5-fold leftward shift in the quinpirole dose-response curve ( Fig.  7A and Table 1) and an increase in the maximal quinpiroleinduced interaction with arrestin3 (53 and 37%, respectively; Fig. 7, A and B). Furthermore, when GRK2 was overexpressed with D 2 -A3, the E max for quinpirole-induced recruitment of arrestin3 to the receptor was indistinguishable from that for D 2 -WT expressed alone (i.e. without GRK2; Fig. 7A). These results suggest that although the A3 mutation partially disrupts interactions with GRK2, GRK2-enhanced recruitment of arres-tin3 remains intact.

Creating an Internalization-defective D 2 Receptor
Overexpression of GRK2 increased the amount of agonistinduced internalization of D 2 -WT (Fig. 7D), and internalization was further enhanced by overexpression of both GRK2 and arrestin3. Similar to previous results, quinpirole failed to induce internalization of D 2 -A3 even when GRK2 and arrestin3 were overexpressed. Although overexpression of GRK2 enhanced D 2 -A3-mediated recruitment of arrestin3 to a level comparable with that of D 2 -WT alone (Fig. 7, A and B, and Table 1), overexpression of GRK2 alone or in combination with arrestin3 failed to rescue internalization of D 2 -A3 (Fig. 7D).

The Role of Arrestin Interactions with Clathrin and AP2 in D 2
Receptor Internalization-Arrestin-mediated endocytosis of GPCRs, such as ␤ 2 -adrenergic receptor, has been shown to depend on the interaction of arrestin with clathrin and the endocytic adaptor AP2, which targets the receptors to clathrincoated pits (32). A possible explanation for our results is that the A3 mutation in D 2 disrupts agonist-induced receptor endocytosis by preventing the association of the receptor or arres-tin3 with these endocytic proteins. The primary clathrin binding site in arrestin is an LX(D/E) motif (33), where X is any residue and is a bulky hydrophobic residue, whereas AP2 binds to a positively charged arginine residue; both binding sites are located in the C-terminal region of arrestin (34). Dominant negative variants of arrestin carrying either deletions or mutations of these binding sites are deficient in mediating internalization and have been used to probe the mechanisms of arrestin-dependent internalization of GPCRs (33)(34)(35).

values from G protein activation and recruitment assays
Quinpirole dose-response curves were generated, and the pEC 50 values were determined using GraphPad Prism. Each value represents the mean Ϯ S.E. of 3-7 independent experiments. The figure in which the corresponding dose-response curves are depicted is also indicated.  duced robust internalization of the receptor that was significantly greater than cells expressing the control plasmid (Fig. 8).
In contrast, none of the three arrestin mutants increased D 2 receptor internalization over the amount of internalization observed in the absence of overexpressed arrestin, indicating that, indeed, interactions with both AP2 and clathrin are required for arrestin3-mediated internalization of the D 2 receptor.

␤2-Adaptin
Interaction with the Receptor-Arrestin Complex-Adaptor protein complexes are essential for cargo selection and subsequent clathrin-mediated endocytosis (36). For example, agonist stimulation of some GPCRs has been shown to induce a direct interaction between arrestin3 and ␤2-adaptin to initiate clathrin-mediated receptor endocytosis (25,37), although this has not been studied in the D 2 receptor. We hypothesized that the failure of D 2 -A3 to internalize in response to quinpirole may be due to its inability to induce the interaction between arres-tin3 and ␤2-adaptin or to directly interact with adaptor protein complexes, despite having the ability to partially recruit GRK2 and arrestin3.
To test this hypothesis, the interaction between Arr3-Rluc8 and ␤2-adaptin-EYFP was measured as described previously (25) in cells containing untagged D 2 -WT or D 2 -A3 receptors (Fig. 9A). Similar to the interaction between receptor and arrestin, quinpirole stimulation of D 2 -WT and D 2 -A3 produced a dose-dependent increase in the interaction between arrestin3 and ␤2-adaptin, and the maximal response was significantly less for the mutant receptor (Fig. 9, B and C). The reduced interaction between ␤2-adaptin and arrestin3 mediated by D 2 -A3 receptor is most likely due to the reduced ability of the receptor to interact with arrestin3.  We sought to determine whether the agonist-induced interaction between arrestin3 and ␤2-adaptin produced an interaction between D 2 -WT and ␤2-adaptin, critical for internalization, that might not be observed between D 2 -A3 and ␤2-adaptin. HEK293 cells were transiently transfected with D 2 -WT-Rluc8 or D 2 -A3-Rluc8 and ␤2-adaptin-EYFP, and the interaction between the two proteins in response to quinpirole stimulation was monitored using BRET (Fig. 10A). Quinpirole stimulation of either D 2 -WT or D 2 -A3 produced a negligible increase in the interaction between the receptor and ␤2-adaptin (Fig. 10B). When the receptor-␤2adaptin BRET pair was co-transfected with arrestin3 and GRK2, however, robust recruitment of ␤2-adaptin was seen in response to agonist stimulation (Fig. 10, B and D). The BRET observed was significantly lower for D 2 -A3 than for the wild type D 2 receptor (Fig. 10, C and E). The reduced response for D 2 -A3 is similar in magnitude to the mutant's reduced ability to recruit arrestin3 (Fig.  3, C and D) and promote the interaction between arrestin3 and ␤2-adaptin (Fig. 9, B and C). These data indicate that D 2 -A3 has a reduced ability to recruit arrestin3, resulting in a diminished but still quite substantial ability to bind ␤2-adaptin.

DISCUSSION
We previously identified a four-residue segment of the D2 receptor, residues 212-215 in IL3, that is required for recruitment of arrestin and receptor internalization but not for G protein-dependent signaling (9), a finding that we confirmed in the present study using agonist-induced BRET between the D2 receptor and arrestin3 to measure arrestin recruitment. The D 2 -A4 mutant also demonstrated a considerable reduction in its ability to activate heterotrimeric G proteins, as determined by measuring the inhibition of cyclic AMP accumulation and BRET between G␣ i1 and mVenus-G␤ 1 ␥ 2 . Thus, we made more limited mutations in an attempt to selectively impair recruitment of arrestin. Instead, we identified a mutant that failed to undergo agonist-induced internalization despite retaining a substantial ability to recruit arrestin3.
Mutants D 2 -A3 and D 2 -A2 were created with residues 213-215 or residues 214 and 215, respectively, substituted with alanine. In contrast to D 2 -A4, both of these mutants had only modestly decreased ability to recruit arrestin or induce translocation to the membrane, which was more evident with the synthetic agonist quinpirole than with dopamine and significantly decreased only for quinpirole at D 2 -A3-Rluc8. Both of the mutants were able to fully activate G␣ i .
As we reported previously (9), the agonist quinpirole was unable to induce internalization of the D 2 -A4 receptor, compared with robust internalization of the wild type receptor that was enhanced by overexpression of arrestin3. Treatment with quinpirole induced partial internalization of D 2 -A2 that was commensurate with the partial ability of that mutant receptor to recruit arrestin. In contrast, D 2 -A3 displayed little or no quinpirole-or dopamineinduced internalization, despite retaining substantial ability to recruit arrestin.
We evaluated several hypotheses for the disjunction between the ability of D 2 -A3 to recruit arrestin and its inability to be internalized. One hypothesis was that, although D 2 -A3 recruited a substantial amount of arrestin3 when measured after 10 min of agonist treatment, the interaction between D 2 -A3 and arrestin3 was less stable than that between the wild type D 2 receptor and arrestin3. We determined, however, that the time course of the recruitment of arrestin3 by the wild type and mutant receptors was indistinguishable for both dopamine and quinpirole. A second hypothesis was that the interaction between arrestin3 was taking place somewhere other than at the cell membrane and therefore would not lead to the receptor being internalized; however, the ability of the hydrophilic antagonist sulpiride to inhibit the quinpirole-induced response indicated that the response came from receptors at the plasma membrane. A third hypothesis was that the mutation impaired the ability of GRK2 to bind to the receptor, and, indeed, we determined that the BRET response between the D 2 receptor and GRK2 was decreased by the A3 mutation. In vitro data using fragments of the receptor strongly support the hypothesis that the A3/A4 sequence is required for binding of arrestin3 to IL3 of the D 2 receptor (9); it is, however, unknown whether the same is true for GRK2. Despite the reduction in the agonist-induced BRET response, overexpression of GRK2 enhanced quinpirole-induced arrestin3 recruitment to D 2 -A3 by approximately the same percentage as recruitment of arrestin3 to the wild type D2 receptor, and quinpirole concentration-response curves for both receptors were shifted to the left in the presence of added GRK2, suggesting that altered binding of GRK2 to D 2 -A3 does not underlie its inability to be internalized.
Knockdown of either GRK2 or arrestin3 significantly reduces the amount of agonist-induced D 2 receptor internalization (10,38), and overexpression of either protein enhances the amount of internalization seen in response to agonist (8,20,39,40). Furthermore, this response is potentiated when GRK2 and arrestin3 are overexpressed together (8,20,39). Thus, it is possible that the reduced internalization of D 2 -A3 simply reflects its diminished GRK2 and arrestin3 recruitment, with a partial recruitment deficit somehow translating into a more severe internalization deficit. However, because overexpression of GRK2 increased the amount of arrestin3 recruitment by D 2 -A3 to the level recruited by the wild type receptor, as quantified by the BRET assay, overexpression of GRK2 would be predicted to increase agonist-induced internalization of D 2 -A3 to the same level observed for the wild type receptor in the absence of overexpressed GRK2. This was found not to be the case because little or no internalization of D 2 -A3 was detected even when arrestin3 recruitment was normalized by GRK2 overexpression.
Arrestin-mediated endocytosis via clathrin-coated pits requires binding of arrestin to both clathrin and AP2, and deletion of the clathrin binding motif (Arr3-⌬LIEFD) or mutation of the arginine that is necessary for binding to AP2 (Arr3-R395E) prevents binding to clathrin and AP2, respectively, and greatly impairs agonist-mediated internalization of the ␤ 2 -adrenergic receptor (35). Using these mutants, we determined that arrestin-dependent D 2 receptor internalization was dependent on the interaction of arrestin3 with both AP2 and clathrin and that preventing either interaction produced complete inhibition of arrestin3-mediated internalization.
AP2 is a heterotetramer that consists of two large subunits (␣2 and ␤2), a medium subunit (2), and a small subunit (2); the ␤2 (␤2-adaptin) and 2 subunits determine cargo selection by direct interaction with the cargo or accessory proteins (41). Agonist stimulation of GPCRs induces a direct interaction between receptor-bound arrestin3 and ␤2-adaptin to initiate clathrin-mediated receptor endocytosis (25,37). We tested the hypotheses that the A3 mutation within the D 2 receptor prevents the receptor from promoting the interaction between ␤2-adaptin and arrestin3 or alters an arrestin-mediated interaction between the receptor and ␤2-adaptin. Either effect could underlie this receptor's inability to internalize in response to quinpirole. Thus, we measured BRET between either arrestin3 or the D 2 receptor and ␤2-adaptin-EYFP. The quinpirole-induced interaction between arrestin3 and ␤2-adaptin was reduced but still quite substantial in cells expressing D 2 -A3 compared with D 2 - WT. Further, although a very limited quinpirole-induced change in BRET was observed when receptor and ␤2-adaptin were co-expressed, the addition of arrestin3 and GRK2 enabled a robust quinpirole-or dopamine-induced interaction between these proteins. This is the first time, to our knowledge, that an energy transfer methodology has been used to measure the interaction between a GPCR and ␤2-adaptin. The BRET response between D 2 -A3 and ␤2-adaptin was also robust but significantly less than the response to the wild type receptor. That this closely recapitulated the mutation-induced decrease in arrestin3 recruitment indicates that it reflects the latter process rather than a specific inability of D 2 -A3 to induce an interaction between arrestin3 and ␤2-adaptin. The extremely low BRET response between the D 2 receptor and ␤2-adaptin in the absence of overexpressed arrestin and GRK2 suggests that functional interaction of overexpressed receptor with endogenous arrestins and GRKs in HEK293 cells is very low; nevertheless, agonist-induced internalization of D 2 -WT without overexpressed arrestin/GRK was greater than internalization of D 2 -A3 with overexpressed arrestin/GRK when robust interaction with ␤2-adaptin was observed. This comparison between the wild type and mutant receptors highlights the qualitatively distinct inability of D 2 -A3 to undergo agonist-induced internalization.
In conclusion, D 2 -A3 is a partially signaling-biased receptor that fully activates G protein but has a moderately decreased ability to recruit arrestin3 and GRK2. D 2 -A3 was also deficient in arrestin-dependent interaction with ␤2-adaptin, most likely reflecting diminished recruitment of arrestin3 to the receptor. Despite a significant capability for recruitment of arrestin and ␤2-adaptin, D 2 -A3 displayed virtually no agonist-induced internalization even in the presence of overexpressed arrestin3 and GRK2. Arrestin probably serves as a scaffold to bring the receptor and AP2 sufficiently close to allow BRET between the C terminus of the receptor and ␤2-adaptin. Nonetheless, in the case of D 2 -A3, the precise conformation and interactions between these components may not be sufficient for internalization. Furthermore, despite the proximity between D 2 -A3 and ␤2-adaptin, D 2 -A3 may not be able to directly interact with other subunits of AP2 (42,43) or with another protein or lipid required for wild type receptor internalization. The inability of D 2 -A3 to internalize may also reflect characteristics of the dynamic interaction among GPCR, arrestin, and AP2 that are not captured in these BRET assays. Regardless of the molecular mechanism, the identification of this mutant provides us with a construct suitable for exploring the physiological roles of D2 receptor internalization in more complex systems and in vivo.