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Molecular Endocrinology 21 (9): 2242-2254
Copyright © 2007 by The Endocrine Society

Roles of Protein Kinase C and Actin-Binding Protein 280 in the Regulation of Intracellular Trafficking of Dopamine D3 Receptor

Eun-Young Cho, Dong-Im Cho, Jae H. Park, Hitoshi Kurose, Marc G. Caron and Kyeong-Man Kim

Department of Pharmacology (E.-Y.C., D.-I.C., K.-M.K), College of Pharmacy, Chonnam National University, Kwang-Ju 500-757, Korea; Department of Biochemistry and Cellular and Molecular Biology (J.H.P), University of Tennessee, Knoxville, Tennessee 37996; Department of Pharmacology and Toxicology (H.K.), Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan; and Department of Cell Biology (M.G.C.), Duke University Medical Center, Durham, North Carolina 27710

Address all correspondence and requests for reprints to: Dr. Kyeong-Man Kim, Department of Pharmacology, College of Pharmacy, Chonnam National University, Kwang-Ju 500-757, Korea. E-mail: kmkim{at}chonnam.ac.kr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
D3 dopamine receptor (D3R) is expressed mainly in parts of the brain that control the emotional behaviors. It is believed that the improper regulation of D3R is involved in the etiology of schizophrenia. Desensitization of D3R is weakly associated with G protein-coupled receptor kinase (GRK)/ß-arrestin-directed internalization. This suggests that there might be an alternative pathway that regulates D3R signaling. This report shows that D3R undergoes robust protein kinase C (PKC)-dependent sequestration that is accompanied by receptor phosphorylation and the desensitization of signaling. PKC-dependent D3R sequestration, which was enhanced by PKC-ß or -{delta}, was dynamin dependent but independent of GRK, ß-arrestin, or caveolin 1. Site-directed mutagenesis of all possible phosphorylation sites within the intracellular loops of D3R identified serine residues at positions 229 and 257 as the critical amino acids responsible for phorbol-12-myristate-13-acetate (PMA)-induced D3R phosphorylation, sequestration, and desensitization. In addition, the LxxY endocytosis motif, which is located between residues 252 and 255, was found to play accommodating roles for PMA-induced D3R sequestration. A continuous interaction with the actin-binding protein 280 (filamin A), which was previously known to interact with D3R, is required for PMA-induced D3R sequestration. In conclusion, the PKC-dependent but GRK-/ß-arrestin-independent phosphorylation of D3R is the main pathway responsible for the sequestration and desensitization of D3R. Filamin A is essential for both the efficient signaling and sequestration of D3R.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE DOPAMINE RECEPTORS differ in their pharmacological profiles and cellular distributions. Based on their structural, pharmacological, and functional characteristics, the dopamine receptors are classified into two subfamilies, D1- and D2-like dopamine receptors (1, 2, 3). With the advent of molecular biological techniques, the D1-like receptors have been subdivided into the D1 and D5 receptors (D1R, D5R) (4, 5, 6), whereas the D2, D3, and D4 receptors constitute the D2-like receptors (D2R, D3R, D4R) (7, 8, 9).

Among the dopamine receptor subtypes characterized, D2R and D3R are the main targets of most currently used neuroleptics. Interestingly, these two receptors show different spatial expression patterns within the brain; D2R and D3R are heavily expressed in the regions responsible for motor functions and emotional mental functions, respectively (7, 8, 10). In addition, a number of genetic studies have shown that abnormal functions of D3R are closely related to the etiology of schizophrenia (11, 12, 13, 14). Disturbances in the motor and endocrine functions are the most serious problems caused by the neuroleptics currently used. Hence, the development of specific ligands or the ability to manipulate the specific signaling pathways of D3R has been suggested as a means to separate the desired therapeutic activities from the unwanted side effects of neuroleptics.

D2R and D3R share most of the signaling pathways such as the inhibition of adenylyl cyclase, extracellular acidification, mitogenesis, ERK activation, the inhibition of dopamine synthesis, and ion channel regulation (for review, see Ref. 15), which is possibly due to high similarity in their amino acids composition (46% overall amino acid homology and 78% identity in the transmembrane domain) (16). Furthermore, recent studies have shown that some mesencephalic dopaminergic neurons express both D2R and D3R even though D3R is more densely expressed in the limbic area (8, 10). Because D2R and D3R have virtually the same signaling pathways and are expressed in the same cells, we have been focusing on the differences in their regulatory mechanisms and have found them to have distinct regulatory or signaling mechanisms (17, 18).

The desensitization of G protein-coupled receptors (GPCRs) is generally accomplished through two distinct molecular events: the homologous or heterologous desensitization pathways (19). The homologous pathway involves GPCR kinase (GRK)-mediated receptor phosphorylation followed by an association with arrestin, resulting in the rapid attenuation of GPCR signaling. Arrestins also work as adaptors for the endocytosis of GPCRs at a later stage. In comparison, the heterologous desensitization pathway involves the phosphorylation of the receptor proteins by protein kinase A (PKA) or protein kinase C (PKC), which might be activated by other cellular pathways.

Previous studies have shown that the molecular determinants involved in the intracellular trafficking of D2R and D3R are confined to their relatively large third intracellular loops (17). Nevertheless, D2R and D3R undergo remarkably different degrees of arrestin-directed trafficking to the clathrin-coated pits, i.e. D2R undergoes robust GRK/ß-arrestin-dependent endocytosis, whereas only a negligible fraction of D3R is internalized in clathrin-coated pits (17, 20). The GRK/ß-arrestin-mediated homologous pathway does not play an important role in the regulation of D3R. Therefore, this study investigated roles of the heterologous pathway and shows that that phorbol-12-myristate-13-acetate (PMA)-induced phosphorylation followed by sequestration is the main pathway for the desensitization of D3R. The critical phosphorylation residues and an endocytosis motif that are responsible for the sequestration of D3R were located. In addition, filamin A was identified as the molecular component responsible for D3R sequestration.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PKC-Dependent Sequestration Is the Main Intracellular Trafficking Pathway of D3R
As reported previously (17), only a fraction of D3R was sequestered in response to dopamine treatment only when the exogenous GRK or ß-arrestin was coexpressed (Fig. 1A1Go). D2R, which was used as a positive control, showed clear GRK/ß-arrestin-dependent sequestration in response to the dopamine treatment. As opposed to the unnoticeable dopamine-induced D3R sequestration, the PMA treatment caused intensive D3R sequestration, whereas the same treatment had only a marginal effect on D2R sequestration (Fig. 1A1Go). This suggests that both homologous and heterologous desensitization pathways are responsible for regulating D2R and D3R, respectively. These results were confirmed in two neural-derived cells, Neuro2a and NG108–15. As shown in Fig. 1A2Go, the same patterns of results were observed in Neuro2a cells: D2R and D3R were mainly sequestered by dopamine and PMA treatment, respectively. Essentially the same results were observed in NG108–15 cells (data not shown). These results suggest that the differential intracellular trafficking properties of D2R and D3R, which were observed in human embryonic kidney (HEK)-293 cells, would have physiological relevance in the nervous system.


Figure 1
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  		Fig. 1. Determination of the Major Sequestration Pathway of D3R

A1, Comparison of the sequestration pathways between D3R and D2R in HEK-293 cells. Cells were transfected with either 2 µg of D3R or D2R in pCMV5 using calcium phosphate method together with 2 µg ß-arrestin 2 in pCMV5 or GRK3 in pRK5 per 100-mm dish. The cells were treated with 10 µM dopamine for 1 h or with 1 µM PMA for 30 min. The sequestration of the receptor proteins was measured by flow cytometry, as described previously (17 ). The levels of D2R and D3R expression were approximately 2 pmol/mg protein. *, P < 0.1 compared with Mock/DA group; ***, P < 0.001 compared with Mock/DA group; ###, P < 0.001 compared with Mock/DA or GRK3/DA group. A2, Comparison of the sequestration pathways between D3R and D2R in Neuro2a cells. Cells were transfected with either 10 µg D3R or D2R in pCMV5 using Lipofectamine 2000 (Invitrogen) together with 5 µg GRK2 in pRK5. The cells were treated with 10 µM dopamine for 1 h or with 1 µM PMA for 1 h. The levels of D2R and D3R expression were approximately 1.7 pmol/mg protein. *, P < 0.1 compared with Mock/DA group; ***, P < 0.001 compared with Mock/DA group; ###, P < 0.001 compared with Mock/DA or GRK2/DA group. B, Effects of various GRKs on the phosphorylation of D3R. Phosphorylation studies were carried out using HEK-293 cells transfected with D3R tagged with the hemagglutinin (HA) epitope at the N-terminal end (HA-D3R) with or without 2 µg GRK2, -3, or -5 in pcDNA1/AMP (B1). The negative control cells (left two lanes) were not transfected with HA-D3R. As a positive control, D2R tagged with Flag epitope at N-terminal end was used (B2). Receptor phosphorylation was determined after 5 min stimulation with 10 µM dopamine. Receptor expression was determined using radioligand binding, and the same amount of receptor proteins was loaded for each lane. The phosphorylated receptors were quantified using a phosphor imager or were visualized by autoradiography. C, The effects of PKA and PKC on the phosphorylation of D3R. The cells were treated with 1 µM forskolin (PKA activator) or PMA (PKC activator) for 5 min. The data represent results from three independent experiments with similar outcomes. DA, Dopamine; FSK, forskolin.

 
Because the phosphorylation of the receptor protein is a key cellular event that determines the internalization of GPCR, effects of dopamine or the PMA treatment on the phosphorylation of D3R were tested. In accordance with the sequestration results of D3R, the phosphorylation of D3R was not significantly affected by the dopamine treatment regardless of the coexpression of three different GRK isoforms (Fig. 1BGo), but the level of phosphorylation was strongly increased by PMA (Fig. 1CGo). In contrast, the phosphorylation of D2R, a positive control, was markedly increased by dopamine treatment (Fig. 1B2Go). This suggests that PKC, not GRK, could be the major protein kinase that regulates D3R functions. These results were also confirmed by ß-arrestin translocation assay, which faithfully reflects agonist-induced receptor phosphorylation and sequestration of GPCRs (21). A robust translocation of ß-arrestin was observed in D2R- but not in D3R-expressing cells in response to dopamine (22) or quinpirole treatment (data not shown). The forskolin treatment did not affect D3R phosphorylation (Fig. 1CGo) and sequestration (data not shown).

The sequence of the inhibition of the PKC activities was examined using specific inhibitors to confirm that the PKC-mediated phosphorylation of the D3R is essential for the subsequent PMA-induced sequestration. 3-[1-(3-Dimethylamino-propyl)-5-methoxy-1H-indol-3-yl] 4-(1H-indol-3-yl)pyrrolidine-2,5-dione (Gö6983), a specific PKC inhibitor, blocked the PMA-induced sequestration of D3R in a dose-dependent manner (IC50 ~ 0.6 µM) (Fig. 2Go, A and B). This event was unaffected by 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo [2–3a] pyrrolo [3,4-c]-carbazol (Gö6976), another PKC blocker. Consistent with this result, Gö6983 also suppressed the PMA-induced phosphorylation of D3R (Fig. 2CGo).


Figure 2
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  		Fig. 2. Determination of the PKC Subtype Responsible for the Sequestration and Phosphorylation of D3R

A, Effects of PKC inhibitors on PMA-induced sequestration of D3R. The cells were treated with 1 µM Gö6976 or Gö6983 for 30 min, followed by a PMA treatment for 30 min. ***, P < 0.001 compared with the control group. B, Dose-response curve for the effect of Gö6983 on the inhibition of the PMA-induced sequestration of D3R. The cells were treated with 0–1 µM Gö6983 for 30 min, followed by the PMA treatment (1 µM, 30 min). C, Effects of Gö6983 on the PMA-induced phosphorylation of D3R. The cells were treated with 1 µM Gö6983 for 30 min, followed by the PMA treatment (1 µM, 5 min). The data represent the results from three independent experiments with similar outcomes. D, The roles of the PKC subtypes on the PMA-induced sequestration of D3R. The cells were transfected with the different subtypes of PKC. The cells were treated with 1 µM PMA for 30 min. ***, P < 0.001 compared with the Mock group. DN, Dominant negative; WT, wild type.

 
Gö6983 and Gö6976 selectively inhibit PKC-{alpha}, -ß, -{delta}, -{xi}, and PKC-{alpha}, -ß, respectively. The sequestration of D3R was enhanced significantly in a kinase activity-dependent manner by coexpression of active forms of PKC-ß or -{delta} but not PKC-{alpha} or -{zeta} (Fig. 2CGo). It seems that blockade of both PKC-ß and -{delta} isoforms is needed to inhibit the PMA-induced sequestration of D3R. Overall, the phosphorylation of D3R mediated by PKC is a preliminary step toward the PMA-induced D3R sequestration.

Characterization of PKC-Dependent D3R Sequestration
The binding of [3H]sulpiride, a hydrophilic ligand, but not that of [3H]spiperone, a hydrophobic ligand, decreased in a dose- and time-dependent manner by the PMA treatment (Fig. 3Go, A and B). This suggests that rather than the total amount of D3R decreasing, the extracellularly positioned ligand-binding domain of D3R translocates intracellularly. This was further confirmed by histological analysis, which detected the translocation of green fluorescent protein (GFP)-tagged D3R from the plasma membrane to the cytosolic compartment in response to PMA stimulation (Fig. 3CGo). The effects of the PMA treatment were tested for D3R signaling to further understand the functional meaning of the PKC-mediated phosphorylation and sequestration of D3R. PKC-dependent D3R phosphorylation and sequestration accompanied by the attenuation of D3R signaling, i.e. the D3R-mediated inhibition of cAMP production became less efficient, suggesting that heterologous desensitization processes are involved in regulating the functions of D3R (Fig. 3DGo).


Figure 3
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  		Fig. 3. Characterization of PMA-Induced Sequestration of D3R

A, Dose-response relationship of the PMA-induced sequestration of D3R. The cells expressing D3R were treated with 0–1 µM PMA for 30 min. The sequestration of D3R was measured with [3H]spiperone and [3H]sulpiride at 2 and 7.2 nM, respectively. Ligand binding to the cells treated with the vehicle (dimethylsulfoxide) was defined as 100%. B, Time course of PMA-induced sequestration of D3R. The cells were treated with 1 µM PMA for the indicated times and treated with 7.2 nM [3H]sulpiride as described in Materials and Methods. The time scale was adjusted so that the sequestration at the early-stage scale could be read clearly. C, Confocal microscopy images of the PMA-induced sequestration of D3R. The cells were transfected with D3R tagged with GFP at the carboxy-terminal tail. After 36 h, the cells were treated with 1 µM PMA for 30 min. Each panel shows a representative cell from each experimental group. D, Effects of the PMA treatment on the signaling of D3R. The HEK-293 cells expressing both D3R and adenylyl cyclase type V were treated with either the vehicle or 1 µM PMA for 30 min, followed by increasing concentrations of quinpirole, a specific agonist for the D2-like receptors. The cellular cAMP level was measured by column chromatography.

 
To determine whether there is some overlap between the homologous and heterologous desensitization pathways, the effects of the molecular components involved in the homologous desensitization of GPCR, for example, GRK2, ß-arrestin2, dynamin I, and their corresponding dominant-negative mutants, were tested for the PMA-induced sequestration of D3R. The coexpression of GRK2, ß-arrestin 1 or 2, dynamin I, and their dominant-negative mutants such as K220R-GRK2, V53D-ß-arrestin 1, and V54D-ß-arrestin 2 did not affect the PMA-induced sequestration of D3R (data for ß-arrestin 1 and V53D-ß-arrestin 1 are not shown). On the other hand, the coexpression of K44A-dynamin I, which blocks the clathrin-coated and caveolin-dependent internalization processes, inhibited D3R sequestration (Fig. 4A1Go). In contrast, the sequestration of D2R was significantly increased by coexpression of GRK2 or ß-arrestin 2 (Fig. 1AGo) but was inhibited by dominant-negative mutant of ß-arrestin2 (V54D-ß-arr2) or dynamin I (K44A-dynI) (Fig. 4A2Go).


Figure 4
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  		Fig. 4. Relationship between the PMA-Induced Sequestration of D3R and Clathrin-Coated Endocytic Pathway

A, The roles of the homologous desensitization components in the PKC-induced sequestration of D3R. The cells were cotransfected with D3R (A1) or D2R (A2) together with 2 µg of the DNA constructs encoding various cellular components involved in the intracellular trafficking of GPCRs: GRK2, G protein-coupled receptor kinase 2; K220R-GRK2, dominant negative GRK2; ß-arr2, ß-arrestin2; V54D-ß-arr2, dominant-negative ß-arrestin 2; DynI, dynamin I; K44A-DynI, dominant-negative dynamin I. Cells were treated with 1 µM PMA for 30 min (A1) or with 10 µM dopamine for 1 h (A2). *, P < 0.1; **, P < 0.01; ***, P < 0.001 compared with the Mock group. B, Relationship between caveolin I and PMA-induced sequestration of D3R. The cells were transfected with D3R or D2R, or together with either 2 µg of wild-type (WT) caveolin I or a mutant one lacking the coding region from the 61th to 100th amino acid residues. The cells were treated with either the vehicle (dimethylsulfoxide) or 10 mM MßCD for 30 min, and subsequently with 1 µM PMA for 30 min (D3R) or with 10 µM dopamine for 1 h (D2R). **, P < 0.01 compared with the vehicle group.

 
The coexpression of the caveolin 1 ({Delta}61–100), which lacks the oligomerization domain (23), or a treatment with methyl-ß-cyclodextrin, which destroys the lipid raft membrane fraction (24), inhibited the sequestration of D2R (Fig. 4B2Go) but not that of D3R (Fig. 4BGo). This excludes the possibility of caveolin involvement in the PMA-induced internalization of D3R.

Localization of Receptor Regions and Identification of the Endocytosis Motif that Determines PKC-Dependent Sequestration of D3R
To locate the receptor region that directs the PMA-induced sequestration of D3R, all the possible phosphorylation sites (serine and threonine residues) in the cytoplasmic loops of D3R were grouped into 14 different regions and mutated to either the alanine or valine residues (Fig. 5Go and Table 1Go).


Figure 5
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  		Fig. 5. Diagram of the D3R Mutants in Putative Phosphorylation Sites

Shaded region represents the putative transmembrane region. Site-directed mutagenesis was performed to mutate the serine or threonine residue at the putative phosphorylation sites designated within the cytoplamic loops (mutants 1–14) into alanine or valine, respectively. Detailed information for the mutants is described in Table 1Go.

 

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  		Table 1. Notation and Descriptions for the D3R Mutants of the Possible Phosphorylation Sites in the Intracellular Regions

 
Mutant 1, in which the triplet threonine residues were located within the first cytoplasmic loop were altered to alanine or valine residues, was not further examined because the mutant receptors were not properly expressed on the plasma membrane. Mutant 14 (S400A) in the C-terminal tail, and mutants 2 (T130V) and 3 (T142V, S145A, S146A) in the second cytoplasmic loop did not show any changes in sequestration (data not shown).

Among the D3R mutants created within the third cytoplasmic loop, mutations on the consensus PKA-mediated phosphorylation sites (25) (mutants 11, 13, or in combination) had no effect on D3R sequestration (data not shown). This result is in line with the lack of D3R phosphorylation (Fig. 1CGo) and sequestration after the forskolin treatment. Surprisingly, serine-to-alanine and threonine-to-valine mutations in the regions containing relatively well-conserved PKC-dependent phosphorylation sites (25) (mutants 9, 10, and 12) did not significantly affect the PMA-induced D3R sequestration either individually or in combination (see Fig. 6AGo). Interestingly, PMA-induced D3R sequestration was significantly inhibited in mutants 4 and 6 (Fig. 6BGo). Mutant 4 contained double mutations, T225A and S229A, and it was further determined that S229A not T225V affects PMA-induced D3R sequestration. Region 6 contains two possible phosphorylation sites (S257 and T262); further mutational dissection demonstrated that the PMA-induced D3R sequestration was reduced by S257A but not by T262V. When S229A and S257A were combined, the PMA-induced sequestration of D3R was almost completely abolished in HEK-293 cells that stably express it (Fig. 6CGo). These results were also confirmed in neural-derived cells, Neuro2a and NG108–15. The sequestration of D3R was 29.5 ± 3.2 and 6.04 ± 0.93% in Neuro2a cells that were transiently transfected with the wild-type D3R and S229A/S257A-D3R, respectively. Essentially the same results were observed in NG108–15 cells (data not shown). It should be mentioned that a similar level of sequestration was observed with S229A/S257A-D3R in HEK-293 cells when they were transiently transfected (data not shown).


Figure 6
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  		Fig. 6. Determination of the Amino Acid Residues Responsible for the PMA-Induced Sequestration and Phosphorylation of D3R

HEK-293 cells, which had been transiently (A, B, and D) or permanently transfected (C), were treated with 1 µM PMA for 30 min. The sequestration was determined using [3H]sulpiride, as described in Materials and Methods. A, Sequestration profiles of the mutants of D3R in the consensus PKC phosphorylation sites. B, Effects of the mutations on the putative phosphorylation sites other than the consensus PKA and PKC phosphorylation sites within the third cytoplasmic loop of D3R. C, The identification of the critical amino acid residues responsible for the sequestration of D3R. D, Identification of the endocytosis motif. *, P < 0.1: compared with the wild type (WT); **, P < 0.01 compared with the WT; ***, P < 0.001 compared with the WT. E1, PMA-induced phosphorylation of the wild-type or mutant D3R. Cells expressing the Flag-tagged D3R were treated with 1 µM PMA for 5 min. E2, Quantification of the results in panel E1. ***, P < 0.001 compared with the vehicle group.

 
Because the S257 residue within region 6 is a putative target for Ca2+/calmodulin-dependent protein kinase (CaMK)II-mediated phosphorylation, the cells expressing either the wild-type D3R or S257A mutant were treated with the specific CaMKII inhibitor, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-ty-rosyl]-4-phenylpiperazine. These treatments did not cause any change in sequestration in either construct (data not shown). In addition, the PMA-induced sequestration of D3R was not affected by PD98059, a MAPK kinase inhibitor (data not shown). This excludes the possibility that the PMA-induced sequestration of D3R results from the PMA-induced activation of CaMKII or MAPK.

A close examination of the third cytoplasmic loop of D3R uncovered one YxxL motif (Y212 to L215) and one reverse YxxL motif (L252 to Y255) (Fig. 5Go). The role of the YxxL motif was first described in the internalization of the Varicella zoster virus Fc receptor glycoprotein gE (26). This molecular signature was later found to be the major endocytosis motif governing the endocytosis of low-density lipoprotein receptor-related protein (27). Remarkably, mutations of the key amino acids (L->A and Y->A) in the reverse YxxL motif significantly reduced PMA-induced D3R sequestration (Fig. 6DGo). However, this was not observed with the forward YxxL motif (Fig. 6DGo).

Because the PKC inhibitor, Gö6983, blocked the PMA-induced phosphorylation and sequestration of D3R (Fig. 2AGo), and the sequestration of D3R was greatly reduced in the mutants S229A- or S257A-D3R, the phosphorylation of these mutants was expected to be reduced. As shown in Fig. 6EGo, the level of S257A-D3R phosphorylation was significantly lower than that of wild-type D3R. Similar results were obtained with S229A/S257A-D3R (data not shown). On the other hand, the phosphorylation of the triple mutant in the putative PKC-phosphorylation sites (mutants 9, 10, and 12), the sequestration of which was not altered, occurred normally (Fig. 6Go, panels E1 and E2).

Profiles of PKC-Mediated Heterologous Desensitization of D3R and Roles of Actin-Binding Protein 280, Filamin A
Filamin A is an actin-binding protein that has been reported to interact with the third cytoplasmic loops of D2R or D3R (28, 29). There are contradictory reports regarding the roles of filamin A on the cell surface expression of dopamine D2R. Either it is absolutely essential for correctly positioning the receptor proteins on the plasma membrane (29) or it is needed to form receptor protein clusters on the plasma membrane (28).

The requirement of filamin A was tested to determine the role of filamin A in the PMA-induced sequestration of D3R. Regardless of the presence (A7 cells) or absence (M2 cells) of endogenous filamin A, the transfection of the wild-type D3R DNA clone successfully produced the receptor proteins on the plasma membrane of the M2 and A7 cells (Fig. 7AGo). Surprisingly, as shown in Fig. 7BGo, the PMA-induced sequestration of D3R normally occurred only in the A7 cells but was completely abolished in the M2 cells. This suggests that filamin A is absolutely essential for PMA-induced D3R sequestration.


Figure 7
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  		Fig. 7. Relationship between Filamin A, the Sequestration, and the Signaling of D3R

A, The expression of D3R in M2 and A7 cells. The M2 and A7 cells were transfected with GFP-tagged D3R. B, Comparison of the sequestration of D3R in M2 and A7 cells. The M2 and A7 cells that stably expressed D3R were used for the sequestration assay. C1, Effects of PMA treatment on the interaction between D3R and filamin A. The cells expressing Flag-D3R and filamin A were treated with 1 µM PMA for 5 min and immunoprecipitated with the agarose beads conjugated to the Flag antibodies. C2, The relative values, PMA-treated/vehicle-treated, are shown. The values in the wild-type (WT) and S257A represent lane-2/lane-3 and lane-4/lane-5 in panel C1, respectively. D, The signaling properties of the D3R mutant, which lacks PMA-induced receptor sequestration. The cellular levels of cAMP were measured using a reporter gene assay, as reported for D1R, D2R, and D5R in previous studies (47 48 49 ). IB, Immunoblotting; IP, immunoprecipitation.

 
It was previously shown that filamin A is crucial for the efficient signaling of D3R (22), and the PMA treatment changes the D3R response (Fig. 3DGo). Therefore, the effects of the PMA treatment on the interaction between D3R and filamin A were tested. As shown in Fig. 7Go, panels C1 and C2, PMA had no effect on the interaction between the wild-type D3R and filamin A at the resting state. However, the PMA treatment markedly reduced the interaction with S257A-D3R, suggesting that a continuous interaction with filamin A is required for the sequestration of D3R.

The functional roles of the PMA-induced receptor phosphorylation on D3R signaling were examined by comparatively testing the signaling properties of the wild-type D3R and the sequestration mutant S229A/S257A-D3R. The cells that stably expressed either the wild-type D3R or S229A/S257A-D3R were treated with quinpirole alone or with 1 µM PMA followed by quinpirole. As shown in Fig. 7DGo, the quinpirole treatment produced a similar level of inhibition of forskolin-induced cAMP production in both the wild-type and S229A/S257A-D3R ({circ} vs. {square}). When the cells were pretreated with PMA, the inhibition of the cAMP production by D3R was almost completely abolished in the cells expressing the wild-type D3R, suggesting that heterologous desensitization occurred possibly through PKC stimulation. In contrast, the inhibition of cAMP production was less affected by the PMA treatment in the cells expressing the S229A/S257A-mutant, suggesting that these two amino acid residues are essential for desensitizing D3R possibly through PMA-induced phosphorylation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Relative Importance of Homologous and Heterologous Desensitization Pathways for D2R and D3R
D3R shares most of the signaling pathways with D2R except that the signaling efficiencies of D3R are 2–5 times less efficient than D2R. The homologous desensitization properties have the same pattern. D2R undergoes stronger GRK/ß-arrestin-dependent sequestration than D3R. Because of these reasons, D3R has been recognized as a GPCR that neither dynamically signals nor desensitizes (8, 17, 22).

Even though the GRK/ß-arrestin-mediated pathway plays a minimal role for the intracellular trafficking of D3R, GRK2 and -3 are reported to regulate the constitutive signaling activity of D3R by controlling the stability of the signaling complex formed around D3R (ß-arrestin/filamin) (22). Therefore, homologous and heterologous desensitization pathways might play a dominant role depending on the cellular environments. However, it is interesting that D3R, which is believed to be inert both in agonist-dependent signaling and desensitization, undergoes robust intracellular trafficking processes in a PKC-dependent manner. In this respect, a PKC-dependent regulatory process is likely to be the major desensitization pathway of D3R, and any cellular event that alters the cellular PKC level should heterologously regulate the functions of D3R.

Molecular Mechanism of the PMA-Induced Sequestration of D3R
Because the enzymatic activity of GRK2 can be enhanced when it is phosphorylated by PKC (30, 31), the homologous and heterologous desensitization pathways might overlap at certain points. This appears to be the case with D2R, because the PMA-induced sequestration of D2R is enhanced somewhat by the coexpression of GRK or ß-arrestin (32). However, this principle does not apply to D3R because the sequestration of D3R was not much affected by the coexpression of GRK or ß-arrestin (Fig. 4AGo). Such biochemical differences between the two receptors can be explained by their differences in GRK dependency for receptor internalization, i.e. the sequestration of D2R but not that of D3R noticeably depended on the cellular GRK and ß-arrestin levels.

The mechanistic aspects of PMA-induced D3R sequestration are complicated. The sensitivity to K44A-dynamin I suggests that the PMA-induced sequestration of D3R is mediated through clathrin-coated pits (CCPs) or the caveolin-mediated pathways. However, the PMA-induced sequestration of D3R was not affected by the coexpression of wild-type or dominant-negative mutants of GRK or ß-arrestin. This suggests that the CCP pathway is unlikely to contribute to this type of cellular process. In addition, it was not affected by the expression of caveolin 1 or its negative-dominant mutant or the disruption of the lipid raft with MßCD. This suggests the involvement of certain cellular pathways other than CCP or caveolin-mediated pathway. These results might be explained by the fact that filamin A is required for the sequestration of D3R. Filamin A is known to directly bind to caveolin A (33), and such a molecular interaction between the two proteins might explain the effects of K44A-dynamin I on the sequestration of D3R.

Endocytosis Motif, LxxY
It has been suggested that the well-conserved NPxxY motif, which was originally described for the endocytosis of low-density lipoprotein receptor (34), is involved in the endocytosis of GPCRs (35). Unlike the NPxxY motif, which is located in the transmembrane domain, the LxxY motif is located in the intracellular loop and is likely to bind with other proteins. Sequence analysis showed that this tyrosine-based internalization motif is likely to bind to the µ2 subunit of ß-adaptin. The two middle amino acid residues could be any polar amino acid residues (36), which might explain why the disruption of the second, but not the first, YxxL motif affected the endocytosis of D3R. The two middle residues for the first and second YxxL motifs are valine-valine and arginine-lysine, respectively.

Filamin A, Caveolin, and Receptor Sequestration and Signaling
The filamin A, actin-binding protein with a molecular mass of 280 kDa, plays an important role in the signaling of some GPCRs as well as in the intracellular trafficking of other GPCRs. For example, filamin A binds the metabotropic glutamate receptors type 7 (37) and calcitonin receptors (38). Filamin A also interacts with the µ-opioid receptors, and the internalization of the µ-opioid receptor requires the presence of filamin A (39).

Both D3R and D2R bind to the lipid-raft associated filamin A (actin-binding protein 280) in their third cytoplasmic loop (28, 29, 40). Previously, it was shown filamin A is essential for the efficient signaling of D3R (22). These results show that filamin A is also necessary for the PMA-induced sequestration of the D3R

The functional meaning of the interaction between D3R and filamin A seems to be more complicated than expected. It was reported that filamin A is essential for the efficient signaling of D3R, i.e. signaling still occurs in the absence of filamin A (in M2 cells), but the signaling efficiency is significantly enhanced by the addition of filamin A (22). In the case of the PMA-induced sequestration of D3R, filamin is an essential component for receptor sequestration because the sequestration of D3R was completely abolished in the M2 cells lacking endogenous filamin A (Fig. 7BGo).

According to the results of the sequestration studies from M2 and A7 cells, and the immunoprecipitation studies from HEK-293 cells, a continuous interaction with filamin A is needed for the PMA-induced sequestration of D3R. It is not clear why PMA treatment reduced the interaction of S257A-D3R with filamin A even though this mutant appears not to be phosphorylated by PMA treatment (Fig. 6EGo). It is speculated that the phosphorylation of both D3R and filamin A is required for their proper interactions to occur. Filamin A is an in vitro and in vivo substrate for PKC, and it has been suggested that the PKC regulates the filamin A-associated signaling through the phosphorylation of filamin A (41). Interestingly, these results suggest that the phosphorylation of the serine residue at 257 by PMA treatment enables D3R to interact with filamin A and be sequestrated. These results might explain why S229A/S257A-D3R, which virtually does not sequester, still undergoes partial desensitization in response to PMA treatment.

In conclusion, this paper reports three important discoveries. First, the molecular mechanisms underlying the sequestration of D3R differ from those of the D2R. Second, the desensitization of D3R is initiated by the PMA-induced phosphorylation of two serine residues, S229 and S257, within the third intracellular loop. In particular, PKC-ß and/or -{delta} isoform play an important role. Finally, the physical interaction between filamin A and phosphorylated D3R is likely to be the driving force for the sequestration of D3R.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Material
HEK-293 cells were obtained from the American Type Culture Collection (Manassas, VA). A human melanoma cell line (M2) that does not endogenously express filamin A, and an A7 cell line, a M2 subclone stably transfected with filamin A cDNA, are described elsewhere (42, 43). Two neural-derived cells, NG108–15 and Neuro2a, were kindly provided by Dr. S. H. Yoon (Catholic University Medical School, Seoul, Korea) and Dr. S. C. Park (Sookmyung Women’s University, Seoul, Korea). The cell culture reagents were obtained from either Cellgro (Herndon, VA) or Invitrogen Life Technologies, Inc. (Carlsbad, CA).

The [32P]-orthophosphate, [3H]-sulpiride, [3H]-adenine, [35S]-GTP{gamma}S, and [14C]-cAMP were purchased from NEN (Boston, MA). The [3H]spiperone was obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). Dopamine, (-)quinpirole, methyl-ß-cyclodextrin (MßCD), PMA, forskolin, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, a specific inhibitor of CaMKII, 3-isobutyl-1-methylxanthine, PD98059, anti-FLAG M2 antibody, anti-FLAG-conjugated agarose beads, and horseradish peroxidase-labeled secondary antibodies were obtained from Sigma/Aldrich Chemical Co. (St. Louis, MO). The antifilamin A antibody was obtained from Research Diagnostics, Inc. (Flanders, NJ). The Gö6976 and Gö6983 were purchased from the Calbiochem (San Diego, CA).

Cell Culture and Transfection
The HEK-293 cells were cultured in MEM supplemented with 10% fetal bovine serum (FBS) and 50 µg/ml gentamicin in a humidified atmosphere containing 5% CO2. The transfections were carried out using the calcium phosphate precipitation method. The M2 and A7 cells were grown in MEM supplemented with 8% newborn calf serum and 2% FBS. The A7 cells were maintained in a medium containing G418 (500 µg/ml; Research Products International Corp., Mt. Prospect, IL). The M2 and A7 cells were transfected with Lipofectamine Plus reagent (Invitrogen).

Plasmid Constructs
The wild-type human D2R (short form) and D3R in the mammalian expression vector pCMV5, D3R tagged at the N-terminal tail with the M2-FLAG epitope or HA epitope, and D3R fused at the C-terminal tail with GFP, are described elsewhere (17, 18, 22). The expression constructs for GRKs 2 and 3 in pRK5, ß-arrestin 1 and 2, V53D-ß-arrestin 1, V54D-ß-arrestin 2 in pCMV5, ß-arrestin 2 in pEGFP, K220R-GRK2 in pcDNA 1.1, and dynamin K44A in pRK5 are also described elsewhere (17, 18, 22). The filamin A in pcDNA 3.0 was provided by Dr. Ohta (Harvard University).

Constructs for the wild-type and dominant-negative (kinase inactive) mutants of PKC were provided by J. W. Soh (Inha University, Incheon, Korea), which include the wild-type and dominant-negative of PKC-{alpha} (K368R), PKC-ß (K371R), PKC-{delta} (K376R), and PKC-{zeta} (K281M). The expression and kinase activities of these constructs were previously confirmed.

For the site-directed mutagenesis of the putative phosphorylation sites (intracellular serine and threonine residues) of D3R (refer to Fig. 5Go and Table 1Go), single-stranded phagemid DNA of D3R in pCDNA3.1/Zeo(+) (Invitrogen), were prepared by infecting an Escherichia coli strain CJ236 (dut– ung–) carrying each plasmid with the helper phage M13K07. The oligonucleotide-directed mutagenesis of the single-stranded phagemid DNA of each plasmid was constructed as described previously (44).

Sequestration Assay
The sequestration of D2R and D3R was measured using the hydrophilic properties of sulpiride or by flow cytometry, as described previously (17).

Whole-Cell Phosphorylation
The HEK-293 cells transfected with D3R and D2R constructs tagged with HA and Flag epitope, respectively, were stimulated with 10 µM dopamine, 1 µM PMA, or 1 µM forskolin for 5 min. The 32P-labeled phosphorylated receptors were assessed by autoradiography. The detailed procedures are described elsewhere (17). For the normalization of the loading volumes, the amount of receptor in each sample was determined by saturation binding with 3 nM [3H]spiperone.

Confocal Microscopy
One day after transfection, the cells were seeded onto 35-mm dishes containing a centered, 1-cm well that was formed from a glass coverslip-sealed hole in plastic (confocal dishes) and allowed to recover for 1 d. The cells were incubated with 2 ml MEM containing 20 mM HEPES, pH 7.4, and examined by Zeiss laser scanning confocal microscopy (Carl Zeiss, Thornwood, NY).

Immunoprecipitation
After 48 h transfection, the cells were lysed in RIPA buffer (150 mM NaCl; 50 mM Tris, pH 8.0; 1% Nonidet P-40; 0.5% deoxycholate; 0.1% sodium dodecyl sulfate) on a rotation wheel for 1 h at 4 C. The supernatants were mixed with 35 µl of 50% slurry of the anti-Flag-agarose beads for 2–3 h on the rotation wheel. The beads were washed with a washing buffer (50 mM Tris, pH 7.4; 137 mM NaCl; 10% glycerol; 1% Nonidet P-40) three times for 10 min each.

Whole-Cell cAMP Assays
The HEK-293 cells expressing the wild-type or mutant D3R plated in 12-well dishes were labeled overnight with 1 µCi/ml [3H]adenine in MEM containing either 10% FBS and gentamicin. Before the assay, the labeling medium was replaced with serum-free MEM containing 10 mM HEPES. The medium containing 10 mM HEPES and 1 mM 3-isobutyl-1-methylxanthine was added with the indicated drugs, and the assay was carried out, as described previously (45). [3H]cAMP accumulation was determined by the sequential chromatography method reported by Salomon (46). The data were normalized by expressing the cAMP levels as a percentage of the forskolin-stimulated cAMP for each experiment. The dose-response curves were fitted using GraphPad Prism (GraphPad Software, Inc., San Diego, CA).

In some experiments, cellular cAMP was measured using an indirect method with reporter gene induction. This method had been used to determine D2R signaling (47, 48). The cells were transfected with D2R or D3R, along with a reporter plasmid containing the firefly luciferase gene under the transcriptional control of multiple cAMP-responsive elements or the control vector (Promega Corp., Madison, WI). The cells were stimulated for 4 h with 1 µM forskolin with or without the dopamine agonist, quinpirole. The relative luciferase activity was then measured using a dual luciferase assay kit (Promega).


   ACKNOWLEDGMENTS
 
We thank Elias Y. Kim for manuscript proofreading. We also thank Dr. S. H. Yoon and Dr. S. C. Park for providing NG108–15 and Neuro2a cells.


   FOOTNOTES
 
This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (A040042). H.K. was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Korea-Japan Joint Project). J.H.P. was supported in part by National Institutes of Health Grant MH-66197 and National Science Foundation Grant IBN-0133538.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 29, 2007

Abbreviations: CaMKII, Ca2+/calmodulin-dependent protein kinase II; CCP, clathrin-coated pit; D3R, D3 dopamine receptor; FBS, fetal bovine serum; GFP, green fluorescent protein; Gö6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo [2–3a] pyrrolo [3,4-c]-carbazol; Gö6983, 3-[1-(3-dimethylamino-propyl)-5-methoxy-1H-indol-3-yl] 4-(1H-indol-3-yl)pyrrolidine-2,5-dione; GPCR, G protein-coupled receptor; GRK, GPCR kinase; HEK, human embryonic kidney; MßCD, methyl-ß-cyclodextrin; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate.

Received for publication April 23, 2007. Accepted for publication May 24, 2007.


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