JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M403899200 on April 21, 2004

J. Biol. Chem., Vol. 279, Issue 27, 28304-28314, July 2, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/27/28304    most recent
M403899200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Beom, S.
Right arrow Articles by Kim, K.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Beom, S.
Right arrow Articles by Kim, K.-M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Comparative Studies of Molecular Mechanisms of Dopamine D2 and D3 Receptors for the Activation of Extracellular Signal-regulated Kinase*

SunRyeo Beom{ddagger}, Dawoon Cheong{ddagger}, Gonzalo Torres§, Marc G. Caron§, and Kyeong-Man Kim{ddagger}

From the {ddagger}Department of Pharmacology, College of Pharmacy, Chonnam National University, Kwang-Ju, 500-757 Korea and the §Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, April 8, 2004 , and in revised form, April 21, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Dopamine D2 and D3 receptors (D2R/D3R), which have similar structural architecture as well as functional similarities, are expressed in the same brain dopaminergic neurons. It is intriguing that two receptor proteins with virtually the same functional roles are expressed in the same neuron. Recently we have shown that D2R and D3R possess different regulatory processes including intracellular trafficking properties, which implies that they might employ different signaling mechanisms for regulation of the same cellular processes. Here we studied the signaling pathways of ERK activation mediated by D2R and D3R in HEK-293 cells and corroborated them with concomitant studies in COS-7 cells and C6 cells. Our results show that Src, phosphatidylinositol 3-kinase, and atypical protein kinase C were commonly involved in D2R-/D3R-mediated ERK activation. However, {beta}-arrestin and sequestration of D2R/D3R were found not to be involved. ERK activations mediated by D3R, but not D2R, were blocked by {beta}ARK-CT, AG1478 epidermal growth factor receptor (EGFR) inhibitor, and by dominant negative mutants of Ras and Raf, suggesting the involvement of the G{beta}{gamma}i pathway. The {alpha}-subunit of Go (G{alpha}o) was able to couple with D3R to mediate ERK activation. We conclude that D3R mainly utilizes the {beta}{gamma} pathway of Gi protein, which involves the transactivation of EGFR in HEK-293 cells. In contrast, the {alpha}-subunit of the Gi protein plays a main role in D2R-mediated ERK activation. Our study suggests one example of intricate cellular regulations in the brain, that is, dopaminergic neurons could regulate ERK activity more flexibly through alternative usage of either the D2R or D3R pathway depending on the cellular situation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Extracellular signal-regulated kinase 1 (ERK1)1 and ERK2 (p44ERK and p42ERK) are key cellular components that control cell proliferation and differentiation (1). The regulation of ERK through G protein-coupled receptors (GPCRs) is a complicated process. Various signaling components play different roles in ERK activation depending on the GPCR and cell types involved (2).

There has been substantial progress in the understanding of cellular events that link the activation of GPCR and ERK (for review, see Ref. 1). These signaling events can be classified into several distinct pathways. They include: (i) Ras-dependent activation of ERK via transactivation of receptor-tyrosine kinases (RTKs) such as EGFR, (ii) Ras-independent ERK activation via protein kinase C (PKC) that converges with RTK signaling at the Raf level (iii) activation or inhibition of ERK via the cAMP/protein kinase A (PKA) pathway, in which the direction of regulation depends on the type of Raf involved, and (iv) the recently substantiated {beta}-arrestin-mediated pathway proven in certain classes of GPCRs (3). However, it should be mentioned that these signaling pathways are deduced from the limited sets of individual receptors or cell types, and more extensive and systemic studies are needed for these signaling models to be generalized (4).

Brain dopamine receptors differ in their pharmacological profiles and in brain distribution. Based on their structural, pharmacological, and functional characteristics, the dopamine receptors have been classified into two pharmacological subfamilies: D1- and D2-like dopamine receptors. With the advent of molecular biological techniques, the D1-like receptors were subdivided into the D1 and D5 receptors, while the D2, D3, and D4 receptors comprise the D2-like receptors.

Among all the dopamine receptor subtypes characterized, it is generally accepted that D2R and D3R are related to schizophrenia. Possibly because of high similarity in their amino acid composition (46% overall amino acid homology and 78% identity in the transmembrane domains) (5), D2R and D3R share most signaling pathways such as adenylyl cyclase, extracellular acidification, mitogenesis, ERK activation, inhibition of dopamine synthesis, and ion channel regulation (K+, Ca2+) (for review, see Ref. 6). Furthermore, recent studies show that although D3R is more densely expressed in the limbic area, mesencephalic dopaminergic neurons express both D2R and D3R (7–9). Because D2R and D3R are virtually the same in functional aspect and are expressed in the same cells, it can be speculated that signaling routes or regulatory mechanisms for functions of effectors could be different. To address this issue, we decided to focus on a specific cellular function and conduct a detailed study on signaling mechanisms in order to see if their signaling or regulatory pathways differ. ERK activation was selected as a model experimental system, because GPCR-mediated ERK regulation consists of multiple complex steps, which are relatively well established.

D2R-mediated ERK activation has been reported from different cell types. It is pertussis toxin (PTX)-sensitive and partially blocked by the dominant negative mutant of Ras, N17Ras, in C6 cells (10) and CHO cells (11, 12). Phosphatidylinositol (PI) 3-kinase is involved in CHO cells, where the platelet-derived growth factor receptor mediates this through a pathway other than the G{beta}{gamma} pathway (11, 12). Even though molecular details are not yet characterized, it has been reported in CHO cells that the D3R-mediated ERK activation, which involves the same signaling pathways as D2R, is PTX-sensitive, and PI 3-kinase and atypical PKC are involved (13). However, there has not been a single comparative study for signaling mechanisms addressing differences in the D2R- and D3R-mediated ERK activation in the same cell type. Considering that signaling of GPCR-mediated ERK activation could greatly vary depending on the cell types involved, it is necessary to conduct well controlled comparative studies of D2R- and D3R-mediated ERK activation in the same cell type to understand the details of the signaling mechanism.

In this study, we investigated the molecular mechanism by which D2R and D3R activates ERK1/2, side by side in the same cell types. In this regard, we designed our experiments to determine: (i) which isotype of G proteins are involved in these processes, (ii) the putative signaling mediators involved using various specific inhibitors and dominant negative mutants, (iii) whether internalization of D2R or D3R is a prerequisite for activation of ERK1/2, (iv) whether transactivation with receptor-tyrosine kinase is involved in the activation of ERK1/2, (v) whether {beta}-arrestin plays a role in ERK activation, and (vi) whether D2R- and D3R-mediated ERK activation accompany the internalization of EGFR.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Dopamine (3-hydroxytyramine HCl), Haloperidol (4-[4-(p-chlorophenyl)-4-hydroxy-piperidino]-4'-fluorobutyrophenone), sulpiride, tyrphostin AG1478 (N-(3-chlorophenyl)-6,7-dimethoxy-4-quinazolinamine), PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), and PMA (phorbol 12-myristate 13-acetate) were purchased from RBI/Fluka/Sigma. Gö6976 (12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indol[2,3-a]pyrrollo[3,4-c]carbazole), Gö6983 (3-[1-(3-dimethylamoni-propyl)-5-methoxy-1H-indol-3-yl]4-(1H-indol-3-yl)pyrrolidine-2,5-dione), Pertussis toxin, U-73122 (1-[6-((17{beta}-3-methoxyestra-1,3,5-(10)-trien-17-yl)-amino)-hexyl]-1H-pyrrole-2,5-dione), and PD98059 (2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) were purchased from Calbiochem (San Diego, CA). The enhanced chemiluminescence kit was purchased from Bio-Rad. The monoclonal mouse anti-phospho-ERK antibody and polyclonal goat anti-ERK2 were purchased from Sigma, Santa Cruz Biotechnology (Santa Cruz, CA), or Cell Signaling Technology (Beverly, MA). Common antibodies such as alkaline phosphatase-conjugated anti-mouse IgG and anti-goat IgG were purchased from Sigma.

Cell Cultures and Transfection—HEK-293 cells, COS-7 cells, and C6 glioma cells were cultured in Eagle's minimum essential medium or Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (Hyclone, Logan, UT) and 10 µg/ml gentamicin (Sigma) at 37 °C with 5% CO2 in air atmosphere. Transfections were performed on a ~70–80% confluent monolayer in 100-mm dishes, using calcium phosphate co-precipitation methods or LipofectAMINE (Invitrogen).

Plasmids—Wild-type human dopamine D2 receptor (short splice variant, D2SR; long splice variant, D2LR) and D3 receptor (D3R) subtypes in mammalian expression vector pCMV5, G protein-coupled receptor kinases (GRKs) 2 and 3, {beta}-arrestins 1 and 2, {beta}-arrestin 1-GFP, {beta}-arrestin 2-GFP, V53D-{beta}-arrestin 1, and K44A-dynamin I have been described in our previous studies (14, 15–18). The pEGFP-N1 expression plasmid encoding GFP-tagged ERK2 (19) was provided by K. DeFea and N. Bunnett (University of California at San Francisco), and EGFR-GFP was from A. Sorkin (University of Colorado Health Sciences Center). Dominant negative p21Ras, RasN17, was from Drs. D. Aultschuler and M. Ostrowski (University of Ohio). Sos-Pro was from Dr. R. J. Lefkowitz (Duke University Medical Center). Dominant negative p74Raf-1, {Delta}NRaf was from Dr. L. T. Williams (University of San Francisco). The cDNA of pertussis toxin-insensitive C351I-G{alpha}o was provided by Dr. De Vries (Centre de Recherche Pierre Fabre).

ERK Measurement—After transfection, cells were cultured in 6-well plates. If necessary, they were starved overnight in a serum-free culture medium containing 0.1% BSA. Cells were treated with dopamine dissolved in 10 µM ascorbic acid, medium was aspirated, and SDS sample buffer was directly added to culture wells. After incubating 20 min at 65 °C, samples were sonicated to shear genomic DNA. Proteins were separated by SDS-PAGE (10% running gel, 5% stacking gel) and electroblotted onto polyvinylidene difluoride or nitrocellulose membrane. The membranes were incubated for 1 h at room temperature in TBS-T containing 5% nonfat dry milk or 4% BSA, followed by 1 h of incubation with antibodies to phospho-ERK (1:1,000 dilution) and 1 h with alkaline phosphatase-conjugated secondary antibodies (1:5,000) in 2% nonfat dry milk. Blots were visualized with chemiluminescent Western blotting kit. The same samples were processed as in phospho-ERK detection except that the membranes were probed with antibodies for ERK.

Immunocytochemistry—The same experimental procedures were used as previously reported (Tohgo et al., Ref. 45). For the co-localization studies between {beta}-arrestin and phospho-ERK, HEK-293 cells transiently expressing D2R or D3R and {beta}-arrestin 2-GFP were grown in collagen-coated 35-mm glass bottom dishes (confocal dishes). Following dopamine stimulation for 5 min, cells were fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with absolute methanol for 10 min at –20 °C, and blocked with phosphate-buffered saline containing 1% BSA, 5% fetal bovine serum for 2 h at room temperature. Immunofluorescent labeling of endogenous phospho-ERK1/2 was done with a 1:500 dilution of rabbit polyclonal anti-phospho-ERK1/2 antibody in phosphate-buffered saline containing 1% BSA, 1% fetal bovine serum for 18 h at room temperature, followed by a 1:500 dilution Texas Red®-conjugated polyclonal anti-rabbit IgG antibody in 1% BSA, 1% fetal bovine serum for 1 h at room temperature. Confocal microscopy was performed using a Zeiss LSM510 laser scanning microscope dual line switching excitation (488 nm for GFP and 568 nm for Texas Red®) and emission (515–540 nm for GFP and 590–620 nm for Texas Red®) filter sets.

EGFR Sequestration Assay Using Confocal Microscopy—HEK-293 cells were transfected with the EGFR-GFP with or without D2R or D3R. One day after transfection, cells were seeded onto confocal dishes and allowed to recover for 1 day. Cells were treated either with 10 ng/ml EGF or 1 µM dopamine for 30 min in 2 ml of MEM containing 20 mM HEPES, pH 7.4, and viewed on a Zeiss laser scanning confocal microscope.

Receptor Sequestration Assay—Three different strategies were used as described previously (18).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of D2R- and D3R-mediated Activation of ERK1/2 in HEK-293 Cells—The time course and dose response relationship of D2R- and D3R-mediated ERK activation were studied in HEK-293 cells, which were transiently transfected with cDNAs encoding D2R or D3R (because D2SR was mainly used in this study, D2R refers to D2SR unless specified). As shown in Fig. 1, the phosphorylation of p44 and p42 forms of ERK (ERK1/2) was transient and dose-dependent. It reached a maximum at 1 µM dopamine at around 5 min, returning to basal levels after 30 min. Pretreatment of cells with haloperidol (10 µM) or sulpiride (10 µM, data not shown) abolished both D2R- and D3R-mediated ERK1/2 activation, indicating that the activation of ERK1/2 is truly mediated by D2RorD3R (Fig. 1C).



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 1.
Time course and dose response of D2R- and D3R-induced ERK activation in HEK-293 cells. HEK-293 cells were transiently transfected with plasmid DNA encoding D2R or D3R (2 µg/100-mm dish). A, time course of D2R- and D3R-mediated activation of ERK. Serum-starved cells were incubated in the presence of DA (1 µM) for the indicated times (0–30 min). B, dose response of D2R- and D3R-mediated ERK activation. Serum-starved cells were incubated with DA (0–10 µM) for 5 min. Cell extracts were prepared as described under "Experimental Procedures" and analyzed on 10% polyacrylamide gels. Levels of activated ERK1/2 were detected by a monoclonal antibody raised against the phosphorylated form of ERK1/2 (pERK). C, specificity of the dopamine receptor involvement for ERK activation. Serum-starved cells were preincubated with or without haloperidol (10 µM) for 5 min, and then DA (1 µM) was added for 5 min to stimulate ERK1/2. Activated ERK1/2 was detected by anti-pERK (upper panel) and anti-ERK2 (lower panel).

 
Involvement of PTX-sensitive G Protein in the D2R- and D3R-mediated Activation of ERK—Although D2R and D3R couple to PTX-sensitive G proteins for the inhibition of adenylyl cyclase, it is possible that GPCRs could couple to different G proteins for different effector proteins. For example, the {beta}2-adrenergic receptor couples to Gs to activate adenylyl cyclase but couples to Gi for the activation of ERK in HEK-293 cells (20).

PTX is known to catalyze ADP-ribosylation of {alpha}-subunits in the Gi and Go subfamilies of heterotrimeric G proteins, and this covalent modification prevents the Gi/o proteins from interacting with receptors (21, 22). Overnight treatment with PTX (100 ng/ml) completely blocked the phosphorylation of ERK1/2, suggesting the involvement of Gi and/or Go proteins in D2R- and D3R-mediated ERK1/2 activation (Fig. 2A). Similar results were obtained from transiently transfected COS-7 cells and C6 cells that stably express D2LR or D3R (data not shown).



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 2.
Characterization of G proteins involved in D2R- and D3R-mediated ERK activation. A, effect of PTX treatment on D2R- and D3R-mediated ERK activation. HEK-293 cells transfected with D2R or D3R were pretreated overnight with or without 100 ng/ml PTX and then incubated with 1 µM DA for 5 min. Immunoblotting was conducted as described in Fig. 1. B, effect of PTX-insensitive Go on D2R- and D3R-mediated ERK activation B1, HEK-293 cells were transfected with D2R or D3R together with GFP-ERK2 and C351I-G{alpha}o, PTX-insensitive G{alpha}o. Cells were treated with PTX as in A. B2, Student's t test was used for the statistical comparison between control and PTX-treated groups. **, p < 0.01 compared with the control group. C, involvement of G{beta}{gamma} subunits in the D2R- and D3R-mediated ERK activation. C1, cells were transiently transfected with plasmid DNAs encoding {beta}ARK1-CT (8 µg/dish), GFP-ERK2 (1 µg/dish), together with D2R (2 µg/dish) or D3R (2 µg/dish). C2, Student's t test was used for the statistical comparison between control and {beta}ARK1-CT groups.

 
It has been reported that both D2R and D3R couple with Go. Co-expression of the {alpha}-subunit of Go (G{alpha}o) increased the G protein coupling of D2R and D3R about six times (data not shown), suggesting that both receptors could effectively couple to Go. To test whether Gi or Go is involved in the activation of ERK, the PTX-insensitive G{alpha}-subunit, G{alpha}o-C351I (23), was co-expressed and tested for ERK activation. D3R-mediated ERK activation from the cells co-transfected with G{alpha}o-C351I was less effectively blocked by PTX treatment than ERK activation from cells not expressing G{alpha}o-C351I (lower two panels in Fig. 2, B1, compare lane 3 versus 4 and 7 versus 8; also see Fig. 2, B2), suggesting the involvement of G{alpha}o for ERK activation. On the other hand, the PTX sensitivity of D2R-mediated ERK activation was not significantly altered by co-expression of C351I-G{alpha}o (upper two panels in Fig. 2, B1, compare lane 3 versus 4 and 7 versus 8; also see Fig. 2, B2). These results suggest that even though Go is able to couple to both D2R and D3R effectively, D2R mediates ERK activation mainly through the Gi protein, whereas both Gi and Go could mediate ERK activation by D3R in HEK-293 cells.

The involvement of {beta}{gamma}-subunits of G proteins in the activation of ERK has been reported for various GPCRs including D2R in CHO cells (24). The carboxy terminus of the {beta}-adrenergic receptor kinase 1 ({beta}ARK1-CT) contains the binding domain for G{beta}{gamma}, which has allowed {beta}ARK1-CT to be used to distinguish the G{alpha} and G{beta}{gamma} signaling pathways (25). As shown in Fig. 2, C1, phosphorylation of co-expressed GFP-ERK2 (upper panel) was effectively attenuated by {beta}ARK1-CT in cells expressing D3R but not in cells expressing D2R (compare lane 1 versus 2 and 3 versus 4 for D2R; lane 5 versus 6 and 7 versus 8 for D3R). Statistical analyses of these results are shown in Fig. 2, C2.

Receptor Sequestration Is Not a Requirement for ERK Activation by D2R and D3R—Recently, it has been suggested for certain GPCRs that receptor internalization may be required for the activation of the ERK cascades under certain conditions (26, 27). On the other hand, receptor sequestration is not necessary for ERK activation of some GPCRs (28). We wanted to test whether receptor internalization is required for D2R- and D3R-mediated ERK activation. We examined the relationship between receptor internalization and ERK activation by blocking receptor internalization using dynamin dominant negative mutant K44A and sucrose. In addition, we modulated the receptor sequestration by co-expressing GRK/{beta}-arrestins, and tested their effect on ERK activation.

Dynamin is a critical component involved in the clathrincoated pit formation. It pinches off vesicles from the plasma membrane, and inhibition of this process by K44A blocks receptor internalization (29). As shown in Fig. 3, A1, in HEK-293 cells, D2R sequestered about 8% in response to agonist treatment (10 µM, 1 h), and this was potentiated by a co-expression of GRK2 up to 23%. Co-expression of the dominant negative V53D {beta}-arrestin 1 or dynamin I K44A reduced the sequestration of D2R, suggesting that D2R sequesters in a clathrin-dependent manner. In COS-7 cells (Fig. 3, B1), sequestration of D2R did not occur in the absence of GRK or {beta}-arrestin but could be increased with the co-expression of {beta}-arrestin 2 up to 12.6 ± 1.2% for the short splice variant (D2SR) and 22.4 ± 3.2% for the long splice variant (D2LR) (data not shown). Co-expression of GRK3/{beta}-arrestin 2 increased the sequestration up to 39 and 54% for D2SR and D2LR, and dynamin I K44A significantly blocked the sequestration of both splice variants of D2R (Fig. 3, B1). On the other hand, D3R sequestered less than 10% in both HEK-293 cells and COS-7 cells, only in the presence of exogenous GRK (18). ERK activation was still observed in COS-7 cells (Fig. 3, B2, lanes 2 and 6) in which D2R or D3R does not undergo agonist-induced receptor sequestration (Fig. 3, B1, Mock). In addition, D2R- or D3R-induced ERK activation was not significantly reduced by co-expression of dynamin I K44A (lane 2 versus 4 and lane 6 versus 8 in Fig. 3, A2 and B2). This was further confirmed by HEK-293 cells that were treated with sucrose (Fig. 3, A3), which is one of the nonspecific but strong inhibitors of receptor sequestration. Sucrose treatment did not affect D2R- or D3R-mediated ERK activation (lane 2 versus 3 and lane 5 versus 6). These results suggest overall that agonist-induced receptor sequestration of D2R or D3R is not the main determinant for ERK activation.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 3.
Role of receptor endocytosis in D2R- and D3R-mediated ERK activation. A, studies in HEK-293 cells. A1, roles of {beta} -arrestin and dynamin on D2R sequestration. HEK-293 cells were transfected with D2R-pCMV5 (2 µg), GRK2-pCDNA1.1 (2 µg), together with 3 µgof {beta}-arrestin 1 V53D or dynamin I K44A. Receptor sequestration was measured using [3 H]spiperone/sulpiride competition methods. Each bar represents mean ± S.E. Student's t test was used for the statistical analysis. *, p < 0.1 compared with Mock/GRK2 group; **, p < 0.01 compared with Mock/Mock group. A2, effects of dominant negative dynamin I on D2R- and D3R-mediated ERK activation. Cells were transfected with 3 µg of dynamin I or dynamin I K44A, GFP-ERK2 (1 µg) and 2 µg of D2R or D3R. Cells were stimulated with 1 µM DA for 5 min. A3, effects of sucrose treatment on D2R- and D3R-mediated ERK activation. HEK-293 cells were transfected with D2R or D3R. Cells were treated with 0.45 M sucrose for 20 min at 37 °C, followed by stimulation with 1µM DA for 5 min. B, studies in COS-7 cells. B1, COS-7 cells were transfected with 2 µg of GRK3-pRK5, {beta}-arrestin 2-pCMV5, dynamin I K44A, and D2R or D3R. Receptor sequestration was assessed by measuring the disappearance of [3H]sulpiride binding sites after dopamine treatment (1 h, 10 µM). **, p < 0.01 compared with GRK3/{beta}-arrestin 2 group. B2, cells were transfected with dynamin I K44A (3 µg) with 2 µg of D2R or D3R. Cells were treated with 1 µM DA for 5 min.

 
Roles of Phospholipase C and Protein Kinase C in the D2R- and D3R-mediated ERK Activation—In HEK-293 cells, one or more isoforms of PLC are involved in the {alpha}1B-AR- and {alpha}2A-AR-mediated ERK activation via the G{beta}{gamma}-subunit (30). The activated PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), which induces the activation of protein kinase C and the release of Ca2+ from intracellular Ca2+ stores. These second messengers subsequently activate a number of cellular processes leading to the modification of cell proliferation and differentiation (31).

There have been studies that showed the regulation of inositol phosphate production by D2-like receptors (32, 33), and we tested whether PLC activation was required for D2R- and D3R-mediated ERK phosphorylation using the pharmacological PLC inhibitor, U-73122. As shown in Fig. 4A, U-73122, did not have a significant effect on D2R- and D3R-mediated activation of ERK (compare lane 3 versus 5 and lane 8 versus 10).



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 4.
Roles of phospholipase C and protein kinase C on D2R- and D3R-mediated ERK activation. A, effect of PLC inhibitor on D2R- and D3R-mediated ERK activation. HEK-293 cells were transiently transfected with plasmid DNA encoding D2R or D3R. Before agonist stimulation, serum-starved cells were treated for 1 h with vehicle (0.75% Me2SO, 0.25% chloroform) and U73122 [GenBank] (10 µM, dissolved in the same solvent). Cells were stimulated with DA (1 µM) for 5 min and ERK1/2 was detected by immunoblotting. Data represent results from three independent experiments. B, effect of PMA-induced PKC depletion on D2R- and D3R-mediated ERK activation. HEK-293 cells were transiently transfected with plasmid DNA encoding D2R or D3R. Serum-starved cells were preincubated overnight with phorbol ester (PMA, 1 µM) or vehicle, and then cells were treated with DA (1 µM, 5 min) or PMA (1 µM, 30 min). Activated ERK1/2 was detected by immunoblotting. Data represent results from two independent experiments. C, effect of PKC subtype-specific inhibitors on D2R- and D3R-mediated ERK activation. HEK-293 cells were transiently transfected with plasmid DNA encoding D2R or D3R. Serum-starved cells were preincubated with increasing concentrations of PKC inhibitors, Gö6976 or Gö6983 (0–5 µM) for 30 min. Cells were treated with DA (1 µM) for 5 min and phosphorylated ERK1/2 was detected by immunoblotting. Data represent results from two independent experiments.

 
The involvement of PKC in D2R- and D3R-mediated ERK activation was tested by either depletion of endogenous PKC by overnight pretreatment with phorbol ester (PMA, 1 µM) or by treatment with specific PKC inhibitors. As shown in Fig. 4B, overnight PMA treatment abolished the subsequent activation of ERK by PMA (30-min treatment), suggesting that PKC depletion was successful. However, D2R- or D3R-mediated ERK activation was not affected by overnight PMA treatment (Fig. 4B, left and right panels, compare lanes 1 and 2 versus lanes 5 and 6), and an additional 30-min PMA treatment did not overshadow agonist effects (compare lanes 5 and 6 versus 7 and 8). These results exclude the involvement of PMA-sensitive PKCs in the D2R- or D3R-mediated activation of ERK. This was further confirmed by studies using the PKC inhibitors, Gö6976 and Gö6983 (34, 35). Gö6983, which inhibits the atypical PKC isoform (aPKC) PKC-{zeta} (IC50, 60 nM), blocked both D2R- and D3R-mediated ERK activations (Fig. 4C, left and right panels, lanes 6–8). In contrast, Gö6976, which inhibits typical PKCs did not have any effect at 5 µM (Fig. 4C, left and right panels, lanes 3–5). Together, these results suggest that PMA-insensitive atypical PKCs are involved in the D2R- and D3R-mediated ERK activation, which is characteristic of ERK activation mediated by Gi but not Go (36). Also it is known that PKC-{zeta} directly phosphorylates and activates MEK (37, 38).

Roles of PI 3-Kinase and Src in D2R- and D3R-mediated ERK Activation—It is known that the atypical PKC subtype, PKC-{zeta}, is activated by PIP3 and PIP2 (39), both of which are products of PI 3-kinase. We next determined the involvement of PI 3-kinase on D2R- and D3R-mediated activation of ERK because D2R and D3R may mediate aPKC activation through PI 3-kinase. Wortmannin blocked both D2R- and D3R-induced ERK activations in a dose-dependent manner (Fig. 5A). These results agree with the signaling of other receptors, such as Gi-coupled LPA receptor, which activates ERK through PI 3-kinase/atypical PKC in CHO cells (40).



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 5.
Roles of PI 3-kinase and Src on the D2R- and D3R-mediated ERK activation. Cells transfected with D2R or D3R were with starved overnight with serum-free media and pretreated with wortmannin (1–10 µM, 30 min) (A) or Src inhibitor, PP2 (10 µM, 30 min) (B), and then treated with DA (1 µM) for 5 min. Phosphorylated ERK1/2 was detected by immunoblotting. Data represent results from three independent experiments.

 
Src is a cytosolic tyrosine kinase that is involved in the initiation of receptor-tyrosine kinase activation. Src is activated by interaction with Ca2+-dependent kinase, Pyk2 (related focal adhesion tyrosine kinase), which is identified as a component of the Gi/o-coupled receptor pathway in ERK activation (41). Treatment of cells with PP2, a Src inhibitor, partially attenuated both D2R- and D3R-induced activation of ERK (Fig. 5B). These results suggest that Src is involved in both D2R- and D3R-mediated ERK activation in HEK-293 cells.

Roles of MEK, Ras, and Raf-1 in the D2R- and D3R-mediated ERK Activation—Since MEK is an immediate signaling component that directly controls ERK activity, its role on the ERK activation was tested using a specific MEK inhibitor, PD98059. Pretreatment of cells with PD98059 effectively suppressed ERK1/2 activity induced by D2R and D3R (Fig. 6A) and excluded the possibility of the involvement of cross-talk between other kinase pathways activated by D2R or D3R, that might lead to MEK-independent activation of ERK.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 6.
Roles of MEK, Ras, and Raf-1 on the D2R- and D3R-mediated ERK activation. A, effects of MEK inhibitor on D2R- and D3R-mediated ERK activation. HEK-293 cells were transiently transfected with plasmid DNA encoding D2R or D3R. Serum-starved cells were preincubated for 30 min with vehicle (0.1% Me2SO) or a MEK inhibitor PD98059 (30 µM, prepared in the same solvent). B, effects of dominant negative mutant of Ras on D2R- and D3R-mediated ERK activation. B1, cells were transfected with 3 µg of dominant negative N17-Ras, together with plasmid DNA encoding D2R or D3R. B2, Student's t test was used for the statistical comparison between control and N17-Ras groups. *, p < 0.1 compared with the control group. C, effects of dominant negative mutant of Raf-1 on D2R- and D3R-mediated ERK activation. C1, cells were transfected with 3 µg of dominant negative {Delta}N-Raf, starved overnight with serum-free media and then treated with DA (1 µM) for 5 min. C2, Student's t test was used for the statistical comparison between control and {Delta}N-Raf groups. **, p < 0.01 compared with the control group.

 
In order to determine the role of Ras in the D2R- and D3R-mediated ERK activation, we used dominant negative, N17Ras. In HEK-293 cells, D3R- but not D2R-mediated ERK activation was partially blocked by N17Ras (Fig. 6, B1, lanes 5 and 6 versus 7 and 8 and Fig. 6, B2). The same results were observed in COS-7 cells (data not shown).

Since Raf-1 is a nearby upstream signaling component of MEK, we tested its role on D2R- and D3R-mediated ERK activation. In HEK-293 cells, transient transfection with the cDNA of dominant negative {Delta}N-Raf effectively abolished only D3R-mediated ERK activation (Fig. 6, C1, lanes 5 and 6 versus 7 and 8; Fig. 6, C2). The same results were also observed in COS-7 cells (data not shown). Results from Ras and Raf studies suggest that divergent Ras-dependent and Ras-independent (probably protein kinase C) pathways are involved in D3R-mediated ERK activation, and they converge at the Raf level.

Transactivation with EGFR and D3R-mediated ERK1/2 Activation—Several groups have reported that mechanisms of GPCR-mediated activation of the ERK cascade closely relate to those employed by receptor-tyrosine kinases, and GPCR-stimulated tyrosine phosphorylation of EGFR involves release of a soluble EGFR ligand, heparin-binding EGF (42). The involvement of G{beta}{gamma} subunits in the D3R-mediated ERK activation pathway suggests the possibility of transactivation with EGFR.

We tested whether D2RorD3R transactivates with EGFR for ERK activation using a selective inhibitor of tyrosine kinase of EGFR, tyrphostin AG1478. AG1478 effectively inhibited D3R- but not D2R-mediated ERK activation in HEK-293 cells (Fig. 7, A1, lanes 5 and 6 versus 7 and 8; Fig. 7, A2), suggesting that the transactivation of EGFR is specifically involved with D3R-mediated ERK activation. Transactivation between D3R and EGFR has not been reported; however, our results are in agreement with previous reports; dopamine-induced ERK1/2 activation in CHO cells was not due to cross-talk between D3R and FGF or insulin receptors (13). Similar results were obtained from COS-7 cells; AG1478 more efficiently inhibited D3R-mediated ERK activation than D2R (data not shown).



View larger version (85K):
[in this window]
[in a new window]
  		FIG. 7.
Role of cross-talk with EGFR on the D2R- and D3R-mediated ERK activation. A, effect of EGFR inhibitor on D2R- and D3R-mediated ERK activation. HEK-293 cells transiently transfected with plasmid DNA encoding D2R or D3R were serum-starved, treated with a selective inhibitor of tyrosine kinase of EGFR, tyrphostin AG1478 (300 nM), for 30 min. ERK1/2 activation was induced by DA (1 µM) for 5 min and phosphorylated ERK1/2 was detected by immunoblotting. B, Student's t test was used for the statistical comparison between control and AG1478 groups. **, p < 0.01 compared with the control group. C, HEK-293 cells were transfected with 1 µg of EGFR-GFP, together with 3 µg of D2R or D3R. Cells were treated with EGF (10 ng/ml) or 1 µM DA for 30 min. Images were taken from the same cells.

 
It has been suggested that transactivation of EGFR through GPCR accompanies the internalization of EGFR. When cells expressing D2R and EGFR-GFP were stimulated with EGF, significant amounts of EGFR-GFP were internalized (Fig. 7B, panel 1, positive control); however, dopamine treatment did not have an effect on EGFR sequestration in the same experimental group (Fig. 7B, panel 2, negative control). In contrast to what we expected from AG1478 results, stimulation of cells expressing D2R/EGFR-GFP or D3R/EGFR-GFP with 1 µM dopamine did not cause noticeable endocytosis of EGFR (Fig. 7B, panels 3 and 4). According to Pierce et al. (43), effects of negative dominant dynamin I K44A mutant on ERK activation result from the blockade of the internalization of EGFR. Considering that K44A did not have any effect on ERK activation in D2R- or D3R-expressing cells, it was not expected that stimulation of D2R or D3R would cause the endocytosis of EGFR.

Role of {beta}-Arrestin for the Activation of D2R- and D3R-mediated Activation of ERK—Recently, the roles of {beta}-arrestin, a signal-terminating protein, have been expanded. For example, in angiotensin II type 1A receptor (AT1AR) {beta}-arrestin 2 works as a receptor-regulated MAPK scaffold for the activation of JNK3 (44). It was further reported that AT1AR-activated ERK activation depends on the stability of the interaction between GPCR and {beta}-arrestin (45, 46), and that {beta}-arrestin 1 and 2 have reciprocal roles for activation of ERK (47). However, it is not clear whether the new role of {beta}-arrestin can be extended for other GPCRs. In this study, we tested the involvement of {beta}-arrestins for D2R- and D3R-mediated ERK activation by co-expressing {beta}-arrestins or dominant negative {beta}-arrestin 1 V53D mutant. We also tested whether agonist-activated phospho-ERK (pERK) and translocated {beta}-arrestin co-localize at the same subcellular regions as reported in AT1AR.

As shown in Fig. 8, A and B, co-expression of {beta}-arrestin 1/2 or dominant negative {beta}-arrestin 1 V53D mutant did not significantly affect the D2R- or D3R-mediated ERK activation. Since it has been reported that the subcellular distribution of agonist-activated ERK and agonist-induced translocated {beta}-arrestin coincide (45), the co-localization of pERK and translocated {beta}-arrestin 2 was compared. As shown in Fig. 8C, phosphorylation of ERK (red color image) was not observed in resting cells. Dopamine treatment caused the intracellular translocation of {beta}-arrestin 2-GFP (green color image) from cytoplasm to plasma membrane, suggesting that these cells express D2R. pERKs were observed mainly in the cytoplasm and plasma membrane, and they did not necessarily co-localize with translocated {beta}-arrestin 2-GFP. These results show that role of {beta}-arrestin in the activation of ERK as observed in AT1AR does not apply to D2R and D3R.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 8.
Role of {beta}-arrestin on D2R- and D3R-mediated ERK activation. A, HEK-293 cells were transiently transfected with 2 µg of plasmid DNA encoding {beta} -arrestin 2 together with D2R or D3R. B, COS-7 cells were transiently transfected with plasmid DNA encoding {beta}-arrestin 1, {beta}-arrestin 1-V53D, together with D2R or D3R. Cells were serum-starved, treated with a selective inhibitor of EGFR tyrosine kinase, tyrphostin AG 1478 (300 nM) for 30 min. ERK1/2 activation was induced by DA (1 µM) for 5 min and phosphorylated ERK1/2 was detected by immunoblotting. C, HEK-293 cells were transfected with 1 µgof {beta} -arrestin 2-GFP together with 3 µg of D2R or D3R. Cells were treated with 1 µM DA for 5 min. Cells were treated as described under "Experimental Procedures" and labeled with antibodies for phospho-ERK1/2. The green and red image represent {beta}-arrestin 2-GFP and phosphorylated ERK1/2, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The ERK activation cascade is relatively well understood in receptor-tyrosine kinases. For example, EGFR is phosphorylated on tyrosine residues by EGF stimulation, and phosphorylated EGFR binds to its adaptor proteins, such as Grb2, and then to SOS that has a stimulatory effect on the GDP/GTP exchange of Ras (48, 49). EGFR-adaptor complex activates Raf and MEK, which is followed by phosphorylation of ERK. ERK then translocates to the nucleus, where it phosphorylates a number of transcription factors, which ultimately regulate the gene expression essential for cell proliferation (50). Since the {beta}{gamma}-subunit of Gi proteins are known to transactivate EGFR, the detailed signaling pathway of EGFR described above might be considered as a {beta}{gamma}-subunit pathway of the Gi protein (51, 52).

According to our studies in HEK-293 cells, profiles of signaling pathways for the D2R- and D3R-mediated ERK activation in terms of G protein involvement are as follows: the G protein is PTX-sensitive, implying Gi/o proteins are involved; and D3R-mediated ERK activation seems to follow the G{beta}{gamma}i pathway, whereas D2R-mediated ERK activation follows the G{alpha}i pathway. D3R-mediated ERK activation has been reported (13); however, molecular details such as the involvement of the G{beta}{gamma} pathway, Ras, and Raf have not been reported yet. In contrast to D3R-mediated ERK activation, D2R-mediated ERK activation has been more extensively studied. Characteristics of D2R-mediated ERK activation are somewhat complicated. Previous studies in GH4ZR7 cells and BALB/c 3T3 cells showed the involvement of G{alpha}o and G{alpha}i3/G{alpha}i2 for D2R-mediated ERK regulation (53–55). However, the involvement of G{beta}{gamma} is different depending on the cell types tested, and the usage of Raf isoforms also seems to vary (53).

If one of the critical signaling components of the G{beta}{gamma}-subunit pathway of the Gi protein ({beta}{gamma}-subunit -> EGFR -> Shc-Grb2-SOS -> Ras -> Raf) is blocked, G{alpha}i pathway might predominate for ERK activation. Indeed when endogenous SOS was blocked by its dominant negative mutant {Delta}SOS, LPA which activates ERK activation through Gi-coupled receptor, activated ERK in a Ras-independent, atypical PKC and PI 3-kinase-dependent manner in CHO cells (40). Similar results were observed for D2R-mediated ERK activation in CHO-K1 cells (12): G{beta}{gamma} was not involved (not blocked by {beta}ARK1-CT), ERK activation was insensitive to N17Ras, and PI 3-kinase and atypical PKC were involved. Therefore, the signaling pathways involved in D2R-mediated ERK activation observed in our study, are virtually the same G{alpha}i pathway as LPA-induced ERK activation in {Delta}SOS-expressing CHO cells.

The activation of ERK through the Go protein is different from the Gi protein (36); in G{alpha}o, PMA-sensitive PKCs are involved in a Ras-independent manner. In many cases, Gq, which mediates PLC activation, is involved in the PKC-induced activation of ERK. The Gq-mediated PKC activation is sensitive to PMA (56), suggesting the involvement of "classical" and/or "novel" PKC isoforms ({alpha}, {beta}1, {beta}2, {gamma}/{delta}, {epsilon}, {theta}, {eta}), which are sensitive to DAG and phorbol ester (57). On the other hand, these pathways are not pertinent to the present system in HEK-293 cells, because D2R- and D3R-mediated activation of ERK is abolished by PTX, which selectively inhibits the involvement of Gi/o proteins, excluding Gq. The PKC involved in D2R- and D3R-mediated activation of ERK1/2 was insensitive to PMA or to Gö6976, but sensitive to Gö6983, again excluding the involvement of Go. However, it should be noted that D3R could mediate ERK activation if G{alpha}o is co-expressed. Therefore, depending on the expression level of the {alpha}-subunit of Gi or Go, D3R might utilize completely different signaling pathways to activate ERK. On the other hand, the Gi pathway seems to be more favorable for ERK activation of D2R in HEK-293 cells even though Go can couple to D2R.

The requirement of receptor endocytosis for the activation of ERK has been controversial (58). For example, dominant negative dynamin I K44A reduced {beta}2AR-mediated ERK activation in HEK-293 cells (26) but not in COS-1 cells (28). Since activation of the ERK signaling pathway by internalized EGFRs located to the endosomal compartments (59), the original proposed role of GPCR endocytosis in ERK activation now seems to be re-interpreted as the effect of endocytosis inhibitors on post-GPCR events (3, 43). Recent studies in D2R-mediated ERK activation showed that ERK activation mediated by the short isoform of D2R, but not the long isoform, can be effectively inhibited by dynamin K44A or ConA treatment (60). In our studies, the internalization of D2R and D3R seems to be not required for ERK activation. In HEK-293 cells, neither the dominant negative dynamin I K44A nor sucrose treatment significantly affected D2R-/D3R-mediated ERK activation. In COS-7 cells, the endocytosis of D2R or D3R does not occur without co-expression of GRK or {beta}-arrestin; however, ERK activation is obvious, and co-expression of K44A did not affect ERK activation as expected. We do not have a clear answer for these discrepancies at this point. However, considering that effects of blocking endocytosis on ERK activation could be connected with postreceptor cellular events, these discrepancies might arise from differences in cell types. The extent of influence of receptor endocytosis could be different in different cell types (58).

Previous studies in HeLa cells showed that EGF-induced ERK activation is decreased when the endocytosis of EGFR is blocked by dynamin I K44A (61). However, this issue is still far away from being clear (58) and there is also another possibility that activated MEK rather than receptor endocytosis is blocked by dynamin mutant (62). A recent study, which is more directly related with our study, reported that in HEK-293 cells stimulation of {beta}2AR with 10 µM isoproterenol activates ERK through transactivation of EGFR, and causes the endocytosis of EGFR in 30 min (63). Considering that D3R-mediated endocytosis was inhibited by AG1478, EGFR kinase inhibitor, endocytosis of EGFR was expected in D3R-expressing cells by dopamine treatment. However, as shown in Fig. 7, a noticeable endocytosis of EGFR-GFP was not observed by dopamine treatment (1 µM,30 min). We do not think these discrepancies are contradictory to what were observed from the {beta}2-adrenergic receptor; rather they reflect the differences in the choice of the range of observation windows. D3R-mediated ERK activation was relatively weak in HEK-293 cells even when the receptor expression levels were around 2–3 pmol/mg membrane protein. At similar receptor expression levels, the amplitude of ERK activation through D3R was ~60% of D2R-mediated ERK activation, which is about 20% of vasopressin type 2 receptor-mediated ERK activation (data not shown). Also lower doses of dopamine (1 µM rather than 10 µM) was used throughout our studies. In contrast to the {beta}2-adrenergic receptor, which might be exposed to high concentrations of circulating epinephrine for prolonged periods, it is unlikely that this would happen with brain D3R. It is possible that the synaptic dopamine concentration can momentarily reach up to 200 nM (64); however, it is unlikely that 10 µM of dopamine will be maintained around D3R over 30 min. The question is then what is the overall contribution and stimulus window to induce EGFR endocytosis? According to our results, the endocytosis of EGFR through GPCR-transactivation seems to require more intense and persistent activation of GPCR compared with the induction of EGFR kinase activity.

Recent studies in AT1AR suggest new roles for {beta}-arrestin in the activation of ERK. {beta}-arrestin 2 acts as a scaffold protein, which brings the spatial distribution and activity of this MAPK module under the control of a GPCR (AT1AR) (44, 45). The stability of the G protein-coupled receptor-{beta}-arrestin interaction determines the mechanism and functional consequence of ERK activation (46), and {beta}-arrestin-mediated ERK activation is qualitatively different from classical G protein-mediated ERK activation rather than simple quantitative differences (65). Furthermore, {beta}-arrestin 1 and {beta}-arrestin 2 seem to have reciprocal roles in ERK activation (47). Judged from the characteristics of {beta}-arrestin-mediated ERK activation observed in AT1AR and our results, we conclude that {beta}-arrestins do not mediate G protein-independent D2R- and D3R-mediated ERK activation, which was observed with AT1AR.

In conclusion, both D2R and D3R mediate ERK activation. D2R follows the {alpha}-subunit pathway and D3R follows the {beta}{gamma}-subunit pathway of the Gi protein; D3R also appears to be able to couple to Go protein for ERK activation. Since both D2R and D3R are expressed in the same brain dopaminergic neurons, cells could modulate ERK activation in a more flexible way depending on the intracellular situations. Further studies are needed to see whether ERK activation mediated by D2RorD3R possesses different cellular roles.


   FOOTNOTES
 
* This work was supported by Korean Research Foundation Grant KRF-2001-FP0125. 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. Back

To whom correspondence should be addressed: Dept. of Pharmacology, College of Pharmacy, Chonnam National University, Kwang-Ju, 500-757 Korea. Tel.: 82-62-530-2936; Fax: 82-62-530-2949; E-mail: kmkim{at}chonnam.ac.kr.

1 The abbreviations used are: ERK, extracellular signal-regulated kinase; GPCR, G protein-coupled receptor; EGFR, epidermal growth factor receptor; PKC, protein kinase C; PI, phosphatidylinositol; BSA, bovine serum albumin; GFP, green fluorescent protein; PTX, pertussis toxin; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate. Back


   ACKNOWLEDGMENTS
 
We thank Ayça Akal-Strader for sincere discussion and proofreading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gutkind, J. S. (2000) Sci. STKE 2000, RE1
  2. Lefkowitz, R. J., Pierce, K. L., and Luttrell, L. M. (2002) Mol. Pharmacol. 62, 971–974[Free Full Text]
  3. Pierce, K. L., Luttrell, L. M., and Lefkowitz, R. J. (2001) Oncogene 20, 1532–1539[CrossRef][Medline] [Order article via Infotrieve]
  4. Liebmann, C. (2001) Cell Signal. 13, 777–785[CrossRef][Medline] [Order article via Infotrieve]
  5. Giros, B., Martres, M. P., Sokoloff, P., and Schwartz, J. C. (1990) C. R. Acad. Sci. III 311, 501–508[Medline] [Order article via Infotrieve]
  6. Missale, C., Nash, S. R., Robinson, S. W., Jaber, M., and Caron, M. G. (1998) Physiol. Rev. 78, 189–225[Abstract/Free Full Text]
  7. Sokoloff, P., Giros, B., Martres, M. P., Bouthenet, M. L., and Schwartz, J. C. (1990) Nature 347, 146–151[CrossRef][Medline] [Order article via Infotrieve]
  8. Diaz, J., Pilon, C., Le Foll, B., Gros, C., Triller, A., Schwartz, J. C., and Sokoloff, P. (2000) J. Neurosci. 20, 8677–8684[Abstract/Free Full Text]
  9. Gurevich, E. V., and Joyce, J. N. (1999) Neuropsychopharmacology 20, 60–80[CrossRef][Medline] [Order article via Infotrieve]
  10. Luo, Y., Kokkonen, G. C., Wang, X., Neve, K. A., and Roth, G. S. (1998) J. Neurochem. 71, 980–990[Medline] [Order article via Infotrieve]
  11. Welsh, G. I., Hall, D. A., Warnes, A., Strange, P. G., and Proud, C. G. (1998) J. Neurochem. 70, 2139–2146[Medline] [Order article via Infotrieve]
  12. Oak, J. N., Lavine, N., and Van Tol, H. H. (2001) Mol. Pharmacol. 60, 92–103[Abstract/Free Full Text]
  13. Cussac, D., Newman-Tancredi, A., Pasteau, V., and Millan, M. J. (1999) Mol. Pharmacol. 56, 1025–1030[Abstract/Free Full Text]
  14. Robinson, S. W., Jarvie, K. R., and Caron, M. G. (1994) Mol. Pharmacol. 46, 352–356[Abstract]
  15. Barak, L. S., Ferguson, S. S., Zhang, J., and Caron, M. G. (1997) J. Biol. Chem. 272, 27497–27500[Abstract/Free Full Text]
  16. Zhang, J., Barak, L. S., Winkler, K. E., Caron, M. G., and Ferguson, S. S. (1997) J. Biol. Chem. 272, 27005–27014[Abstract/Free Full Text]
  17. van der Bliek, A. M., and Meyerowitz, E. M. (1991) Nature 351, 411–414[CrossRef][Medline] [Order article via Infotrieve]
  18. Kim, K. M., Valenzano, K. J., Robinson, S. R., Yao, W. D., Barak, L. S., and Caron, M. G. (2001) J. Biol. Chem. 276, 37409–37414[Abstract/Free Full Text]
  19. DeFea, K. A., Zalevsky, J., Thoma, M. S., Dery, O., Mullins, R. D., and Bunnett, N. W. (2000) J. Cell Biol. 148, 1267–1281[Abstract/Free Full Text]
  20. Daaka, Y., Luttrell, L. M., and Lefkowitz, R. J. (1997) Nature 390, 88–91[CrossRef][Medline] [Order article via Infotrieve]
  21. Stryer, L., and Bourne, H. R. (1986) Annu. Rev. Cell Biol. 2, 391–419[Medline] [Order article via Infotrieve]
  22. Gilman, A. G. (1987) Annu. Rev. Biochem. 56, 615–649[CrossRef][Medline] [Order article via Infotrieve]
  23. Diverse-Pierluissi, M. A., Fischer, T., Jordan, J. D., Schiff, M., Ortiz, D. F., Farquhar, M. G., and De Vries, L. (1999) J. Biol. Chem. 274, 14490–14494[Abstract/Free Full Text]
  24. Choi, E. Y., Jeong, D., Won, K., Park, and Baik, J. H. (1999) Biochem. Biophys. Res. Commun. 256, 33–40[CrossRef][Medline] [Order article via Infotrieve]
  25. Koch, W. J., Hawes, B. E., Inglese, J., Luttrell, L. M., and Lefkowitz, R. J. (1994) J. Biol. Chem. 269, 6193–6197[Abstract/Free Full Text]
  26. Luttrell, L. M., Daaka, Y., Della Rocca, G. J., and Lefkowitz, R. J. (1997) J. Biol. Chem. 272, 31648–31656[Abstract/Free Full Text]
  27. Daaka, Y., Luttrell, L. M., Ahn, S., Della Rocca, G. J., Ferguson, S. S., Caron, M. G., and Lefkowitz, R. J. (1998) J. Biol. Chem. 273, 685–688[Abstract/Free Full Text]
  28. DeGraff, J. L., Gagnon, A. W., Benovic, J. L., and Orsini, M. J. (1999) J. Biol. Chem. 274, 11253–11259[Abstract/Free Full Text]
  29. van der Bliek, A. M., Redelmeier, T. E., Damke, H., Tisdale, E. J., Meyerowitz, E. M., and Schmid, S. L. (1993) J. Cell Biol. 122, 553–563[Abstract/Free Full Text]
  30. Della Rocca, G. J., van Biesen, T., Daaka, Y., Luttrell, D. K., Luttrell, L. M., and Lefkowitz, R. J. (1997) J. Biol. Chem. 272, 19125–19132[Abstract/Free Full Text]
  31. Berridge, M. J., and Irvine, R. F. (1984) Nature 312, 315–321[CrossRef][Medline] [Order article via Infotrieve]
  32. Pizzi, M., D'Agostini, F., Da Prada, M., Spano, P. F., and Haefely, W. E. (1987) Eur. J. Pharmacol. 136, 263–264[CrossRef][Medline] [Order article via Infotrieve]
  33. Rasolonjanahary, R., Gerard, C., Dufour, M. N., Homburger, V., Enjalbert, A., and Guillon, G. (2002) Endocrinology 143, 747–754[Abstract/Free Full Text]
  34. Martiny-Baron, G., Kazanietz, M. G., Mischak, H., Blumberg, P. M., Kochs, G., Hug, H., Marme, D., and Schachtele, C. (1993) J. Biol. Chem. 268, 9194–9197[Abstract/Free Full Text]
  35. Gschwendt, M., Dieterich, S., Rennecke, J., Kittstein, W., Mueller, H. J., and Johannes, F. J. (1996) FEBS Lett. 392, 77–80[CrossRef][Medline] [Order article via Infotrieve]
  36. van Biesen, T., Hawes, B. E., Raymond, J. R., Luttrell, L. M., Koch, W. J., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 1266–1269[Abstract/Free Full Text]
  37. Diaz-Meco, M. T., Dominguez, I., Sanz, L., Dent, P., Lozano, J., Municio, M. M., Berra, E., Hay, R. T., Sturgill, T. W., and Moscat, J. (1994) EMBO J. 13, 2842–2848[Medline] [Order article via Infotrieve]
  38. Schonwasser, D. C., Marais, R. M., Marshall, C. J., and Parker, P. J. (1998) Mol. Cell. Biol. 18, 790–798[Abstract/Free Full Text]
  39. Nakanishi, H., Brewer, K. A., and Exton, J. H. (1993) J. Biol. Chem. 268, 13–16[Abstract/Free Full Text]
  40. Takeda, H., Matozaki, T., Takada, T., Noguchi, T., Yamao, T., Tsuda, M., Ochi, F., Fukunaga, K., Inagaki, K., and Kasuga, M. (1999) EMBO J. 18, 386–395[CrossRef]