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Molecular Endocrinology, doi:10.1210/me.2003-0066
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Molecular Endocrinology 18 (3): 640-652
Copyright © 2004 by The Endocrine Society

Distinct Regulation of Internalization and Mitogen-Activated Protein Kinase Activation by Two Isoforms of the Dopamine D2 Receptor

Sung Jae Kim, Myeong Yoon Kim, Eun Jin Lee, Young Soo Ahn and Ja-Hyun Baik

School of Life Sciences and Biotechnology (S.J.K., M.Y.K., E.J.L., J.-H.B.), Korea University, Seoul 136-701, South Korea; and Brain Korea 21 Project for Medical Science (S.J.K., E.J.L.) and Department of Pharmacology (Y.S.A.), College of Medicine, Yonsei University, Seoul 120-752, South Korea

Address all correspondence and requests for reprints to: Dr. Ja-Hyun Baik, Molecular Neurobiology Laboratory, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, South Korea. E-mail: jahyunb{at}korea.ac.kr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Two isoforms of the dopamine D2 receptor, D2L (long) and D2S (short), differ by the insertion of a 29-amino acid specific to D2L within the putative third intracellular loop of the receptor. Here, we examined D2 receptor-mediated MAPK activation in association with receptor internalization. Overexpression of ß-arrestin 1 and 2 increased the D2S-mediated activation of MAPK, whereas it did not affect the activation of MAPK by D2L. Expression of a dominant negative ß-arrestin 2 (319–418) mutant and of a dominant negative dynamin I (K44A) mutant inhibited the activation of MAPK by D2S, but not the activation of MAPK by D2L. Treatment with inhibitors of internalization, i.e. concanavalin A and monodansylcadaverin, blocked D2S-mediated MAPK activation but not D2L-mediated activation. By confocal microscopy, we observed ß-arrestin 1 and 2, translocated to the plasma membrane and colocalized with D2L and D2S receptors upon stimulation with dopamine, and this was followed by the translocation of receptors into endocytic vesicles. Moreover, the expression of the ß-arrestin 2 (319–418) mutant blocked the internalization of both D2L and D2S. In addition, although K44A dynamin mutant expression did not alter D2L internalization, it completely blocked the internalization of D2S. The stimulation of D2L induces activation of MAPK via transactivation of the platelet-derived growth factor receptor, whereas D2S does not. Taken together, these data suggest that D2L activates MAPK signaling by mobilizing the growth factor receptor, platelet-derived growth factor receptor, whereas D2S appears to activate MAPK signaling by mobilizing clathrin-mediated endocytosis in a ß-arrestin/dynamin-dependent manner.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE DOPAMINE D2 receptor belongs to a family of seven transmembrane domain G protein-coupled receptors (GPCRs) and is highly expressed in the central nervous system and in the pituitary gland (1, 2). The binding of dopamine to the D2 receptor is crucial for the regulation of diverse physiological functions, such as the control of locomotor activity and the synthesis of pituitary hormones (3). Recently, dopamine D2 receptor knockout mice were created (4, 5), and the absence of the D2 receptors was later found to be associated with a severely impaired locomotor activity and abnormal pituitary development, demonstrating that the D2 receptor plays a key role in dopaminergic nervous function (4, 5, 6).

Two alternatively spliced transcripts are generated from the D2 receptor gene and code for the D2L (long) and D2S (short) isoforms, which are 444 and 415 amino acids in length, respectively (7, 8). The D2L isoform differs from D2S by the insertion of 29 amino acids in the putative third intracellular loop of the receptor. This loop is believed to be involved in the coupling of the receptor with different G proteins. Several studies suggest that the two isoforms might employ different signaling pathways (9, 10, 11), and experiments with D2L-deficient mice support the hypothesis that D2L and D2S might have different functions in vivo (12, 13).

The MAPKs are a group of serine/threonine kinases, which are activated by a cascade of protein kinases to induce responses such as proliferation, differentiation, apoptosis, and long-term potentiation. This signaling cascade is a prominent cellular pathway and is used by many growth factors, hormones, and neurotransmitters to regulate diverse physiological functions. It is now generally accepted that numerous GPCRs can also activate MAPK and that this may allow plasma membrane receptor systems to influence diverse cellular processes, ranging from the regulation of neuronal survival to cell differentiation and gene expression (14, 15).

Recent studies suggest that proteins mediating GPCR internalization, such as ß-arrestins and dynamin, may also mediate signaling leading to MAPK activation. Recent studies on the ß2 adrenergic receptor have demonstrated that clathrin/dynamin-mediated receptor internalization may be essential for the activation of the MAPK pathway by GPCRs (15, 16, 17). It has been proposed that the stimulation of the ß2 adrenergic receptor results in the assembly of a protein complex containing activated c-Src and ß-arrestin, and that the formation of this complex is crucial for MAPK activation (16). In addition, it has been reported that the dominant negative forms of dynamin can inhibit MAPK activation by GPCRs (18, 19), suggesting that GPCR internalization is a requirement either for MAPK activation (20) or for the dynamin-dependent internalization of downstream proteins, such as MAP/ERK kinase (MEK) (21).

We showed previously that D2L- and D2S-mediated MAPK activation involves predominantly Gß{gamma} subunit-mediated signaling, and that protein kinase C and tyrosine phosphorylations are involved in these signaling pathways (10). However, it is unclear how these two D2 dopamine receptors couple to the MAPK signaling pathway and, furthermore, whether there are any subtype-related regulations in this signaling pathway.

In the present study, we investigated the regulation of the MAPK pathway by these two dopamine D2 receptors in association with agonist-induced receptor internalization.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Role of Src Kinase Inhibitor in D2L- and D2S-Mediated MAPK Activation
Recent studies have confirmed that certain GPCRs use ß-arrestin as a clathrin adaptor to mediate internalization of receptor signaling complexes (16, 17). By binding to both the nonreceptor tyrosine kinase, c-Src, and to the agonist-occupied GPCRs, ß-arrestin can confer tyrosine kinase activity upon the receptor (17, 18). We assessed the role of protein tyrosine kinases, including c-Src kinase, in D2L- and D2S-mediated MAPK activation in stable Chinese hamster ovary (CHO) cell lines expressing two isoforms of the dopamine D2 receptor, D2L and D2S (CHOD2L and CHOD2S, respectively) (10). As previously demonstrated, pretreatment with a general tyrosine kinase inhibitor, genistein, inhibited both D2L- and D2S-mediated MAPK activation by up to 58% and 42%, respectively. The Src family tyrosine kinase, herbimycin A, and the Src-specific tyrosine kinase inhibitor, 4-amino-5 (4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP2), significantly abrogated D2L- and D2S-mediated MAPK activation to a similar extent (Fig. 1Go, A and B). These results demonstrated that D2L- and D2S-mediated MAPK activation requires Src-kinase activity.



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  		Fig. 1. Effect of Tyrosine Kinase Inhibitors on MAPK Activity in CHOD2L and CHOD2S Cells

Cells were preincubated for 30 min with PP2 (10 µM), 2 h with herbimycin A (1 µM), and 2 h with genistein (100 µM). Phospho-ERK and total ERK levels was analyzed in cell lysates by immunoblotting. CHOD2L (A) and CHOD2S (B) cells were treated with dopamine (1 µM) for 1min. Data indicate mean ± SE from at least three independent experiments. (**, P < 0.01 as compared with dopamine nonstimulated control; $, P < 0.05; $$, P < 0.01 as compared with inhibitor nontreated control). DA, Dopamine; HERBI, herbimycin A; GEN, genistein.

 
Effects of ß-Arrestin 1 and 2 Overexpressions on Dopamine-Stimulated MAPK Activity by D2L and D2S Receptors
It has been proposed that ß-arrestin binding to certain GPCRs plays a dual role. By sterically blocking the GPCR-G protein interaction, G protein-dependent signaling is arrested. Simultaneously, by recruiting ERK to the agonist-occupied receptor, ß-arrestin-dependent activation of the ERK cascade is initiated (16).

We assessed the role of ß-arrestins in D2L- and D2S-mediated MAPK activation, by overexpressing ß-arrestin 1 or ß-arrestin 2 in CHOD2L and CHOD2S cells. As already proposed elsewhere, if ß-arrestins function as scaffolds to facilitate receptor-mediated ERK activation, then the overexpression of ß-arrestin might be expected to increase D2 receptor-mediated ERK phosphorylation (15, 16). In this study, the overexpression of neither ß-arrestin 1 nor ß-arrestin 2 affected D2L-mediated MAPK activation (Fig. 2AGo). However, as shown in Fig. 2BGo, the overexpressions of both ß-arrestin 1 and ß-arrestin 2 significantly increased D2S-mediated MAPK activation by up to 22–34% and 27–43%, respectively. These results suggest that ß-arrestin is differentially involved in the process of MAPK activation by the D2L and D2S receptors.



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  		Fig. 2. Effects of ß-Arrestin 1 and ß-Arrestin 2 on D2L- and D2S-Mediated MAPK Activation

Cells were transiently transfected with plasmid containing the ß-arrestin 1, ß-arrestin 2, or mock (control). CHOD2L (A) and CHOD2S (B) cells were treated with dopamine (1 µM) for the indicated periods of time, and phospho-ERK and total ERK levels was analyzed in cell lysates by immunoblotting. Data indicate mean ± SE from at least four independent experiments. (*, P < 0.05 as compared with control). DA, Dopamine; Arr1, ß-arrestin 1; Arr2, ß-arrestin 2; NS, dopamine nonstimulated control.

 
Effects of Dominant Negative ß-Arrestin 2 (319–418) Mutant and Dominant Negative Dynamin I (K44A) Mutant on Dopamine-Stimulated MAPK Activity by D2L and D2S Receptors
To determine whether the interaction of the D2L/D2S receptors with ß-arrestin or dynamin is required for MAPK activation by the two D2 receptors, we analyzed the effect of the dominant negative ß-arrestin 2 (319–418) mutant and of the dominant negative dynamin I (K44A) mutant in D2L- and D2S-mediated MAPK activation. ß-Arrestin 2 (318–419) is a truncated form of ß-arrestin 2 that interferes with GPCR sequestration by interacting with clathrin (22, 23) and the dominant negative dynamin I (K44A) mutant, which is void of GTPase activity (24, 25, 26). Through GTP binding and hydrolysis, dynamin pinches off clathrin-coated invaginations to form endocytotic vesicles. As shown in Fig. 3AGo, the expression of neither the dominant negative mutant of ß-arrestin nor that of dynamin affected D2L-mediated MAPK activation. However, D2S-mediated MAPK activation was markedly reduced by the expression of the dominant negative ß-arrestin 2 (319–418) mutant and the dominant negative dynamin I mutant (K44A), in both case being reduced by approximately 60%. These results show that ß-arrestin and dynamin are not essential for D2L-mediated MAPK activation, but that these proteins are crucial for D2S-mediated MAPK activation.



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  		Fig. 3. Effects of a Dominant Negative ß-Arrestin 2 (319–418) Mutant and a Dominant Negative Dynamin I (K44A) Mutant on MAPK Activation in CHOD2L and CHOD2S Cells

Cells were transiently transfected with plasmid containing the ß-arrestin 2 (319–418), dynamin I (K44A), or mock (control). CHOD2L (panel A) and CHOD2S (panel B) cells were treated with dopamine (1 µM) for the indicated periods of time. Data indicate mean ± SE from at least four independent experiments. (*, P < 0.05; **, P < 0.01 as compared with control). DA, Dopamine; NS, dopamine nonstimulated control.

 
Effect of Inhibitors of Internalization on D2L- and D2S-Mediated MAPK Activation after Dopamine Stimulation
The internalization of GPCRs can be inhibited by diverse agents, such as lectin and concanavalin A (con A) (27), which block receptor clustering, and by monodansylcadaverine (MDC) (28), which prevents clathrin association. The pretreatment of cells with either of these compounds has been shown to inhibit clathrin-mediated GPCR internalization without affecting signal transduction (27). Therefore, we investigated whether D2L- or D2S-mediated MAPK activation still occurs in the presence of these GPCR internalization-inhibiting compounds, to explore the association between internalization and MAPK activation with respect to these two dopamine D2 receptors. As shown in Figs. 4AGo and 5AGo, we observed that D2L-mediated MAPK activation was not affected by the presence of either of these compounds. However, in the case of D2S, MAPK activation was severely affected by pretreatment with con A or MDC (Figs. 4BGo and 5BGo). These results suggest that agonist-induced internalization may be differentially involved in D2L- and D2S-mediated MAPK activation and that the internalization event may play an important role in D2S-mediated MAPK activation.



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  		Fig. 4. Effect of an Internalization Inhibitor, con A on MAPK Activity in CHOD2L and CHOD2S Cells

Cells were preincubated for 30 min with con A (0.25 µg/ml). Phospho-ERK and total ERK levels was analyzed in cell lysates by immunoblotting. CHOD2L (panel A) and CHOD2S (panel B) cells were treated with dopamine (1 µM) for the indicated periods of time. Data indicate mean ± SE from at least three independent experiments. (*, P < 0.05; **, P < 0.01 as compared with con A nontreated control). DA, Dopamine; NS, dopamine nonstimulated control.

 


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  		Fig. 5. Effect of an Internalization Inhibitor, MDC, on MAPK Activity in CHOD2L and CHOD2S Cells

Cells were preincubated for 20 min with MDC (300 µM). Phospho-ERK and total ERK levels was analyzed in cell lysates by immunoblotting. CHOD2L (panel A) and CHOD2S (panel B) cells were treated with dopamine (1 µM) for the indicated periods of time. Data indicate mean ± SE from at least three independent experiments. (*, P < 0.05; **, P < 0.01 as compared with MDC nontreated control). DA, Dopamine; NS, dopamine nonstimulated control.

 
Internalization Kinetics of D2L and D2S
We examined the dopamine-induced internalization of D2L and D2S in the absence or presence of ß-arrestin. We incubated CHOD2L and CHOD2S cells with [3H]spiperone, and internalization was measured as the decrease in [3H]spiperone binding activity on the cell surface, as described previously (29). Time course studies revealed that the internalization of D2L and D2S receptors was observed from 30 sec and that about 10% of D2L and 20% of D2S internalization occurred within 1 min and reached a maximum for both D2L and D2S at this time point (Fig. 6Go). The presence of ß-arrestin 1, as demonstrated by Western blot analysis (Fig. 6CGo), strongly enhanced the dopamine-induced receptor internalization for both the D2L and D2S receptors (D2L, 31.5%; D2S, 49.2%, respectively). Therefore, ß-arrestin stimulates the internalization of both D2L and D2S, whereas ß-arrestin appears to influence the D2S-mediated MAPK activation, but not the D2L-mediated one.



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  		Fig. 6. Internalization Kinetics of D2L and D2S

A, Cells were transiently transfected with plasmid containing the ß-arrestin 1 or mock (control). CHOD2L and CHOD2S cells were treated with dopamine (1 µM) for the indicated periods of time. The internalization was assessed by measuring the disappearance of [3H]spiperone binding from the cell surface. B, The diagram indicates the degrees of dopamine-induced internalization at 1 min. Data indicate mean ± SE from at least four independent experiments. (**, P < 0.01 as compared with dopamine nonstimulated control; $, P < 0.05 as compared with D2L). C, ß-Arrestin 1 levels was analyzed in cell lysates by immunoblotting from CHOD2L and CHOD2S cells. Arr 1, ß-Arrestin 1.

 
Confocal Microscopic Analysis of the Cellular Distributions of D2L-Red Fluorescent Protein (RFP) and D2S-RFP after Dopamine Stimulation in Human Embryonic Kidney (HEK)-293 Cells
To determine the effect of dopamine stimulation on the cellular distributions of D2L and D2S, we transiently expressed D2L-RFP and D2S-RFP into HEK293 cells. In parallel with this, we analyzed the regulation of MAPK activation by the aforementioned inhibitor of internalization in HEK293 cells by transiently transfecting D2L and D2S. The experiments performed in HEK293 cells exhibited results similar to those observed in CHOD2L and CHOD2S cells, indicating that our results are a measure of the differential signaling properties of the two receptors, rather than the clonal difference (data not shown). As shown in Fig. 7Go, the fluorescence distributions of the receptors were almost exclusively localized in the plasma membrane in the unstimulated cells. During the initial 5-min period after stimulation with dopamine, the cellular distributions of D2L-RFP and D2S-RFP were unchanged, but after 30 min, they had both been redistributed to the endocytic vesicles.



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  		Fig. 7. The Cellular Distribution of D2L- and D2S-RFP after Dopamine Stimulation in HEK-293 Cells

HEK-293 cells were transiently transfected with plasmid DNA encoding D2L- (A, C, E, and G) and D2S-RFP (B, D, F, and H). Cells treated with vehicle (A and B) or dopamine (DA, 10 µM) for 1 min (C and D), 5 min (E and F), and 30 min (G and H). Data shown are representative of four independent experiments. DA, Dopamine; NS, dopamine nonstimulated control.

 
The Cellular Distributions of D2L-RFP/D2S-RFP and Green Fluorescent Protein (GFP)-ß-Arrestin 1/2 after Dopamine Stimulation in HEK-293 Cells
The involvement of ß-arrestins in the regulation of the internalization of D2L and D2S was investigated using GFP-conjugated arrestins (GFP-ß-arrestins; see Materials and Methods) together with D2L-RFP or D2S-RFP. HEK 293 cells were transiently cotransfected with D2L-RFP or D2S-RFP along with GFP-ß-arrestin 1 or GFP-ß-arrestin 2. In the absence of the ligand, D2L-RFP and D2S-RFP were distributed along the plasma membrane, whereas the GFP-ß-arrestins were diffused in the cytosol [Fig. 8Go, A (a–c) and B (a–c) and Fig. 9AGo (a–c) and B (a–c)]. The stimulation of D2L and D2S resulted in the rapid recruitment of ß-arrestin 1 or ß-arrestin 2 from the cytoplasm to agonist-occupied receptors on the plasma membrane 5 min after dopamine stimulation (Fig. 8Go, A (d–f) and B (d–f) and Fig. 9Go, A (d–f) and B (d–f). After 30 min of exposure to dopamine, D2L-RFP and either GFP-ß-arrestin 1 or GFP-ß-arrestin 2 were colocalized to the endocytic vesicles [Fig. 8Go, A (g–i) and B (g–i)]. Similarly, D2S-RFP and either GFP-ß-arrestin 1 or GFP-ß-arrestin 2 underwent a dramatic translocations to the endocytic vesicles [Fig. 9Go, A (g–i) and B (g–i)]. These results demonstrate that ß-arrestins are recruited to the agonist-activated D2L and D2S receptors at the plasma membrane and are translocated with them into endocytic vesicles.



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  		Fig. 8. The Cellular Distribution of D2L-RFP and GFP-ß-Arrestin 1/2 after Dopamine Stimulation in HEK-293 Cells

HEK-293 cells were transiently transfected with plasmid DNA encoding D2L-RFP and GFP-ß-arrestin 1 (panel A)/2 (panel B). Cells treated with vehicle (a–c) or dopamine (DA, 10 µM) for 5min (d–f) and 30 min (g–i). Colocalization of D2L-RFP and GFP-ß-arrestin 1/2 is shown in the overlay images (c, f, and i). Data shown are representative of three independent experiments. DA, Dopamine; NS, dopamine nonstimulated control.

 


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  		Fig. 9. The Cellular Distribution of D2S-RFP and GFP-ß-Arrestin 1/2 after Dopamine Stimulation in HEK-293 Cells

HEK-293 cells were transiently transfected with plasmid DNA encoding D2S-RFP and GFP-ß-arrestin 1 (panel A)/2 (panel B). Cells treated with vehicle (a–c) or dopamine (DA, 10 µM) for 5 min (d–f) and 30 min (g–i). Colocalization of D2S-RFP and GFP-ß-arrestin 1/2 is shown in the overlay images (c, f, and i). Data shown are representative of three independent experiments. DA, Dopamine; NS, dopamine nonstimulated control.

 
Effect of the Expressions of a Dominant Negative ß-Arrestin 2 (319–418) Mutant and of a Dominant Negative Dynamin I (K44A) Mutant on the Cellular Distribution of D2L-RFP/D2S-RFP after Dopamine Stimulation in HEK-293 Cells
To confirm the role of arrestins and dynamin in the regulation of the internalization of the D2L and D2S receptors, dominant negative forms of dynamin I (K44A) or ß-arrestin (319–418) were also tested for their effects on dopamine-induced receptor internalization.

Figure 10AGo shows that the D2L-RFP and D2S-RFP receptors remain associated with the plasma membrane in cells expressing the dominant negative ß-arrestin 2 (319–418) mutant even after agonist stimulation (Fig. 10AGo, c and d), indicating that the dominant negative ß-arrestin had blocked the ligand-driven internalizations of D2L and D2S. When the dominant negative mutant of dynamin I was cotransfected, the internalization of D2L was not inhibited, and the D2S-RFP receptors remained at the cell surface (Fig. 10BGo, c and d). These results indicated that both ß-arrestin and dynamin are required for the internalization of D2S, whereas the internalization of D2L is arrestin dependent but dynamin independent.



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  		Fig. 10. Effect of the Expression of a Dominant Negative ß-Arrestin 2 (319–418) Mutant and a Dominant Negative Dynamin I (K44A) Mutant on the Cellular Distribution of D2L- and D2S-RFP after Dopamine Stimulation in HEK-293 Cells

HEK-293 cells were transiently transfected with plasmid DNA encoding D2L- (a and c) or D2S-RFP (b and d) and a dominant negative ß-arrestin 2 (319–418) mutant (panel A) or a dominant negative dynamin I (K44A) mutant (panel B). Cells treated with vehicle (a and b) or 10 µM dopamine (c and d) for 30 min. Data shown are representative of three independent experiments. DA, Dopamine; NS, dopamine nonstimulated control.

 
Platelet-Derived Growth Factor (PDGF) Receptor Activation Is Required for D2L-Mediated MAPK Activation
Given that the activation of MAPK by D2L and D2S was differentially regulated, we were interested in identifying the potential mechanism behind this distinct signaling pathway. In recent years receptor tyrosine kinases, such as the epidermal growth factor (EGF) receptor or PDGF receptor, have been implicated in the signaling mechanism from GPCRs to MAPK (30, 31, 32). Therefore, we tested the hypothesis that the D2 receptor-mediated transactivation of a receptor tyrosine kinase might be responsible for the D2 receptor-mediated MAPK activation. It has recently been reported that D2L receptors activate the MAPK cascade by first mobilizing signaling by the PDGF receptor, followed by the subsequent activation of MAPK by pathways associated with this receptor tyrosine kinase (33).

To test whether ERK activation by dopamine is dependent on PDGF receptor signaling, CHOD2L and CHOD2S cells were preincubated with the selective PDGF receptor tyrosine kinase inhibitor, tyrphostin A9, before stimulation with dopamine. Interestingly, A9 markedly inhibited dopamine-induced MAPK activation in CHOD2L cells (Fig. 11Go). Consistent with its ability to inhibit the PDGF receptor, A9 also inhibited PDGF-BB-induced phosphorylation of MAPK. However, in the case of D2S, pretreatment of tyrphostin A9 did not affect the response of MAPK to dopamine, suggesting that D2L induces the activation of MAPK via the transactivation of the PDGF receptor, whereas D2S does not (Fig. 11Go).



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  		Fig. 11. Effect of a PDGF Receptor Inhibitor, Tyrphostin A9 (A9), on MAPK Activity in CHOD2L and CHOD2S Cells

Cells were preincubated 60 min with tyrphostin A9 (1 µM, black bar) or mock (control, open bar). Phospho-ERK and total ERK levels were analyzed in cell lysates by immunoblotting. CHOD2L (panel A) and CHOD2S (panel B) cells were treated with dopamine (1 µM) for the indicated periods of time. Data indicate mean ± SE from at least three independent experiments. (*, P < 0.05; **, P < 0.01 as compared with tyrophostin A9 nontreated control). DA, Dopamine; NS, dopamine nonstimulated control.

 
As additional evidence of PDGF receptor activation, we measured the tyrosine phosphorylation by immunoprecipitation of the native PDGF receptors from cell lysates, followed by immunoblotting with an antiphosphotyrosine antibody. Indeed, the transient transfection of the PDGF receptor-ß in CHOD2L followed by treatment with dopamine produced an immediate increase in the tyrosine phosphorylation of PDGF receptor (Fig. 12Go), supporting the hypothesis that the dopamine D2L receptor can transactivate this receptor tyrosine kinase. Under similar conditions, however, the transient transfection of the PDGF receptor-ß in CHOD2S followed by treatment with dopamine did not induce the tyrosine phosphorylation of the PDGF receptor-ß (Fig. 12Go).



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  		Fig. 12. Effects of Dopamine Treatment on Tyrosine Phosphorylation of PDGF Receptor-ß (PDGFRß) in CHOD2L and CHOD2S Cells

Cells were transiently transfected with plasmid DNA encoding PDGF receptor-ß. Cells were preincubated 60 min with tyrphostin A9 (1 µM) or mock (control). CHOD2L (panel A) and CHOD2S (panel B) cells were treated with dopamine (1 µM) and PDGF-BB (1 ng/ml) for 1 min. PDGF receptor-ß was immunoprecipitated (IP) from cell lysates, and the phosphotyrosine levels were analyzed by immunoblotting (IB). DA, Dopamine; BB, PDGF-BB; A9, tyrphostin A9; pY, phosphotyrosine.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Considerable evidence now indicates that certain GPCRs can interact with the MAPK signaling pathway, although the molecular basis for this interaction has not yet been elucidated.

It has been proposed that the receptor-ß-arrestin complex initially signals to MAPK, as was proposed in a study of the ß2-adrenergic receptor (16, 17, 18). ß-Arrestin binds to agonist-activated GPCRs after their phosphorylation by receptor kinases and links the activated GPCRs to clathrin, thereby targeting them to the endocytic pathway (34, 35, 36).

Recent studies indicate that the ligand-induced activation of MAPK, both by receptor tyrosine kinases, such as the epidermal growth factor receptor (37), and GPCRs, such as the ß2-adrenergic receptor (18), is also specifically inhibited by dominant negative mutant dynamin.

Similar results have been reported for several other GPCRs. MAPK activation by the m1 muscarinic receptor (38), the {delta}-opioid receptor, serotonin 1A receptors (39, 40), and the proteinase-activated receptor 2 (41) were reported to be sensitive to endocytosis inhibition. However, these findings conflict with other reports, based on studies of the {alpha}2 adrenergic receptor (42, 43), the m3 muscarinic receptor (44), and the B2 bradykinin receptor (45). In the case of these receptors, signaling to MAPK was not blocked by dominant-negative mutant dynamin (42, 43, 44), suggesting that these receptors can signal MAPK through a dynamin-independent mechanism. These reports suggest that receptor endocytosis is not universally essential for MAPK activation by GPCR. Accordingly, the contribution of endocytosis to GPCR signaling through the MAPK pathway is not fully understood.

In the present study, we demonstrated that the internalization is differentially regulated in association with MAPK activation for D2L and D2S, the two isoforms of the dopamine D2 receptor, by using both stable CHOD2L and CHOD2S cells and HEK293 cells transiently expressing these two receptors.

Both D2L- and D2S-mediated MAPK activations were suppressed by herbimycin A and PP2, suggesting that c-Src is involved as an upstream regulator of MAPK in this pathway. It has also been reported that the recruitment of c-Src is essential both for the receptor-mediated activation of the MAPK cascade and for the internalization of the ß2-adrenergic receptor (16). Furthermore, there are several reports which indicate that the c-Src-mediated phosphorylation of components of the endocytic machinery, such as dynamin and clathrin, are important for this internalization process (46, 47). We observed that both D2L and D2S could recruit c-Src after being stimulated by dopamine, in the presence of ß-arrestin; this indicates that their interaction with c-Src is a necessary and indispensable step for both receptors, in order for them to be able activate the ERK cascade, irrespective of their involvement in the internalization of the receptors (data not shown).

The role of the major component of internalization, ß-arrestin, in D2 receptor-mediated MAPK activation, was also explored in this study. D2L-mediated MAPK activation was not significantly affected by the overexpression of ß-arrestins or the expression of the dominant negative ß-arrestin 2 (319–418) mutant, whereas D2S-mediated MAPK activation was significantly increased by the overexpression of the ß-arrestins. Furthermore, D2S-mediated MAPK activation was significantly attenuated by the expression of a dominant negative ß-arrestin 2 (319–418) mutant. These data demonstrate that ß-arrestins are required for D2S-mediated MAPK activation, whereas these components are not required for D2L-mediated MAPK activation.

In addition, the inhibition of D2L receptor internalization by con A or MDC did not affect the ability of the receptor to stimulate MAPK activity, for example, pretreating con A or MDC significantly impaired MAPK activation by D2S. This difference in MAPK activation may imply that agonist-induced internalization involves D2L- and D2S-mediated MAPK activation differentially and strongly suggests that the internalization event is indispensable in D2S-mediated MAPK activation.

Because con A, MDC, and dominant negative dynamin (K44A) mutant inhibit the endocytic pathway by very different mechanisms, it appears that clathrin-dependent endocytosis is essential for D2S-mediated MAPK activation. In this situation, proteins in the MAPK cascade need to assemble at coated pits or endocytic vesicles, to effectively activate MAPK (20, 21). This is probably the case for D2S, but it is likely that a different dynamin-independent mechanism exists for D2L.

Indeed, using confocal microscopy, we observed that both D2L and D2S rapidly recruit ß-arrestin 1 and ß-arrestin 2 after agonist stimulation. These results indicate that ß-arrestins participate in the internalization of D2L; however, this does not necessarily lead to MAPK activation by D2L. Furthermore, a clear difference was observed when the dynamin mutant was coexpressed, as this showed complete inhibition of the internalization of D2S but had no effect on the internalization of D2L. It has been proposed that dynamin controls the MAPK cascade at the level of MEK-induced MAPK activation, with no apparent role for dynamin-regulated receptor internalization, suggesting that there is a proper compartmentalization and dynamin-controlled trafficking of signaling intermediates in the GPCR-mediated MAPK activation cascade (21). In this context, two assumptions can be made. First, that D2L and D2S differ in their regulation of the MAPK activation cascade. This suggestion is compatible with our previous reports (10). Second, that this differential regulation of MAPK activation could be largely due to the differential internalization profiles of D2L and D2S receptors with their differential employment of ß-arrestins and dynamin.

Indeed, in our experiments designed to assess the internalization kinetics of D2L and D2S receptors in CHOD2L and CHOD2S cells, respectively, both receptors displayed a different internalization kinetic profile. D2S exhibits a more efficient internalization profile than D2L and, interestingly, the presence of the ß-arrestin enhanced the internalization of both D2 receptors to the same extent. It is likely that ß-arrestin is a key player in the internalization of both receptors, which, however, interfere differentially with receptor-mediated MAPK activation.

In this context, it is also intriguing that a growth factor receptor could be trans-activated by D2L but not by the D2S receptor. Recently, Oak et al. (33) reported a rapid increase in tyrosine phosphorylation of PDGF receptor-ß upon stimulation of the D4 and D2L receptors. In this study, as a possible mechanism of MAPK activation mediated by two D2 receptors, we assessed whether the transactivation of the PDGF receptor is related to MAPK signaling by D2 receptors. In the present study, we showed that D2L activates MAPK signaling by mobilizing the growth factor receptor, PDGF receptor, whereas D2S appears to activate MAPK signaling by mobilizing clathrin-mediated endocytosis in a ß-arrestin/dynamin dependent manner. D2L-mediated MAPK activation, which is sensitive to inhibitors of the PDGF receptor, is not ß-arrestin-dependent, despite the fact that the internalization of D2L is enhanced by the presence of ß-arrestin. Based on these data, it appears that in the case of D2L, the MAPK activation and internalization involves two distinct signaling pathways whereas in the case of D2S, these two events are part of a single signaling cascade (Fig. 13Go).



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  		Fig. 13. Proposed Mechanism of MAPK Activation by D2L and D2S Receptors

In the case of D2L receptor, PDGF receptor-ß is transactivated by an unknown process that may involve c-Src. Subsequent signaling pathways involved in signal transduction include Ras, MEK, and ERK1/2. The D2S receptor appears to activate MAPK signaling by mobilizing clathrin-mediated endocytosis in a ß-arrestin/dynamin-dependent manner. Different inhibitors, which were used in this study, are indicated.

 
Recently, in separate reports, it was suggested that the internalization of D2L could be dynamin-independent (48) and that that of D2S might be dynamin-dependent (29, 49), which would be in-line with the results of our present study. It has also been reported that there are significant differences in the agonist-induced sequestrations of D2L and D2S (50). In fact, we observed by confocal microscopic analysis that more endocytic vesicles were detected upon D2S stimulation than upon D2L stimulation, suggesting that D2L and D2S differ quantitatively and qualitatively in terms of receptor trafficking.

The D2L and D2S receptors are identical except for the insertion of 29 amino acids in the third intracellular loop of D2L, resulting from alternative splicing. It is therefore tempting to speculate that the third intracellular loop of the D2 receptor might be involved in the differential regulation of internalization, in association with MAPK activation. It has been suggested that there are some specific structural requirements relating to the third intracellular loop or the C-terminal region of GPCR, which are essential for the internalization of GPCR. For example, it has been reported that the third intracellular loops of the m1 and m2 muscarinic acetylcholine receptors are involved in internalization (51, 52). And, interestingly, it has also been reported that the ß2-adrenergic receptor is internalized to a greater extent than the ß1-adrenergic receptor, which contains an additional 24 amino acid residue in the third intracellular loop (53). We will undertake the identification of the structural requirements of these differential endocytic pathways of the two D2 receptors in a future study.

The physiological role served by this differential internalization profile between two isoforms of dopamine D2 receptors also remains to be elucidated. Recent studies with D2L-deficient mice have raised the possibility that the presynaptic effects of dopaminergic ligands are probably mediated by D2S and not by other D2-like receptors (12, 54). Based on these studies, one can hypothesize that the differential endocytosis of D2L and D2S receptors may play an important role in their postsynaptic/presynaptic functions. Indeed, dynamin-dependent endocytosis clearly plays a critical role in synaptic vesicle membrane recycling in presynaptic neurons (55), which may explain the necessity for these two D2 receptors to have different endocytic membrane trafficking properties. Thus, it is possible that the segregation of D2L and D2S in distinct endocytic membranes plays an important role in determining subtype-specific receptor signaling.

Recently, Kotecha et al. (56) reported that D2 class receptors inhibit N-methyl-d-aspartate receptor (NMDAR) activity in the hippocampus by a mechanism that involves transactivation of PDGF receptors and the subsequent enhancement of Ca2+-dependent inactivation. Because PDGF receptors are likely to play a pivotal role in synaptic neurotransmission, it is tempting to speculate that the distinct involvement of the transactivation of PDGF receptor in MAPK activation shared by D2L and D2S may result in a distinct physiological outcome in vivo. Furthermore, their different endocytosis signaling mechanisms might lead to there being a variable combination of differential signaling possibilities for D2L and D2S in vivo. An examination of the physiological roles, dictated by these distinct forms of endocytosis of the two dopamine D2 receptors, might provide fundamental new insights into the diversity of membrane trafficking and its relation with dopaminergic neurotransmission and related disorders.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
ß-Arrestin 1 and ß-arrestin 2 were kindly provided by Dr. R. J. Lefkowitz (Duke University, Durham, NC). A dominant negative ß-arrestin 2 (319–418) mutant and a dominant negative dynamin I (K44A) mutant were kindly provided by Dr. E. Kelly (Bristol University, Bristol, UK) and Dr. Benovic (Thomas Jefferson University, Philadelphia, PA). PDGF receptor-ß was kindly provided by Dr. M. Quon (National Institutes of Health, Bethesda, MD) (57) and Dr. A. Kazlauskas (Harvard Medical School, Boston, MA) (58). pEGFP-C2 and pDsRed1-N1 were purchased from CLONTECH (Palo Alto, CA). FuGene 6 was purchased from Roche Diagnostics (Indianapolis, IN). Herbimycin A and PP2 were obtained from Calbiochem (San Diego, CA). Genistein was obtained from Research Biochemicals, Inc. (Natick, MA). Con A, MDC, tyrphostin A9, and PDGF-BB were obtained from Sigma-RBI (St. Louis, MO). Mouse monoclonal antiphospho-ERK (Tyr204), rabbit polyclonal anti-ERK, and rabbit polyclonal anti-PDGF receptor-ß (958) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal mouse antiphospho-tyrosine antibody (4G10) was from Upstate Biotechnology, Inc. (Lake Placid, NY). Mouse anti-ß arrestin 1 monoclonal antibody was from Research Diagnostics, Inc. (Flanders, NJ). Antimouse-horseradish peroxidase (HRP) and antirabbit-HRP were from Zymed Laboratories, Inc. (South San Francisco, CA). All other biochemicals, including dopamine, were obtained from Sigma.

Plasmid Construction
Constructs were produced by PCR using sets of primers, D2L/S and ß-arrestin 1/2. For D2L/S-RFP, D2L/S fragments were generated by PCR from D2L/S, ligated into the expression vector pFLAG, and sequenced. The forward and reverse primers used were 5'-CAGAGATCTAGCCATGGATCCACTGAACCTGTC and 3'-ACTGAATTCCGCA GTGCAGGATCTTCATGA, respectively. The amplified fragments were fluorescently tagged at the carboxyl terminus using a mutant RFP. The entire D2L and D2S were subcloned into BglII (5')-EcoRI (3') sites of pDsRED1-N1. For GFP-ß-arrestin 1/2, ß-arrestin 1/2 fragments were generated by PCR from ß-arrestin 1/2, ligated into the expression vector pCMV5, and sequenced. The ß-arrestin 1 forward and reverse primers used were 5'-CAGAAGCTTCATGGGCGACAAAGGGACA, 3'-ACTGTCGACTCTAT CTGTTGTTGAGGTG. The ß-arrestin 2 forward and reverse primers used were 5'-CAGAAGCTTCATGGGTGAAAAACCCGGG and 3'-ACTGTCGACCCTAGCAGAACT GGTCATC, respectively. The amplified fragments were fluorescently tagged at the amino terminus using a mutant GFP. The entire ß-arrestin 1 and 2 were subcloned into HindIII (5')-SalI (3') sites of pEGFP-C2.

Cell Culture and Transfection
The generation of the CHO cell lines stably expressing the D2L and D2S dopamine receptors has been described previously (16). The Bmax (maximal binding capacity) value of the clones CHOD2L and CHOD2S, which were used in this study, were found to be 191.4 ± 0.74 and 176.8 ± 0.79 fmol/mg protein, respectively. CHO cells were maintained in F-12 medium supplemented with 10% fetal bovine serum, 100 mg/ml streptomycin sulfate, 100 U/ml penicillin G, and 250 mg/ml amphotericin B. Transient transfections of CHO and HEK cells were performed using the liposome-mediated transfection reagent, FuGene 6. Briefly, 70% ~ 80% confluent monolayers in 30-, 60-, and 100-mm culture plates were incubated at 37 C in serum-free medium with transfection mixture containing the plasmid DNA encoding ß-arrestin 1, ß-arrestin 2, a dominant negative ß-arrestin 2 (319–418) mutant, a dominant negative dynamin I (K44A) mutant, c-src, PDGF receptor-ß, and the plasmid pCH110 carrying the ß-galactosidase gene and liposome reagent. After 6 h, the transfection mixture was then replaced with growth medium. Assays were performed 48 h after transfection. Expressions of ß-arrestin 1, ß-arrestin 2, a dominant negative ß-arrestin 2 (319–418) mutant, a dominant negative dynamin I (K44A) mutant, c-Src, and PDGF receptor-ß were normalized by measuring the ß-galactosidase activity. To detect transfection efficiencies, cells cotransfected with pCH110 were extracted and harvested in Z-buffer (0.06 M Na2HPO4, 0.04 M NaH2PO4, 0.01 M KCl, 0.001 M MgSO4, 0.05M mercaptoethanol). The cell lysates were analyzed using the colorimetric O-nitrophenyl-ß-D-galactopyranoside (Sigma) assay at 37 C for 30 min. Optical density of the reactions was read at a wavelength of 420 nm. One milliunit is defined as the amount of ß-galactosidase that hydrolyzes 1 nmol of O-nitrophenyl-ß-D-galactopyranoside per min at 37 C. A standard curve of ß-galactosidase from 0.05–100 mU was measured with each set of samples. A dilution that produced optical density values in the linear range was used to determine transfection efficiency.

Immunoblotting Analysis
Serum-starved transfected cells were stimulated with dopamine for the indicated time period at 37 C. The media were then aspirated and the cells were lysed in lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerolphosphate, 1 mM Na3VO4, 1 mg/ml leupeptin, and 1 mM phenylmethylsulfonylfluoride. Samples were sonicated four times for 5 sec each and centrifuged at 10,000 x g at 4 C for 10 min and the supernatant was collected. For phospho-specific p44/p42 MAPK (ERK 1/2) and total MAPK (ERK1/2) measurements, cell lysates were prepared as in the assay for MAPK activity. The proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA). The blots were incubated with 5% dried milk powder in TBST (10 mM Tris, pH 8.0; 150 mM NaCl; 0.05% Tween 20; also used for all incubations and washing steps) for 30 min. Next, the blots were incubated for 1 h with monoclonal antiphospho-ERK (Tyr204), and rabbit polyclonal anti-ERK antibody followed by extensive washing. The blots were subsequently incubated with peroxidase-conjugated antirabbit-IgG antibody. After washing, signals were visualized using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Arlington Heights, IL). Western blot analysis for ß-arrestin detection was performed using monoclonal anti-ß-arrestin 1 (1:3000).

Immunoprecipitating Analysis
Immunoprecipitations of myc epitope-tagged D2L and D2S were performed after transient transfection of CHO cells in 100-mm dishes. Cells were transfected with or without plasmids encoding pCMV-ß-arrestin 1 (2 µg/plate) and pUSE-src (2 µg/plate) with expression plasmid encoding myc-D2L, D2S (2 µg/plate), as indicated. Transient transfections of CHO cell lines stably expressing the D2L and D2S dopamine receptors were performed with or without plasmid encoding pcis2-PDGF receptor-ß (3 µg/plate). Stimulation was performed as described in the figure legends. Cells were lysed in lysis buffer, and equal protein aliquots of cell lysates (1 mg) were incubated with a monoclonal anti-myc-antibody (1:1000), a polyclonal anti-PDGF receptor-ß (1 µg/ml) (4 h at 4 C) followed by addition of 20 µl of 50% protein A-sepharose (6 h at 4 C). After washing three times with lysis buffer, the pelleted beads were resuspended in sample buffer and denatured (100 C for 3min). The immunoprecipitated proteins were separated with a 10% SDS-PAGE gel, and Western blotting was carried out using monoclonal anti-src, antiphospho-tyrosine (1 µg/ml)/antimouse-HRP (1:1000).

Confocal Microscopy
For the D2L and D2S translocation assay, HEK-293 cells were transfected with D2L-RFP or D2S-RFP. For the colocalization assay, HEK-293 cells were transfected with D2L-RFP or D2S-RFP and either GFP-ß-arrestin 1 or GFP-ß-arrestin 2. After various treatments, the cells were fixed with 4% paraformaldehyde in PBS for 30 min. Cells were then washed with PBS and mounted for fluorescent confocal microscopic evaluation. Confocal microscopy was performed on a Zeiss LSM-510 laser scanning microscope (Carl Zeiss, Thornwood, NY) by using a Zeiss x100 oil-immersion lens. Fluorescent signals were collected using Zeiss LSM software in the line switching mode with dual excitation (488 nm, 568 nm) and emission (515–540 nm, 590–610 nm) filter sets.

Internalization Assay
Transfected cells were incubated overnight in serum-free medium at 37 C. Serum-starved cells were stimulated with 1 µM dopamine for 0–60 min as indicated. Stimulation was terminated by quickly cooling the dishes on ice, washing the cells two times with ice-cold PBS, and rinsing the cells from the plate in 100 µl ice-cold PBS/EDTA. Lysates (100 µl) were mixed with 400 µl [3H]spiperone (final concentration 3 µM) in binding buffer (120 mM NaCl, 50 mM Tris-HCl, 5 mM KCl, 5 mM MgCl2, 1.5 mM CaCl2). The hydrophobic properties of [3H]spiperone allowed for the measurement of the total cellular levels of D2R (i.e. intra- and extracellular), whereas the displacement of extracellular (i.e. plasma membrane-associated) [3H]spiperone with unlabeled, hydrophilic sulpiride (3 µM) allowed the direct measurement of the internalized receptor. Nonspecific binding was determined in the presence of 10 µM spiperone. The reactions for the purpose of measuring the total, intracellular, and nonspecific binding were performed in triplicate for each time point. Binding reactions were incubated for 4 h at 4 C to prevent receptor recycling and terminated by washing three times with vacuum filtration over GF/C glass fiber filters (Whatman, Clifton, NJ) in a Brandel cell harvester using 50 mM Tris-HCl, pH 7.7. The bound radioligand was detected by liquid scintillation counting.


   ACKNOWLEDGMENTS
 
We thank Dr. Robert J. Lefkowitz (Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC) for providing the ß-arrestin 1 and ß-arrestin 2 plasmids, and also Dr. E. Kelly (Bristol University) and Dr. Benovic (Thomas Jefferson University, Philadelphia, PA) for providing the arrestin mutant and dynamin mutant construct. We are also grateful to Dr. M. Quon (National Institutes of Health, Bethesda, MD) and Dr. A. Kazlauskas (Harvard Medical School, Boston, MA) for providing us with the PDGF receptor-ß DNAs. We also thank Drs. H. I. Kim, M. G. Lee, and J. Kim (College of Medicine, Yonsei University, Seoul, South Korea) for their help and useful advice.


   FOOTNOTES
 
This work was supported by a research grant (Grant R04-2000-00051) from the Korea Science and Engineering Foundation, the Basic Research Program of the Korea Research Foundation (Grant 2000-015-DP0327), the National Neurobiology Research Program (Grant M1-0108-00-0063), and the Molecular and Cellular BioDiscovery Research Program (Grant M1-0311-00-0069) of the Korean Ministry of Science and Technology, and by a Korea University Grant. S.J.K. and E.J.L. are the recipients of a Brain Korea 21 program grant for medical science from the Korean Ministry of Education.

Abbreviations: CHO, Chinese hamster ovary; con A, concanavalin A; D2L, long isoform of dopamine D2 receptor; D2S, short isoform of dopamine D2 receptor; EGF, epidermal growth factor; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; HEK, human embryonic kidney; HRP, horseradish peroxidase; MDC, monodansylcadaverine; MEK, MAP/ERK kinase; PDGF, platelet-derived growth factor; PP2, 4-amino-5 (4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine; RFP, red fluorescent protein;

Received for publication February 28, 2003. Accepted for publication December 9, 2003.


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