Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

G Protein-coupled receptors (GPCRs) play a critical role in regulation of cell proliferation, differentiation, and survival. These receptors transduce signals by coupling to heterotrimeric G proteins. Recent studies have identified members of the mitogen-activated protein kinase (MAPK) family as critical intracellular signaling pathways that are activated by GPCRs. Ras-dependent and-independent mechanisms were demonstrated to be involved in MAPK activation by GPCRs (Garnovskaya et al., 1996; Lopez-Ilasaca et al., 1997; Sugden and Clerk, 1997). The mechanism by which pertussis toxin (PTX)-sensitive Gi-coupled receptors activate MAPK was reported to be Ras-dependent and mediated via G subunits (Gutkind, 1998). Moreover, the linkage between GPCRs and Ras/MAP kinase activation was shown to require tyrosine phosphorylation of the adaptor protein SHC and its subsequent association with Grb2. Tyrosine kinases such as Src play essential roles in mediating the phosphorylation of SHC by GPCRs (Luttrell et al., 1996, 1997) and the SHC-Grb2/Ras/MAP kinase cascades constitute important mitogenic pathway initiated by GPCRs (Sugden and Clerk, 1997; Gutkind, 1998).

GPCRs were also shown to activate nuclear factor NF-B and to regulate the expression of inducible genes (Shahrestanifar et al., 1999; Xie et al., 2000). Under basal conditions, NF-B is a heterodimer composed of 50- and 60-kDa subunits that exist as a complex bound to a member of the IB family of inhibitory proteins in cytosol. A stimulus triggers the dissociation of NF-B from IB; the latter is rapidly degraded. Cytoplasmic NF-B is then translocated into the nucleus, where it regulates gene transcription. MAP kinases and Akt were both shown to regulate NF-B activation (Schulze-Ostehoff et al., 1997; Kane et al., 1999; Ozes et al., 1999; Xie et al., 2000). Many GPCRs regulate Akt (Murga et al., 1998 and 2000) and its downstream transcription factor, NF-B, comprising a major cellular pathway in the regulation of cell survival and differentiation (Marte and Downward, 1997).

Dopamine receptors are members of the GPCR superfamily. The five cloned dopamine receptors are distinguished based on pharmacological and biochemical characteristics (Sibley et al., 1993; Tang et al., 1994; Missale et al., 1998). Members of the D₂-like dopamine receptor family, D₂, D₃, and D₄, couple to Gi protein. Activation of these dopamine receptors initiates a number of signaling events, including inhibition of adenylyl cyclase and opening of K⁺ channels. Stimulation of dopamine receptors was also demonstrated to differentially regulate the MAPKs. Although stimulation of D₂ and D₃ dopamine receptors was shown to activate ERK in vitro and in vivo (Welsh et al., 1998; Cussac et al., 1999; Yan et al., 1999; Cai et al., 2000) and to evoke a mitogenic response through the Ras/ERK pathway (Luo et al., 1998), D₁ dopamine receptor stimulation activates p38 MAPK via a protein kinase A-dependent pathway (Zhen et al., 1998). The D₄ dopamine receptor has garnered attention because of its potential role in schizophrenia and in the psychopharmacology of antipsychotic drugs (Van Tol et al., 1991; Helmeste et al., 1996). However, it remains to be determined whether D₄ dopamine receptors participate in the regulation of the ERK pathway, as do other D₂-like dopamine receptors.

Enhanced NF-B activation was observed in brains from patients with Parkinson's disease (Hunot et al., 1997), suggesting its possible regulation by the dopaminergic system. However, the regulation and signaling mechanism of Akt and its associated NF-B cascade by dopamine receptors has not been addressed. In the present study, we employed a stable D₄ dopamine receptor-transfected MN9D cell line (D₄MN9D) to examine the possible role of the Akt/NF-B and ERK pathways in D₄ dopamine receptor signaling. Our results constitute the first evidence that the D₄ dopamine receptor regulates the Akt/NF-B and ERK pathways via distinct mechanisms.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. PD168077 maleate (a selective D₄ dopamine receptor agonist), L745,870 trihydrochloride (antagonist), and quinpirole (D₂-like receptor agonist) were purchased from Tocris Cookson, Inc. (Baldwin, MO). NF-B consensus oligonucleotide and T4 kinase were from Promega (Madison, WI). PD98059, LY294002, PP2 and PP3 were obtained from CalbioChem (La Jolla, CA). Myelin basic protein (MBP) and PKI were purchased from Sigma (St. Louis, MO). Electrophoresis reagents were obtained from Bio-Rad (Richmond, CA). Anti-phosphotyrosine antibody, anti-SHC, and Crosstide were obtained from Upstate Biotechnology (Lake Placid, NY). Anti-Akt, ERK₂, IB, and anti-Grb2 and proteins A/G PLUS were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-HA 12CA5 antibody was from Roche Molecular Biochemicals (Indianapolis, IN). Horseradish peroxidase-linked anti-rabbit secondary antibodies were obtained from Pierce (Rockford, IL). [³²P]ATP (3000 Ci/mmol) and [³H]thymidine (76 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). LipofectAMINE transfection reagent was obtained from Invitrogen (Carlsbad, CA). Other reagents were purchased from standard laboratory suppliers.

Cell Culture and Cotransfection. Wild-type MN9D cells and D₄ dopamine receptor-transfected MN9D cells (D₄MN9D) were kindly provided by Dr. K. L. O'Malley (Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO). The MN9D cell line is a stable immortalized clonal cell line established by the fusion of the neuroblastoma cell N18TG2 with embryonic mouse mesencephalic dopamine-producing neurons. This cell line does not express native dopamine receptors yet exhibits features of neuronal cells and has been demonstrated to be useful in the study of dopamine receptor-mediated signal transduction (Tang et al., 1994; Yamaguchi et al., 1997). Plasmids expressing Myr-Akt (activated Akt) and kinase-deficient Akt-K179 M (KD-Akt) were gifts from Dr. R. N. Pittman (University of Pennsylvania, Philadelphia, PA). D₄MN9D cells were cultured in DMEM as described previously (Yamaguchi et al., 1997). Cells were transfected by the LipofectAMINE method using the indicated amounts of plasmid encoding N17Ras, Myr-Akt, or KD-Akt. After 48 h, cells were treated with various agents and harvested for assays.

Preparation of Cell Lysates. When the cells were 70 to 80% confluent, the medium was replaced with low serum DMEM (1% FBS) and kept overnight before experiments. After incubation with various agents, the reactions were stopped by aspiration of the medium and the cells were washed twice with cold phosphate-buffered saline. For immunoblot and phosphorylation analyses, cells were lysed in buffer A (50 mM Tris, pH 7.4, 150 mM NaCl, 0.25% sodium deoxycholate, 3 mM Na₃VO₄, 1 mM EGTA, 10 mM NaF, 1 mM PMSF, and proteinase inhibitors). In preparation for the ERK activity assay, cells were lysed in buffer B (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 20 mM -glycerophosphate, 1 mM EGTA, 20 mM NaF, 3 mM Na₃VO₄, 1 mM PMSF, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 1% Nonidet P-40). The lysis buffer for the Akt assay was described previously (Murga et al., 2000). Lysates were centrifuged at 12,000g for 15 min at 4°C and the protein content in the supernatant was determined. Aliquots of supernatants were used in the immunoprecipitation or immunoblotting experiments as described below.

In Vitro Immune Complex Kinase Assays. For immunoprecipitation, 3 µg of anti-ERK2 or anti-Akt antibodies were added to aliquots (200 µg of protein) of cell lysates and incubated overnight at 4°C before the addition of 15 µl of protein A/G PLUS agarose and incubated for 2 h at 4°C. The immune complex was washed three times with buffer B and twice with the corresponding kinase assay buffer (see below). Aliquots of immunoprecipitates (10 µl) were boiled in an equal volume of 2× sample buffer and subjected to SDS-PAGE. After transfer to nitrocellulose, the filter was blotted with anti-ERK2 (which reacts predominantly with ERK2, exhibiting a slight cross-reactivity with ERK1) or Akt antibodies to confirm the equivalence of precipitated enzyme. ERK kinase activity was assessed for 20 min at 30°C in the presence of 50 µM [³²P]ATP (5 µCi), and 0.2 mg/ml MBP as described previously (Zhen et al., 1998). The reaction was terminated by adding an equivalent volume of 2× sample buffer, boiled for 5 min, and the products were resolved by SDS-PAGE. The gels were stained with Coomassie Blue to confirm equivalence of loaded substrate. Phosphorylated MBP was assessed by autoradiography and quantified by densitometry. Alternatively, the radioactivity incorporated into MBP was determined by scintillation counting (see below). The Akt kinase assay was performed in 20 mM HEPES, pH 7.4, containing 10 mM MgCl₂, 10 mM MnCl₂, 20 µM ATP, 1 mM DTT, 10 µCi of [-³²P]ATP, and 30 µM Crosstide in the presence of PKI (a protein kinase A inhibitor) for 20 min at 30°C. The reaction was stopped by addition of 200 mM EDTA-bovine serum albumin solution (15 µl) and 20 µl were spotted onto P81 paper and washed extensively in 0.425% phosphoric acid. Radioactivity incorporated into Crosstide was determined by scintillation counting as described previously (Zhen et al., 1998).

Immunoblotting. For the assay of tyrosine phosphorylation of SHC, 0.5 mg of supernatant protein was incubated overnight at 4°C with 3 µg of anti-SHC polyclonal antibody. After the addition of 15 µl of protein A/G PLUS agarose, immunoprecipitates were collected and washed three times with buffer A. The immune complex was resuspended in 40 µl of sample buffer, separated by SDS gel electrophoresis, and transferred to nitrocellulose membranes. The blots were probed with anti-phosphotyrosine antibody. The signals were visualized with the Supersignal Western Blot Detection System (Pierce). In some experiments, membranes containing SHC immunoprecipitates were stripped and reprobed with anti-Grb2 antibody to assess SHC-Grb2 complex formation. For immunoblots, equal amounts of protein were subjected to 10 or 12% SDS-PAGE, blotted, and the membranes were probed with anti-IB or anti-HA antibody, respectively.

Nuclear Extraction. Nuclear protein extraction was prepared as described previously (Pan et al., 1999; Shahrestanifar, 1999). Briefly, control or treated cells grown in 100-mm² culture dishes were washed in cold phosphate-buffered saline and collected by centrifugation. All procedures were conducted at 4°C. Cells were suspended in buffer (10 mM HEPES, pH 7.8, 10 mM KCl, 0.1 mM EDTA, 2 mM DTT, and 1 mM PMSF, and proteinase inhibitors for 10 min on ice. After adding Nonidet P-40 (to a final concentration of 0.5%) and incubation for an additional 10 min, the lysate was centrifuged for 5 min at 12,000g. The pellet was resuspended in buffer [20 mM HEPES, pH 7.8, 0.42 M NaCl, 5 mM EDTA, 2 mM DTT, 1 mM PMSF, and 10% (v/v) glycerol], and agitated at 4°C for 30 min. After centrifugation at 12,000g for 10 min, the protein content of the supernatant (nuclear fraction) was determined by Bradford assay (Bio-Rad) and aliquots were stored at 80°C.

Electrophoretic Mobility Shift Assay (EMSA). Double-stranded oligonucleotides containing a specific consensus sequence for NF-B were labeled with ³²P using T₄ polynucleotide kinase and purified with G-50 spin columns. The binding reaction was conducted in 20 µl of reaction buffer (10 mM Tris-HCl, 10 mM MgCl₂, 100 mM KCl, 1 mM EDTA, 1 mM DTT, and 10% glycerol) containing 10 µg of nuclear extract and 1 µg of poly(dI.dC), and ³²P-labeled oligonucleotide at room temperature for 20 min. After addition of loading buffer, the samples were separated by electrophoresis on 5% polyacrylamide gels in 0.5× Tris/borate/EDTA buffer. The gel was dried and exposed to film at 80°C. Appropriate positive and negative controls were included in the assay to ensure the specificity of the reaction.

Measurement of Cell Proliferation. Cells were seeded in 24-well plates and grown to 70 to 80% confluence. The medium was replaced with fresh medium containing 0.5% FBS and the cells were incubated overnight before the addition of test agents. The incubation continued for 18 h before the addition of [³H]thymidine (0.5 µCi/ml) and cells were harvested 6 h later. Incorporation of [³H]thymidine was determined by liquid scintillation counting.

Data Analysis. Unless otherwise indicated, data were analyzed by analysis of variance followed by Newman-Keuls test. p < 0.05 was considered significant.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

D₄ Dopamine Receptor Regulates Akt and ERK Pathways in D₄MN9D Cells. Subconfluent D₄MN9D cells were treated with the selective D₄ dopamine receptor agonist PD168077, and Akt or ERK activity was determined. A time-dependent activation of Akt was observed; maximal stimulation was achieved at 5 min and maintained for at least 30 min (Fig. 1A). Stimulation of Akt was detected with 0.1 µM PD168077, reached a maximal 2-fold stimulation at 1 µM, and declined at 10 µM (Fig. 1B). The kinetics of ERK stimulation indicates that maximal stimulation by 1 µM PD168077 occurred at 10 min and returned to basal levels by 30 min (Fig. 2A). As was the case for Akt, maximal stimulation of ERK was achieved with 1 µM PD168077 (Fig. 2B). Quinpirole, an agonist of the D₂ dopamine receptor family, also activated ERK and Akt (Fig. 3A) in these cells. In contrast to this agonist-specific effect in D₄MN9D cells, treatment of MN9D cells that do not express the D₄ dopamine receptor with PD168077 did not stimulate either ERK or Akt (Fig. 3B). The results, therefore, suggest that activation of ERK and Akt are mediated by D₄ dopamine receptors in D₄MN9D cells. Indeed, a selective D₄ receptor antagonist, L745,870, completely blocked the stimulatory effects of PD168077 on ERK and Akt in D₄MN9D cells (Fig. 3C). Moreover, PTX attenuated the PD168077-induced stimulation of ERK and Akt, indicating that a PTX-sensitive G protein is involved in D₄ receptor-mediated activation of Akt and ERK (Fig. 3C).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Time- and dose-dependent activation of Akt by the selective D4 dopamine receptor agonist, PD168077. D4MN9D cells were cultured in DMEM containing 10% FBS and allowed to reach 70 to 80% confluence. The medium was replaced with DMEM containing 1% FBS overnight before addition of PD168077 for the indicated times or doses. Akt activity was assessed as described under Experimental Procedures. A, time course for Akt activation induced by 1 µM PD168077. B, dose response for PD168077-mediated Akt activation during 5 min of stimulation. The results of at least three experiments are summarized; data are expressed in fold stimulation relative to control. *p < 0.05, **p < 0.01.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   D4 dopamine receptor mediates ERK activation in D4MN9D cells. Cells were treated as described in Fig. 1 for the indicated times (A) or with the indicated concentrations of PD168077 for 10 min (B). ERK activity was assessed using MBP as substrate. Representative autoradiograms of three independent experiments that yielded similar results are shown for each experiment.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   PTX-sensitive G protein is involved in activation of ERK and Akt by D4 dopamine receptor stimulation. D4MN9D (A) and MN9D (B) cells were incubated with the indicated concentrations of quinpirole (QP) (A) or PD168077 (B) for 5 min. ERK and Akt activities were measured in cell lysates. C, D4MN9D cells were incubated with vehicle (C), 100 ng/ml pertussis toxin (PTX) for 6 h, or 1 µM L745,870 (L) for 30 min before and during incubation with 1 µM PD168077 for 10 min. Akt and ERK activities were assayed. The results summarize at least three independent experiments and are expressed as mean ± S. E.M. * p < 0.01 compared with control; #p < 0.01 compared with stimulation with PD168077.

Stimulation of the D₄ Dopamine Receptor Elicits Activation of NF-B. We next tested whether NF-B is also activated by stimulation of the D₄ dopamine receptor. Stimulation of the receptor resulted in a time- and dose-dependent activation of NF-B in D₄MN9D cells (Fig. 4, A and B). Increased NF-B binding activity in the EMSA was observed at 30 min and achieved maximal stimulation after 120 min of receptor stimulation. Maximal stimulation of NF-B was achieved at 1 µM PD168077, a dose similar to that observed for Akt activation. In agreement with the increases in NF-B binding activity noted in the EMSA, decreases in IB protein levels were observed upon receptor stimulation (Fig. 4C, PD). Furthermore, pretreatment of cells with 1 µM L745,870 attenuated the PD168077-induced decline in IB (Fig. 4C), indicating that activation of NF-B in D₄MN9D cells is mediated by the D₄ receptor. The data, therefore, indicate that D₄ dopamine receptor stimulation activates NF-B in a time-and dose-dependent manner.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   D4 dopamine receptor mediates the activation of NF-B in D4MN9D cells. D4MN9D cells were treated for the indicated time periods with 1 µM PD168077 (A) or with various concentrations of PD168077 for 2 h (B), and NF-B binding activity in nuclear extracts was assessed by EMSA. C, cells were preincubated with vehicle (C) or 1 µM L745,680 (L) for 30 min before and during the addition of 1 µM PD168077 (PD) for additional 2 h. Cells were lysed, and IB protein was assessed by Western blotting using anti-IB antibody. All experiments were repeated at least three times and a representative autoradiograph or blot is shown.

Requirement of Akt, Not ERKs for D₄ Dopamine Receptor-Mediated NF-B Activation. NF-B is an important downstream signaling molecule in the Akt and MAP kinase pathways. We, therefore, sought to determine whether Akt is involved in the activation of NF-B by the D₄ receptor stimulation. Transiently transfection of plasmids expressing Myr-Akt into D₄MN9D cells enhanced D₄ receptor-stimulated NF-B activation as assessed by the level of IB, while transfection of the KD-Akt into D₄MN9D cells attenuated D₄ dopamine receptor-mediated NF-B activation (Fig. 5A, left panel). Expression of Akt was monitored using anti-HA antibody (Fig. 5A, right panel). In contrast, inhibition of MEK with PD98059, did not change D₄ receptor-mediated NF-B activation, Similarly a selective Src inhibitor (PP2) that blocked D₄ receptor-induced ERK activation (see below) did not alter the receptor-activated NF-B (Fig. 5B). These observations suggest that D₄ dopamine receptor-mediated NF-B activation requires Akt but not ERK activity.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Requirement of Akt but not ERK or Src for D4 dopamine receptor-mediated NF-B activation. A, D4MN9D cells were transiently transfected with 3 µg of plasmids expressing KD-Akt, Myr-Akt, or with empty vector, and 48 h later, cells were incubated in medium containing 1% serum and stimulated with various concentrations of PD168077 for 2 h. Cells were lysed and NF-B activation was assessed by measuring the expression of IB (left panel, anti-IB.). Akt expression was monitored by anti-HA antibody. Representative blots are shown. B, D4MN9D cells were pretreated with either 10 µM MEK inhibitor PD98059 or 20 nM PP2 for 60 min before and during stimulation of 1 µM PD168077 for an additional 2 h. NF-B activation was assessed as described above. The experiments were repeated three times with similar results. C, control; PD, PD168077.

Ras Is Involved in D₄ Dopamine Receptor-Mediated ERK, but Not Akt and NF-B Activation. Ras-dependent and -independent mechanisms were reported to regulate the ERK and Akt/NF-B pathways. To further define the signaling cascades involved in the activation of ERK and Akt/NF-B by the D₄ dopamine receptor, we tested the role of Ras in these pathways. Transient transfection of the Ras dominant negative N17Ras into D₄MN9D cells induced inhibition of D₄ receptor-mediated ERK activation (Fig. 6A). However, neither Akt (Fig. 6A) nor NF-B (Fig. 6B) activation was altered in N17Ras-transfected cells, indicating that D₄ dopamine receptor stimulation activates ERK via a Ras-dependent mechanism, whereas Akt and NF-B activation seems to be independent of Ras. To further check the possible interaction between ERK and Akt/NF-B, we measured D₄ receptor-stimulated ERK activation in D₄MN9D cells transiently expressing Myr-Akt, KD-Akt, or empty plasmid. As shown in Fig. 6C, PD168077-mediated ERK activation was not changed by transfection of any of these plasmids, indicating that Akt is not an upstream signaling molecule in the ERK pathway. Similarly, inhibition of ERK by PD98059 exhibited no effect on D₄ receptor-stimulated Akt activation (Fig. 6D). Taken together, D₄ dopamine receptors seem to regulate the ERK and Akt/NF-B pathways via independent mechanisms in D₄MN9D cells.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Requirement of Ras but not Akt for D4 dopamine receptor-mediated ERK activation in D4MN9D cells. A, D4MN9D cells were transiently transfected with various concentrations of dominant negative Ras, N17Ras; the cells were stimulated with 1 µM PD168077 or vehicle for 5 min, lysates were prepared, and ERK and Akt activities were assessed. The results are expressed as mean ± S. E. of three independent experiments. B, D4MN9D cells transfected with empty vector or N17Ras were stimulated with various concentrations of PD168077 for 2 h. Cell lysates were prepared and blotted with anti-IB antibody. C, D4MN9D cells were transiently transfected with Myr-Akt, KD-Akt, or empty plasmid. The cells were then stimulated with 1 µM PD168077 for 10 min, and ERK activity was then determined in cell lysates. Results are expressed fold change over control and summarized as mean ± S.E. of three independent experiments. D, D4 MN9D cells were preincubated with 10 µM MEK inhibitor PD98059 for 60 min before and during addition of 1 µM PD168077 for 5 min, Akt activity was detected as described in Fig. 1. The experiments were repeated two times with similar results. *p < 0.01, compared with basal control. #p < 0.01, compared with PD168077 stimulation. C, control; PD, PD168077.

Role of Src in D₄ Receptor-Mediated Tyrosine Phosphorylation of SHC and ERK Activation. We have demonstrated that D₄ dopamine receptor stimulation activates ERK via a Ras-dependent but Akt-independent mechanism. To further define the upstream signaling cascades for D₄ dopamine receptor-mediated ERK activation, cells were treated with 1 µM PD168077 and cell lysates were immunoprecipitated with anti-SHC antibody. The immune complex was resolved on SDS-PAGE and blotted with anti-phosphotyrosine antibody. As seen in Fig. 7A, a rapidly increase in tyrosine-phosphorylated SHC (both 52- and 46-kDa isoforms) was observed at 2 min. The 52-kDa SHC band was the predominant type activated in response to D₄ receptor stimulation with PD168077. Moreover, a parallel increase in the association of SHC with Grb2 was also observed when the blots were stripped and reprobed with anti-Grb2 antibody (Fig. 7A, bottom), suggesting that D₄ dopamine receptor-mediated ERK activation involves SHC-Grb2 association. We next tested the role of the tyrosine kinase Src in D₄ receptor-mediated SHC phosphorylation and ERK activation. PP2, a selective Src inhibitor (20 nM), abolished PD168077-stimulated SHC phosphorylation, whereas PP3 (20 nM), a negative control for PP2, did not affect SHC phosphorylation (Fig. 7B). Moreover, 20 nM PP2 that was sufficient to abolish SHC phosphorylation also blocked PD168077-mediated ERK activation. In contrast, the PI-3 kinase inhibitor LY294002 (20 µM) did not change D₄ receptor-stimulated ERK activity (Fig. 7C). These observations clearly indicate that Src is responsible for D₄ dopamine receptor-stimulated SHC phosphorylation and ERK activation.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   D4 dopamine receptor-mediated activation of ERK involves Src-mediated SHC-Grb2 association. A, D4MN9D cells were incubated with 1 µM PD168077 for the indicated times; cells were lysed and equal aliquots of lysate protein were precipitated with anti-SHC antibody. Immunoblots were performed and tyrosine-phosphorylated SHC was detected with anti-phosphotyrosine antibody (anti-p-Tyr). Membranes from A were stripped and reprobed with anti-Grb2 antibody (bottom). B, D4MN9D cells were preincubated with either PP2 or PP3 for 60 min before and during stimulation with PD168077 for additional 5 min. The tyrosine phosphorylation of SHC was analyzed as described in A. C, D4MN9D cells were preincubated with either PP2 or PP3 or LY294002 for 60 min before and during stimulation with PD168077 for 10 min. ERK activity was detected. The experiment was repeated three times with similar results. PD, PD168077; LY, LY294002.

ERK Activation Is Responsible for the Mitogenic Action of D₄ Dopamine Receptor Stimulation. The data summarized in Fig. 8 illustrate that the selective MEK inhibitor PD98059 (10 µM), which blocks the activation of ERK, abolished cell proliferation induced by 1 µM PD168077 in D₄MN9D cells. Moreover, selective inhibition of Src by 20 nM PP2, which blocked SHC phosphorylation and ERK activation, also attenuated D₄ receptor-stimulated cell proliferation. In contrast, SN50 (50 µg/ml), a cell-permeable inhibitor of NF-B translocation, exhibited no effect on D₄ receptor-stimulated cell proliferation in D₄MN9D cells. The results clearly implicate Src kinase-dependent ERK activation in mediating the mitogenic response to D₄ dopamine receptor stimulation.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Src/ERK activation mediates D4 dopamine receptor-stimulated cell proliferation. A,D4MN9D cells were cultured in 24-well plates and pretreated with control or 10 µM PD98059 for 2 h before incubation with 1 µM PD168077 for 22 h. B, D4MN9D cells were pretreated with control or 20 nM PP2, PP3, or SN50 (50 µg/ml) for 60 min before PD168077 stimulation for 22 h. The incorporation of [3H]thymidine into DNA during the last 6 h of the incubation was measured. The data are expressed as mean ± S.E. and a summary of three experiments with six determinations for each point is presented. *p < 0.01, compared with control; #p < 0.01, compared with stimulation with PD168077, Student's t test. C, control; PD, PD168077.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The data presented demonstrate that stimulation of the D₄ dopamine receptor activates the ERK and Akt/NF-B pathways via a PTX-sensitive G protein in D₄ dopamine receptor-expressing D₄MN9D cells. Activation of ERK is mediated via Src and Ras-dependent mechanisms and requires the association of the signaling molecules SHC and Grb2. In contrast, activation of NF-B depends on Akt but does not require Src or Ras, indicating that separate mechanisms mediate ERK and Akt/NF-B activation by D₄ receptor stimulation. Lastly, this communication demonstrates that activation of the ERK signaling pathway is responsible for cell proliferation in response to D₄ dopamine receptor stimulation.

Ras is a key signaling intermediate in many cellular pathways involved in cell proliferation and differentiation (Marte and Downward, 1997). Stimulation of ERK via pertussis toxin-sensitive G protein-coupled receptors, including angiotensin II, lysophosphatidic acid, and D₂ dopamine receptors were shown to be dependent on Ras and seem to be mediated by G released from G proteins (Faure et al., 1994; Luttrell et al., 1996, 1997; Gutkind, 1998; Luo et al., 1998). Moreover, it has been demonstrated that the nonreceptor tyrosine kinase, Src, links G to the activation of the Ras/MAPK pathway via phosphorylation of SHC and formation of SHC-Grb2 complex (Gutkind, 1998). In agreement with these observations, our data indicate that Src is also involved in D₄ dopamine receptor-mediated SHC phosphorylation and ERK activation (Fig. 7). Although previous studies implicated PI-3 kinase, activated by the G complex, in the activation of MAPK in response to Gi-coupled receptor stimulation (Lopez-Ilasaca et al., 1997; Gutkind, 1998), as in D₃ dopamine receptor-stimulated MAPK (Cussac et al., 1999), D₄ receptor-mediated ERK activation does not seem to be mediated through PI-3 kinase because inhibition of PI-3 kinase by LY294002 did not interfere with the activation of the ERK pathway (Fig. 7C). However, notwithstanding the precise mechanism, the activation of the ERK pathway is shared by the three dopamine receptors that constitute the D₂ family of dopamine receptors (Luo et al., 1998; Cussac et al., 1999; Yan et al., 1999; Cai et al., 2000). In addition, blocking Ras function has variable effects on Akt activity (Marte and Downward, 1997). In the present study, a dominant negative Ras had no effect on D₄ receptor-stimulated Akt activation, although ERK activation was attenuated (Fig. 7), clearly indicating that Ras is not upstream in this cascade that leads to Akt activation. Thus, the detailed mechanism underlying Akt activation in D₄MN9D cells remains to be determined.

It is generally believed that receptor-stimulated tyrosine phosphorylation plays an important role in mitogenesis initiated by GPCRs. In the case of the growth factor receptor, binding of the adapter protein SHC to the receptor is believed to be critical for mitogenic signaling. Here we show that the phosphorylation of SHC by Src kinase is responsible for D₄ dopamine receptor-induced ERK activation and cell proliferation (Figs. 7 and 8). It seems, therefore, that the D₄ dopamine receptor shares a common signaling pathway with growth factor receptors in regulating cell proliferation. Indeed, recent evidence indicates that some GPCRs may activate such growth factor receptors as platelet-derived growth factor and epidermal growth factor (Luttrell et al., 1997; Gutkind, 1998). It will be interesting to determine whether D₄ receptor-stimulated SHC phosphorylation and subsequent activation of ERK are associated with trans-activation of the platelet-derived growth factor or epidermal growth factor receptors.

In D₄MN9D cells, Gt₂ couples to D₄ receptors and regulates cAMP formation, although the cells also express Gi₂ and Go (Tang et al., 1994; Yamaguchi et al., 1997). D₄ dopamine receptor-mediated stimulation of Akt/NF-B and ERK are sensitive to PTX, which suggests the involvement of Gi/t₂ protein in these responses.

In summary, the present data identify ERK and Akt/NF-B cascades as distinct signal transduction pathways that are activated by D₄ dopamine receptors. These receptors have been implicated in the pathogenesis of neuropsychiatric disorders and in the pharmacology of antipsychotic drugs. Both ERK and Akt/NF-B pathways play important roles in neuronal plasticity (Impey et al., 1999; Mattson et al., 2000). The present results, therefore, provide new insights into the signaling and function of the D₄ dopamine receptor that may be important in understanding its role in the CNS during health and disease. However, the present findings, obtained in transfected cells that overexpress D₄ dopamine receptor, may not fully reflect the role these pathways play in all physiologic situations. Future studies, therefore, will have to address the question of relevance of these results in physiologic situations in which the density of receptors may differ.