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Vol. 60, Issue 1, 92-103, July 2001
Laboratory of Molecular Neurobiology, Centre for Addiction and Mental Health, Toronto, Ontario, Canada; and Departments of Pharmacology, Psychiatry, and Institute of Medical Science, University of Toronto
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The ability of dopamine D4 and D2 receptors to
activate extracellular signal-regulated kinases (ERKs) 1 and 2 was
compared using Chinese hamster ovary (CHO-K1) cells transfected with
D4.2, D4.4, D4.7, and
D2L receptors. Dopamine stimulation of D4 or
D2L receptors produced a transient, dose-dependent increase
in ERK1/2 activity. Receptor-specific activation of the ERK
mitogen-activated protein kinase (MAPK) pathway was confirmed using the
D2-like receptor-selective agonist quinpirole, whereas the
specific antagonist haloperidol blocked activation. MAPK stimulation
was dependent on a pertussis-toxin-sensitive G protein
(Gi/o). trans-Activation of the
platelet-derived growth factor (PDGF) receptor was an essential step in
D4 and D2L receptor-induced MAPK activation.
PDGF receptor-selective tyrosine kinase inhibitors tyrphostin A9 and
AG1295 abolished or significantly inhibited ERK1/2 activation by
D4 and D2L receptors. Dopamine stimulation of
the D4 receptor also produced a rapid increase in tyrosine
phosphorylation of the PDGF receptor-
. The Src-family tyrosine
kinase inhibitor PP2 blocked MAPK activation by dopamine; however, this
drug was also found to inhibit PDGF-BB-stimulated ERK activity and
autophosphorylation of the PDGF receptor-. Downstream signaling
pathways support the involvement of a receptor tyrosine kinase. The
phosphoinositide 3-kinase inhibitors wortmannin and LY294002, protein
kinase C inhibitors GF109203X and Calphostin C, dominant-negative
RasN17, and the MEK inhibitor PD98059 significantly attenuated or
abolished activation of MAPK by dopamine D4 and D2L receptors. Our results indicate that D4 and
D2L receptors activate the ERK kinase cascade by first
mobilizing signaling by the PDGF receptor, followed by the subsequent
activation of ERK1/2 by pathways associated with this receptor tyrosine kinase.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Dopamine is the predominant catecholamine neurotransmitter in the central nervous system and is involved in neurological and psychiatric disorders, including Parkinson's disease, Tourette's syndrome and schizophrenia. The D2-like dopamine receptors (including D2, D3 and D4) share a high affinity for antipsychotic drugs and couple through pertussis toxin-sensitive G proteins (Gi/o). Functionally, all D2-like receptors have been shown to inhibit adenylyl cyclase, stimulate the Na+/H+ antiporter, activate K+-channels, produce Cl- influx, alter neural morphology and stimulate mitogenesis. Additional intracellular effects of D2 or D4 receptors include arachidonic acid release, phosphoinositide hydrolysis, Ca2+-channel inhibition and apoptosis.
It is now established that numerous G protein-coupled receptors can
also activate mitogen-activated protein kinases (MAPKs), such as p38,
ERK, and c-Jun NH2-terminal kinase (Gutkind,
1998
). The mitogenic potential of dopamine
D2-like receptors was recognized by Lajiness et
al. (1993), who observed that the D2 receptor
could stimulate [3H]thymidine incorporation in
CHO cells by a mechanism that was independent of cAMP inhibition but
sensitive to the Gi/o-inactivator pertussis toxin
and the tyrosine kinase inhibitor genistein. This effect was later
observed with D3 and D4
receptors as well (Lajiness et al., 1995; Pilon et al., 1994). These
reports were in contrast to studies with pituitary tumor tissue and
cell lines, where the D2 receptor was found to
have an antiproliferative role (Florio et al., 1992) and reduced
thyrotropin-releasing hormone-induced MAPK activation (Ohmichi et al.,
1994). In neuronal MN9D cells, D2,
D3, and D4 receptors were
shown to alter neural morphology (Swarzenski et al., 1994), whereas
others reported both apoptosis and differentiation as a result of
D2 receptor signaling in neuronal cells (Coronas
et al., 1997). A unifying element of these effects may be MAPK
pathways, which are capable of mediating diverse cellular actions.
Recent studies have reported that dopamine D2,
D3, and D4 receptors can
independently stimulate the activity of the MAPKs ERK1/2 or c-Jun
NH2-terminal kinase (Luo et al., 1998; Oldenhof et al., 1998; Welsh et al., 1998; Cussac et al., 1999).
The MAPKs are a group of serine/threonine kinases that are activated by a cascade of protein kinases to induce responses such as proliferation, differentiation, apoptosis, and long-term potentiation. Classically, activation of MAPK pathways have been attributed to the activity of growth factor receptors. Binding of growth factors such as EGF or PDGF to receptor tyrosine kinases results in receptor dimerization and autophosphorylation. Binding of Grb2 (either directly or via other adapter proteins) to activated growth factor receptors promotes translocation of the Grb2-associated protein Sos to the membrane. The guanine nucleotide exchange factor Sos catalyzes the activation of Ras by promoting GTP/GDP exchange. Ras participates in the activation of the cytosolic serine/threonine kinase Raf-1 (MAPK kinase kinase), which activates MEK1/2 (MAPK kinase) and ultimately stimulation of ERK1/2. Activated ERK1/2 can phosphorylate cytosolic substrates and is translocated to the nucleus, leading to the activation transcription factors.
Among Gi/o-coupled receptors, activation of MAPK
has been attributed to both G
and G
, and the mechanism of
activation can differ among specific Gi/o-coupled
receptors and cell lines. Some G protein-coupled receptors seem to
activate MAPK by trans-activation of a receptor tyrosine
kinase such as the EGF receptor (Daub et al., 1996). Once
trans-activated by an as-yet poorly defined mechanism, these
receptors can recruit effectors, such as the Shc/Grb2/Sos pathway of
Ras activation, to stimulate MAPK. The
Ca2+-dependent kinase Pyk2 (related focal
adhesion tyrosine kinase) has been identified as a component of the
Gi/o-coupled receptor pathway to ERK activation
(Dikic et al., 1996). Ras-independent mechanisms of MAPK activation by
Gi-coupled receptors have also been described. In
CHO cells, Gi-coupled lysophosphatidic acid receptors seem to signal through PI3-kinase/PKC
to activate MEK
in a manner that is unaffected by dominant negative Sos (Takeda et al.,
1999).
We have demonstrated that both dopamine D4 and
D2L receptors activate the ERK MAPK pathway
through Gi/o. No observable differences were
apparent in the magnitude or duration of MAPK activation by
D4.2, D4.4,
D4.7, and D2L receptors.
Activation of ERK1/2 by dopamine involves trans-activation
of the PDGF receptor- and is also dependent on PKC, PI3-kinase,
Ca2+, Ras, and MEK. These results suggest that
cross-talk with a growth factor receptor is a required upstream
component in the activation of the ERK MAPK pathway by
D4 and D2L receptors.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Reagents.
CHO-K1 cells were purchased from American Type
Culture Collection (Manassas, VA). -MEM was purchased from Central
Media Preparation Service (University of Toronto, Toronto, ON, Canada).
Fetal bovine serum, horse serum, Geneticin (G418), T4 DNA ligase, T4
polynucleotide kinase, sequencing oligonucleotides and myelin basic
protein were bought from Invitrogen (Carlsbad, CA). All
restriction endonucleases, calf intestinal alkaline phosphatase, rabbit
polyclonal anti-phosphoERK(Tyr204), mouse monoclonal anti-phosphoERK
E10 (Thr202/Tyr204), rabbit polyclonal anti-phosphoElk1 (Ser383) and
anti-rabbit-HRP were obtained from New England BioLabs (Beverly, MA).
[3H]Spiperone (98 Ci/mmol) and protein
A-Sepharose were from Amersham Pharmacia Biotech (Baie d'Urfé,
PQ, Canada). Dopamine, forskolin, pertussis toxin, phorbol 12-myristate
13-acetate (PMA), LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one HCl], and
polyclonal rabbit anti-mouse-HRP were purchased from Sigma Chemical Co.
(St. Louis, MO). 3-Isobutyl-1-methyl xanthine was obtained from Aldrich Chemical Co. (Milwaukee, WI). Haloperidol, tyrphostin A9, AG1295 (6,7-dimethyl-2-phenylquinoxaline), AG1478
[N-(3-chlorophenyl)-6,7-dimethoxy-4-quinazolinamine], wortmannin, BAPTA-AM, and PDGF-BB were purchased from Sigma/RBI (St.
Louis, MO). [32P]ATP (3000 Ci/mmol) and
adenosine 3',5'-cyclic phosphoric acid, 2'-O-succinyl
[125I]iodotyrosine methyl ester
([125I]cAMP) (3300 Ci/mmol) were bought from
PerkinElmer Life Science Products (Mississauga, ON). PD98059
[2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one], GF109203X
(bisindolylmaleimide I),
4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), Gö 6976, and Calphostin C were from Calbiochem (San Diego, CA). Rabbit anti-ERK1 (C-16), rabbit anti-ERK2 (C-14), and rabbit polyclonal anti-PDGFR (958) were acquired from Santa Cruz
Biotechnology (Santa Cruz, CA). Monoclonal mouse anti-phosphotyrosine
antibody (4G10) was from Upstate Biotechnology (Lake Placid, NY).
D4 and D2 Plasmid Constructs.
The
vector pcDNA3 (Invitrogen) containing the dopamine
D2L or D4 receptor cDNA
with an amino-terminal, cleavable signal sequence (MKTIIALSYIFCLVFA)
and the hemagglutinin (HA) epitope (DYPYDVPDYA), were produced by Dr.
R. Vickery (University of California at San Francisco).
D2L was excised from pcDNAI by a blunted
NcoI/XhoI digest. The plasmid pBCSSHA
was
digested with BamHI to excise the -opioid receptor. After
blunting the vector and a XhoI digest, the
D2 sequence was inserted to create pBCSSHAD2L.
The modified D2L receptor was removed from pBC
with a HindIII/XhoI digest and subcloned into
pcDNA3 to produce pcSSHAD2L. Using a partial
NcoI/BamHI digest, the dopamine
D4.7 receptor was cut from pBD4.7 (Asghari et
al., 1994) and cloned into the NcoI/BamHI site of
pBCSSHA to create pBCSSHAD4.7. The tagged D4
receptor was cut with HindIII/BamHI from
pBCSSHAD4.7 and subcloned into pcDNA3. Partial
NotI/XbaI digest of the
D4.2 and D4.4 receptors in
pBluescript (Asghari et al., 1994) resulted in 1.2- and 1.3-kilobase
pair fragments that were ligated into pcSSHAD4.7 at
NotI/XbaI to produce pcSSHAD4.2 and pcSSHAD4.4.
Cell Culture.
Cells were grown in supplemented -MEM media
(2.5% fetal bovine serum and 2.5% horse serum) as monolayers
at 37°C in a humidified, 5% CO2 atmosphere.
Stable CHO-K1 cell lines of pcDNA3 (vector control), pcSSHAD4.2,
pcSSHAD4.4, pcSSHAD4.7, and pcSSHAD2L were transfected by
electroporation as described previously (Asghari et al., 1995).
Transient transfections and stable transfection of pBK-PDGFR were
carried out with LipofectAMINE reagent (Invitrogen) as described by the
manufacturer. Individual CHO-K1 clones were grown in media containing
500 µg/ml G418.
Radioligand Binding. Cells were resuspended in binding buffer (120 mM NaCl, 50 mM Tris-HCl, 5 mM KCl, 5 mM MgCl2,1.5 mM CaCl2, 0.5 mM EDTA, pH 7.4) and mechanically homogenized (Pro Scientific Inc., Monroe, CT; maximum, 15 s, on ice). Cell membranes were pelleted by centrifugation (34,000g for 20 min at 4°C) and resuspended by homogenization (maximum, 5 s, on ice) in binding buffer. Binding assays were carried out by mixing 25 µg of membrane protein with 1 ml of binding buffer containing [3H]spiperone and unlabeled drugs, in duplicate. After incubation (2 h at room temperature), membranes were harvested onto glass-fiber filters using a combicell harvester (Skatron Instruments Inc., Sterling, VA) and washed with 50 mM Tris-HCl, pH 7.5 (2 × 9 s). Bound radioligand was detected by liquid scintillation counting.
Saturation binding experiments were carried out with 0.01 to 1 nM [3H]spiperone. The receptor Kd (nanomolar) and Bmax (disintegrations per minute) were determined by nonlinear curve fitting using GraphPad Prism v2.0 (GraphPad Software, San Diego, CA). Saturation binding data were fit to the equation Y = [Bmax × (X
Y)] / [Kd + (X Y)] + (X Y) × NS, where X is the total amount of ligand (disintegrations per minute), Y is the total binding (disintegrations per minute), and
NS is the nonspecific binding constant. NS was determined experimentally by coincubating with 1 µM haloperidol and fitting the
data to the equation Y = X × [NS / (NS + 1)]. Protein
concentration was determined using the bicinchoninic acid assay
(Pierce, Rockford, IL).
cAMP Assay.
Cells stably expressing receptors were plated on
six-well plates and grown to ~80% confluence. Cells were treated
with 1 µM dopamine and the cAMP concentration in the lysate was
determined by [125I]cAMP radioimmunoassay as
described previously (Asghari et al., 1995) using a standard curve fit
to a sigmoidal dose-response curve [Y = Min + (Max Min) / (1 + 10(LogEC50 
X) ×
nH, where X is the logarithm of the
concentration, Y is the response, and nH is
the Hill slope] using GraphPad Prism v2.0.
MAPK Assay.
Cells were plated on six-well plates, grown to
~80% confluence, rinsed with serum-free -MEM and incubated
overnight in serum-free -MEM. Cells were preincubated with various
drugs before activation with 1 µM dopamine/quinpirole or 10 ng/ml
PDGF-BB. Drug incubation was ended by washing the cells with 2 ml of
ice-cold PBS followed by the addition of either 250 µl of lysis
buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%
Triton X-100, 2.5% sodium pyrophosphate, 1 mM -glycerolphosphate, 1 mM sodium orthovanadate, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and
1 mM phenylmethylsulfonyl fluoride) for Elk or MBP kinase assays or 100 to 200 µl of sample buffer [62.5 mM Tris-HCl, pH 6.8, 2% (w/v) SDS,
10% glycerol, 50 mM DTT, and 0.1% (w/v) bromphenol blue] to assay
the phosphorylation state of ERK1/2.
-glycerolphosphate, 2 mM DTT, 0.1 mM sodium orthovanadate, and 10 mM MgCl2); beads were resuspended in 50 µl of
Elk kinase buffer containing 0.1 mM ATP and 1 µg of Elk1-GST fusion
protein and incubated at 30°C for 30 min. The reaction was stopped by addition of 25 µl of 3× sample buffer, denatured (95°C for 5 min), microcentrifuged for 2 min and separated by 10% SDS-PAGE gel (Novex, San Diego, CA). After electrophoresis, proteins were transferred from
the gel to a polyvinylidene difluoride membrane (Novex). The membrane
was incubated for 2 h in blocking buffer [TBS (20 mM Tris-HCl,
137 mM NaCl, pH 7.6) and 0.1% Tween-20, 5% nonfat dry milk, and
0.02% NaN3] and probed overnight with
anti-phospho-Elk1 (1:1000 in blocking buffer) at 4°C. After washing
three times with TBS-T (TBS with 0.1% Tween-20) for 5 min, the blot
was probed with peroxidase-conjugated secondary antibody (1:2000
anti-rabbit-HRP in blocking buffer without NaN3)
for 1 h at room temperature. The membrane was washed three times
with TBS-T for 5 min and phosphorylated Elk1 was detected by enhanced
chemiluminescence with ECL+plus (Amersham Pharmacia Biotech, Oakville, ON).
ERK1/2 activity was also measured by the in vitro phosphorylation of
myelin basic protein (MBP). Cleared cell lysates (see above) were
incubated overnight with 1:100 dilutions of anti-ERK1 (C-16) and
anti-ERK2 (C-14). After collection with protein-A Sepharose, the beads
were washed twice with lysis buffer and twice with MBP kinase buffer
(20 mM HEPES, pH 7.5, 10 mM MgCl2, and 1 mM DTT). Beads were resuspended in 50 µl of MBP kinase buffer containing 12.5 µg of myelin basic protein, 2 µCi of
[-32P]ATP, and 20 µM unlabeled ATP and
incubated at 30°C for 20 min. A 10-µl aliquot was spotted onto P81
phosphocellulose filter paper (Whatman), washed with cold 0.5%
phosphoric acid (5 × 5 min), rinsed with ethanol and dried. The
incorporation of 32P into myelin basic protein
was measured by liquid scintillation counting.
To detect phosphorylated forms of ERK1/2, cells were scraped after
addition of sample buffer, sonicated for 20 s and denatured (95°C for 5 min). Samples were microcentrifuged for 5 min, and 20 µl was loaded onto an 8 to 16% Tris-glycine gel. After SDS-PAGE, Western blotting, and blocking, the polyvinylidene difluoride membranes
were incubated with mouse monoclonal anti-phospho-MAPK E10
(Thr202/Tyr204) (1:1000 in blocking buffer). The blots were incubated 1 to 2 h at room temperature, washed with TBS-T (4 × 5 min),
and subsequently incubated for 1 h with anti-mouse-HRP (1:4000
blocking buffer without NaN3) at room
temperature. Membranes were washed with TBS-T (4 × 5 min) and the
phosphorylated forms of ERK1 and ERK2 were detected by chemiluminescence.
Western Blotting of Phosphorylated PDGFR.
CHO-K1 cells
stably expressing PDGF receptor- were transiently transfected with
pcSSHAD4.4 and/or treated with drugs and cell lysates were prepared as
described previously (Domin et al., 1996). Receptors were
immunoprecipitated by incubating with 1 µg of anti-PDGFR- (3 h at
4°C) followed by addition of 20 µl of 50% protein A-Sepharose (3 h
at 4°C). After washing three times with lysis buffer, pelleted beads
were resuspended in sample buffer and denatured (95°C for 2 min).
Immunoprecipitated proteins were separated with a 4 to 12% SDS-PAGE
gel and Western blotting was carried out using anti-phosphotyrosine (1 µg/ml)/anti-mouse-HRP (1:8000).
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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CHO-K1 Cell Lines Expressing HA-tagged D4 or
D2L Receptors.
Wild-type human dopamine
D4.2, D4.4,
D4.7, and D2L receptors
were modified by the addition of a cleavable, membrane-targeted signal
sequence and an amino terminal HA-epitope tag. Clonal CHO-K1 cell lines
expressing 0.78 to 1.6 pmol of receptor/mg of membrane protein were
selected (Table 1). All cell lines except
the control CHO-pcDNA3 inhibited forskolin-stimulated cAMP in response
to 1 µM dopamine (Fig. 1).
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Activation of ERK1/2 by haD4 and haD2L
Receptors.
To determine whether various polymorphic variants of
the dopamine D4 receptor can stimulate the MAPK
pathway, the activity of immunoprecipitated ERK1/2 was measured by an
in vitro Elk1 kinase assay. Dopamine treatment stimulated
ERK1/2-dependent phosphorylation of Elk1 in cells expressing
haD4.2, haD4.4,
haD4.7, and haD2L, but had
no effect on CHO-K1 cells transfected with the vector, pcDNA3 (Fig.
2A). Increased ERK activity was also
distinguished by an increase in the level of the phosphorylated
(pThr202/pTyr204) forms of
ERK1/2 (Fig. 2B), whereas the total level of ERK1/2 was unaffected
(Fig. 2C). Dopamine stimulation of ERK1/2 phosphorylation and activity
was transient, with ERK kinase activity returning to basal levels by 30 min (Fig. 3B), whereas residual ERK2
phosphorylation was still observable after 60 min (Fig. 3A). The three
polymorphic variants of the dopamine D4 receptor
and the D2L receptor all displayed a similar
pattern of transient ERK activation. Dopamine stimulation of CHO-haD4.4
and CHO-haD2L cells (1 µM for 5 min) produced increases of 355 ± 71% (mean ± S.D., n = 12) and 330 ± 105% (n = 13), respectively, in the activity of ERK1/2
as measured by in vitro myelin basic protein kinase activity compared
with unstimulated cells.
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MAPK Activation by D4 and D2L Receptors
Involves G Protein-Coupled Stimulation of Ras and MEK.
To study
the mechanism of ERK activation by dopamine D4
and D2 receptors, CHO-haD4.4 and CHO-haD2L cell
lines were preincubated with pertussis toxin (100-200 ng/ml), which
blocks receptor coupling through Gi/o, or with
the MEK inhibitor PD98059 (50 µM) (Fig. 6, A and B). Both pertussis toxin and
PD98059 abolished ERK activation by dopamine. To further characterize
the pathway, wild-type CHO-K1 cells were transiently cotransfected with
pcSSHAD4.4 or pcSSHAD2L along with the dominant negative constructs
ARK1ct or RasN17 (Fig. 7).
Coexpression of ARK1ct and RasN17 significantly attenuated the
activation of ERK by haD4. Coexpression of
haD2L with RasN17 also reduced ERK activation,
whereas ARK1ct did not show a statistically significant effect. The
effect of haD4 and haD2L
receptors on ERK activity was independent of cellular cAMP levels,
because pretreatment of cells with 8 bromo-cAMP (0.01 to 1 mM) did not block activation of this MAPK pathway (data not shown).
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Inhibitors of PKC and PI3-Kinase Block Activation of ERK.
To
determine whether ERK activation by D4 and
D2L receptors is dependent on PKC, CHO-haD4.4 and
CHO-haD2L cells were preincubated with the PKC inhibitors GF109203X
(bisindolylmaleimide I) (2 µM), Gö 6976 (2 µM), or Calphostin
C (1 µM) (Fig. 8). MAPK activation by
both haD4 and haD2L
receptors was sensitive to the broad spectrum PKC inhibitors GF109203X
and Calphostin C, but was not reduced by Gö 6976, which is
selective for PKC- and PKCI. Pretreatment of cells with the
PI3-kinase inhibitors wortmannin (1 µM) and LY294002 (30 µM) also
blocked >50% of ERK stimulation by dopamine (Fig.
9).
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Intracellular Ca2+ Is Required for MAPK Activation by
Dopamine D4 and D2L Receptors.
Pretreatment of CHO-haD4.4 and CHO-haD2L cells with the intracellular
Ca2+ chelator BAPTA-AM (100 µM) abolished
dopamine-mediated ERK1/2 phosphorylation (Fig.
10A). This observation was confirmed by
an in vitro kinase assay, where it was found that BAPTA-AM was only effective at concentrations above 10 µM (Fig. 10B).
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ERK Activation by Dopamine Is Dependent on PDGF Receptor
trans-Activation.
To test whether growth factor
receptor signaling is recruited by dopamine D4
and D2L receptor MAPK signaling, cells were
preincubated with the selective receptor tyrosine kinase inhibitors
AG1478 (EGF receptor), tyrphostin A9 (PDGF receptor), and AG1295 (PDGF receptor). Although AG1478 (1 µM) had no effect on
dopamine-stimulated ERK phosphorylation, tyrphostin A9 pretreatment (1 µM) eliminated the response of ERK to dopamine (Fig.
11A). The in vitro kinase assay
confirmed that both tyrphostin A9 and a second PDGF receptor tyrosine
kinase inhibitor, AG1295 (10 µM) potently blocked ERK stimulation by
both haD4.4 and haD2L
receptors in CHO-K1 cells (Fig. 11, B and C).
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cells (stably expressing an
increased level of PDGF receptor-) with pcSSHAD4.4 followed by
treatment with dopamine (1 µM for 1-5 min) produced an immediate increase in the tyrosine phosphorylation of PDGF receptor- (Fig. 12), confirming that dopamine receptors
can trans-activate this receptor tyrosine kinase.
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(Fig. 14A). In addition, PP2 also
strongly suppressed PDGF receptor- autophosphorylation in response
to PDGF-BB (Fig. 14B).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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This study examined the activation of the ERK MAPK pathway by
dopamine D4 and D2L
receptors to understand and compare the mechanism they use. Dopamine
stimulation of both receptors resulted in a rapid, transient, and
dose-dependent increase in ERK phosphorylation and activity. The
magnitude of activation was similar between D4.2,
D4.4, D4.7, and
D2L receptors. The EC50
value of ERK activation (9-10 nM) was similar to values reported
previously for the inhibition of intracellular cAMP by
D4.4 (12 nM) and D2L (5 nM)
receptors (Asghari et al., 1995; Guiramand et al., 1995).
ERK activation by both D4 and
D2L receptors required a PTX-sensitive G protein
(Gi/o), whereas sequestering G with
ARK1ct peptide significantly attenuated ERK activation by the
D4 receptor. Activation of the ERK MAPK pathway
by dopamine D2S, D2L, and
D3 receptors has previously been attributed to
PTX-sensitive G proteins (Cussac et al., 1999; Luo et al., 1998). The
dominant-negative mutant RasN17 and the MEK inhibitor PD98059 also
reduced or eliminated activation of ERK by both
D4 and D2L receptors,
suggesting that dopamine activates the Ras 
Raf MEK ERK pathway.
Because it has emerged that G protein-coupled receptors can recruit
growth factor receptors to facilitate MAPK signaling (Daub et al.,
1996), we tested selective inhibitors of EGF receptor and PDGF receptor
tyrosine kinase activity. Although EGF receptor-selective AG1478 failed
to block dopamine-stimulated ERK phosphorylation, PDGF
receptor-selective tyrphostin A9 and AG1295 potently inhibited ERK
phosphorylation and kinase activity, indicating that this receptor
tyrosine kinase is a required intermediate. CHO-K1 cells are known to
endogenously express a low level of the PDGF receptor- (Duckworth
and Cantley, 1997), and the PDGF receptor can act as a scaffold for
numerous other kinases and adapter proteins, including Src, SH2
domain-containing phosphotyrosine phosphatase-2, Shc, Grb2/Sos,
PLC-, PI3-kinase, and Ras-GAP (reviewed by Heldin et al., 1998). The
role of trans-activation is supported by a rapid increase in
tyrosine phosphorylation of PDGF receptor- upon stimulation of the
D4.4 receptor, demonstrating that activation of
the growth factor receptor occurs in a timeframe consistent with its
involvement in MAPK signaling by dopamine.
The Src-family tyrosine kinase inhibitor PP2 blocked the activation of
MAPK by dopamine and PDGF receptor-. PP2 also inhibited PDGF-BB-stimulated autophosphorylation of PDGF receptors overexpressed in CHO-K1 cells. These results are in agreement with a recent report by
Waltenberger et al. (1999) that indicated that PP1, a Src-family kinase
inhibitor closely related to PP2, inhibits the tyrosine kinase activity
of the PDGF receptor- but did not block activation of EGF,
fibroblast growth factor-1, or insulin-like growth factor-1 receptors.
Conversely, tyrphostin A9 blocks Src kinase activity only partially at
a concentration of 10 µM (Lakkakorpi et al., 2000), whereas AG1296, a
less hydrophobic analog of AG1295, does not inhibit the kinase activity
of Src (Kovalenko et al., 1994). Therefore, it seems that the Src
kinase inhibitor PP2 acts at the PDGF receptor- to block MAPK
activation by both dopamine and PDGF-BB.
Nonselective PKC inhibitors and the Ca2+-chelator
BAPTA-AM (100 µM) also abolished MAPK activation. CHO-K1 cells are
known to possess PKC, , , 
, and isotypes (Tippmer et
al., 1994; Shirai et al., 2000). Cussac et al. (1999) proposed that an
atypical PKC, PKC , was involved in ERK activation by the
D3 receptor in CHO cells. Because the reported
IC50 value of PKC inhibition by GF109203X is
5.8 µM (Martiny-Baron et al., 1993), it is unlikely that activation
of this PKC isoform contributes significantly to ERK activation by
D4 or D2L receptors; we
observed complete inhibition of ERK activation with 2 µM GF109203X.
These observations point to the involvement of PKC, , or in
ERK activation by D4 and
D2L. Of these, classical PKC is activated by
intracellular Ca2+, although its sensitivity to
Gö 6976 is unknown.
The PI3-kinase inhibitors wortmannin and LY294002 both attenuated
dopamine-stimulated ERK activity by approximately 50%, as was
previously observed with D2S and
D3 cells expressed in CHO cells (Welsh et al.,
1998; Cussac et al., 1999). PI3-kinases are known to act upstream of
Sos in G-mediated MAPK activation (Hawes et al., 1996), but
D4 and D2L do not use the
same Ras-independent signaling pathway through PI3-kinase and PKC
that was described for lysophosphatidic acid receptors in CHO cells
(Takeda et al., 1999), because we found that both dopamine receptors
utilize Ras and a PKC other than PKC. However, we do not presently
know the identity of the PI3-kinase involved in MAPK signaling by dopamine.
Figure 15 shows a model of pathways
leading from D4/D2L to ERK
activation based on our data. Grb2/Sos recruitment by phosphorylated PDGF receptor- represents one mechanism for Ras activation by D4 and D2L receptors.
Activated Shc or SH2 domain-containing phosphotyrosine phosphatase-2
may also facilitate the binding of the Grb2/Sos complex to the PDGF
receptor. In addition, PLC- and PI3-kinase have all been implicated
in the transduction of mitogenic signals by this receptor tyrosine
kinase (Duckworth and Cantley, 1997). Both PLC- and PI3-kinase can
activate PKC after activation by the PDGF receptor (Moriya et al.,
1996), and GF109203X has been shown to fully block mitogenic signaling
by PDGF receptor- (Alimandi et al., 1997). The mechanism by which
Gi/o trans-activates the PDGF receptor
has not yet been identified.
|
It has recently emerged that the mechanism of MAPK activation by the
2-adrenergic receptor seems to involve the
formation of a complex between receptor, -arrestin, dynamin, Src,
and the EGF receptor (Maudsley et al., 2000). Activation of MEK
requires dynamin-dependent endocytosis, where phosphorylation of
dynamin by Src is required (Daaka et al., 1998). This process may
involve G protein-coupled receptor endocytosis, as seen with the
2-adrenergic receptor, although
internalization of the 2A-adrenergic receptor did not occur after EGF receptor trans-activation (Pierce et
al., 2000). In this regard, D2L receptors are
known to undergo agonist-dependent internalization in CHO cells
(Itokawa et al., 1996). Although Vickery and von Zastrow (1999)
reported that D2 receptors are internalized by a
dynamin-independent mechanism, others have found that
D2 undergoes dynamin-dependent sequestration when
coexpressed with GRK2 or GRK5 (Ito et al., 1999). Because GRK2 is
abundantly expressed in CHO cells, the
-arrestin/Src/dynamin-dependent mechanism of
trans-activation may be involved. Although we do not observe agonist-stimulated internalization of the D4
receptor (R. Vickery, M. von Zastrow, and H. H. M. Van Tol,
unpublished observations), results with the
2-adrenergic receptor have demonstrated that G
protein-coupled receptor endocytosis is not required for
dynamin-dependent trans-activation (Pierce et al., 2000).
Although 2-adrenergic receptor
trans-activation was independent of
Ca2+ (Maudsley et al., 2000), serotonin
5-HT1A-dependent activation of ERK in CHO cells
was dependent of Ca2+/calmodulin- and
clathrin-dependent endocytosis (Della Rocca et al., 1999). In this
case, the Ca2+-dependent tyrosine kinase Pyk2
seems to mediate EGF receptor activation by bradykinin, presumably
through activation of Src and downstream kinases (Blaukat et al.,
1999). Therefore, the Ca2+ dependence of ERK
signaling by D4 and D2L
receptors may originate at the level of PDGF receptor endocytosis or
Pyk2 activation.
The physiological role of dopamine receptor-mediated
trans-activation is not fully understood; however, J. F. MacDonald (Dept. of Physiology, University of Toronto;
unpublished observations) found that the D4
receptor uses PDGF receptor- trans-activation to block
NMDA currents in hippocampal neurons. In addition, the emergence of
MAPK pathways as an element of G protein-coupled receptor signaling has
also vastly increased the range of cellular processes that dopamine
receptors may affect. Quinpirole was recently found to stimulate ERK
phosphorylation in rat neurons in a PKC- and
Ca2+-dependent manner (Yan et al., 1999) and
dopamine D2-like receptor activation has been
shown to contribute to long-term depression (LTD) in rat prefrontal
cortex through a mechanism involving MAPK (Otani et al., 1999). These
finding suggest that dopamine D2-like receptor
activation of MAPK pathways may play an important role in the brain,
possibly through a mechanism involving trans-activation of
receptor tyrosine kinases such as the PDGF receptor.
| |
Acknowledgments |
|---|
We thank Dr. R. Vickery and Dr. M. von Zastrow (University of
California, San Francisco, California) for constructing pcSSHAD4.7 and
pcSSHAD2L, Dr. R. Lefkowitz (Duke University Medical Center, Durham,
NC) for providing the plasmids containing RasN17 and ARK1ct, and Dr.
J. F. MacDonald (University of Toronto, Toronto, Ontario, Canada)
for providing pBK-PDGFR.
| |
Footnotes |
|---|
Received September 8, 2000; Accepted March 14, 2001
This work was supported by Canadian Institutes of Health Research Grant 14573.
Dr. Hubert H. M. Van Tol, Center for Addiction and Mental Health, 250 College St. W., Toronto ON, Canada, M5T 1R8. E-mail: hubert.van.tol{at}utoronto.ca
| |
Abbreviations |
|---|
MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated kinase;
CHO, Chinese hamster ovary;
EGF, epidermal growth factor;
PDGF, platelet-derived growth factor;
Sos, son
of sevenless guanine nucleotide exchange factor;
MEK, mitogen-activated
protein kinase/extracellular signal-regulated kinase kinase;
Ras, rat
sarcoma virus p21 GTPase;
Pyk2, proline-rich tyrosine kinase;
PI3-kinase, phosphoinositide 3-kinase;
PKC, protein kinase C;
MEM, minimal essential medium;
HRP, horseradish peroxidase;
tyrphostin A9, [[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-methylene]propanedinitrile;
BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid acetoxymethyl ester;
PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-day-]pyrimidine;
HA, hemagglutinin;
DTT, dithiothreitol;
PAGE, polyacrylamide gel
electrophoresis;
TBS, Tris-buffered saline;
MBP, myelin basic protein;
haD2, HA epitope-tagged dopamine D2 receptor;
haD4, HA epitope-tagged dopamine D4 receptor;
ARK1ct, -adrenergic receptor kinase 1 carboxyl terminus;
PTX, pertussis toxin;
DMSO, dimethyl sulfoxide;
PDGFR, platelet-derived
growth factor receptor-;
PLC, phospholipase C;
Shc, SH2-containing
adapter protein.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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) and CHO-haD2L
(
) with dopamine (1 µM) for up to 60 min, ERK1/2 was
immunoprecipitated and their activity was quantified with an in vitro
kinase assay using MBP as a substrate. Data are presented as mean ± S.D. (n = 3).
