Abstract
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-β. 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-β. 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.
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
- βARK1ct
- β-adrenergic receptor kinase 1 carboxyl terminus
- PTX
- pertussis toxin
- DMSO
- dimethyl sulfoxide
- PDGFRβ
- platelet-derived growth factor receptor-β
- PLC
- phospholipase C
- Shc
- SH2-containing adapter protein
- Received September 8, 2000.
- Accepted March 14, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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