1. D3 dopamine receptor signals to activation of phospholipase D through a complex with Rho.
- Author
-
Everett PB and Senogles SE
- Subjects
- Cell Line, Transformed, Dopamine Agonists pharmacology, Dose-Response Relationship, Drug, Enzyme Activation drug effects, Enzyme Activation physiology, Gene Expression Regulation drug effects, Humans, Immunoprecipitation methods, Myristic Acid metabolism, Receptors, Dopamine D3 genetics, Signal Transduction drug effects, Tetrahydronaphthalenes pharmacology, Transfection, Tritium metabolism, cdc42 GTP-Binding Protein metabolism, rhoA GTP-Binding Protein genetics, Gene Expression Regulation physiology, Phospholipase D metabolism, Receptors, Dopamine D3 metabolism, Signal Transduction physiology, rhoA GTP-Binding Protein metabolism
- Abstract
Dopamine acts through a family of G protein-coupled receptors to exert its myriad effects. The D3 Dopamine receptor is one member of the D2-like dopamine receptors. We have previously demonstrated in human embryonic kidney (HEK293) cells that D3 receptor stimulation of phospholipase D (PLD) activity is pertussis toxin insensitive [Everett and Senogles. Neurosci. Lett. 371 (2004), 34]. We hypothesized that a low molecular weight G protein was involved in the agonist-mediated activation of PLD. When the D3 receptor was coexpressed with RhoA in HEK293 cells, agonist-induced stimulation of PLD activity was increased. However, co-expression of Rac or Cdc42 with the D3 receptor did not change the PLD activity. As well, expression of a dominant-negative construct of RhoA, N19 Rho completely ablated D3 receptor-mediated PLD activation, when co-expressed with the D3 receptor in HEK293 cells. In contrast, expression of dominant-negative constructs of Rac or Cdc42 had no effect. Treatment of HEK293 cells transfected with the D3 receptor and treated with a D3 preferring agonist R+-hydroxy(dipropylamino)tetralin hydrobromide, results in an agonist-induced physical complex of D3 receptor and either endogenous Rho or transfected hemaglutinin (HA)-RhoA that can be detected by immunoprecipitation and western blotting. Treatment of cells transfected with D3 receptor with R+-hydroxy(dipropylamino)tetralin hydrobromide also results in agonist-dependent Rho activation, as measured by a Rho effector pull-down assay. The data suggest that D3 receptor/RhoA association and activation is necessary for D3 receptor-mediated PLD activation.
- Published
- 2010
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