D₃ dopamine receptor signals to activation of phospholipase D through a complex with Rho

J. Neurochem. (2009) 112, 963-971. 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.