title: D3 dopamine receptor signals to activation of phospholipase D through a complex with Rho.
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.
authors.affiliation: Department of Molecular Sciences, College of Medicine, The University of Tennessee Health Science Center, Memphis, Tennessee, USA.
authors.forename: P Benjamin
authors.initials: PB
authors.lastname: Everett
authors.forename: Susan E
authors.initials: SE
authors.lastname: Senogles
mesh_terms: D002461:Cell Line, Transformed; D018491:Dopamine Agonists / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D019814:Myristic Acid / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D050637:Receptors, Dopamine D3 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013764:Tetrahydronaphthalenes / Q000494:pharmacology; D014162:Transfection; D014316:Tritium / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D018491:Dopamine Agonists; D050637:Receptors, Dopamine D3; D013764:Tetrahydronaphthalenes; D019814:Myristic Acid; D014316:Tritium; D010739:Phospholipase D; D020764:cdc42 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; C031349:7-hydroxy-2-N,N-dipropylaminotetralin