title: Dopamine D2 receptor-induced heterologous sensitization of adenylyl cyclase requires Galphas: characterization of Galphas-insensitive mutants of adenylyl cyclase V.
abstract: Whereas acute stimulation of Galphai/o-coupled receptors inhibits the activity of adenylyl cyclase, a delayed consequence of persistent activation of the receptors is heterologous sensitization, an enhanced responsiveness of adenylyl cyclase to activators such as forskolin or agonists of Galphas-coupled receptors. Galphas-insensitive mutants of adenylyl cyclase type V were used to test the hypothesis that heterologous sensitization requires Galphas-dependent activation of adenylyl cyclase. When adenylyl cyclase was stably expressed in human embryonic kidney (HEK) 293 cells with the D2L dopamine receptor, basal, forskolin-stimulated, and isoproterenol-stimulated cyclic AMP accumulation were all enhanced by 2-h pretreatment with the D2 receptor agonist quinpirole. Transient expression of wild-type adenylyl cyclase and three Galphas-insensitive mutants (F379L, R1021Q, and F1093S) in HEK293 cells stably expressing the D2L receptor demonstrated that all three mutants had little or no responsiveness to beta-adrenergic receptor-mediated activation of Galphas but that the mutants retained sensitivity to forskolin and to D2L receptor-mediated inhibition. Transiently expressed adenylyl cyclase V was robustly sensitized by 2-h pretreatment with quinpirole. In contrast, the Galphas-insensitive mutants displayed no sensitization of forskolin-stimulated cyclic AMP accumulation, indicating that responsiveness to Galphas is required for the expression of heterologous sensitization.
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907, USA. wattsv@pharmacy.purdue.edu
authors.forename: V J
authors.initials: VJ
authors.lastname: Watts
authors.forename: R
authors.initials: R
authors.lastname: Taussig
authors.forename: R L
authors.initials: RL
authors.lastname: Neve
authors.forename: K A
authors.initials: KA
authors.lastname: Neve
mesh_terms: D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D002478:Cells, Cultured; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D009154:Mutation; D017448:Receptors, Dopamine D2 / Q000378:metabolism*
chemical_list: D000318:Adrenergic beta-Agonists; D007527:Isoenzymes; D017448:Receptors, Dopamine D2; D020962:Heterotrimeric GTP-Binding Proteins; D000262:Adenylyl Cyclases; C419510:adenylyl cyclase type V; D007545:Isoproterenol