title: Activation of G proteins and extracellular signal-regulated kinase 1/2 phosphorylation via human dopamine D4.4 receptors: differential pathway-dependent potencies of receptor agonists.
abstract: Agonist activity at recombinant human dopamine D4.4 receptors was compared in stably transfected CHO cells using two functional readouts: G protein activation by [35S]GTPgammaS binding and phosphorylation of extracellular signal-regulated kinase 1/2 (pERK1/2). Results with a large series of agonists reveal markedly higher relative agonist efficacy in the pERK1/2 assay compared with [35S]GTPgammaS binding, while potencies were generally higher in the latter readout. Whereas efficacies were highly correlated when comparing both tests, potencies determined using the pERK1/2 assay were neither correlated with those for G protein activation nor with binding affinities. In order to examine if these differences may be attributable to distinct assay conditions (5 min incubation for pERK1/2 compared with binding equilibrium conditions for [35S]GTPgammaS), selected compounds were tested in a modified short-duration [35S]GTPgammaS binding assay. In these experiments, potencies were generally reduced; however, compounds exhibiting comparably high potency in the pERK1/2 assay were not affected by this duration-dependent potency shift. We conclude that assay parameters such as signal amplification and incubation time have to be considered with respect to the appropriate choice of experimental approaches that best reflect agonist activity at dopamine D4 receptors in vivo.
authors.affiliation: Department of Cellular and Molecular Biology, Centre de Recherche Pierre Fabre, 17, avenue Jean Moulin, F-81106, Castres Cedex, France. peter.heusler@pierre-fabre.com.
authors.forename: Peter
authors.initials: P
authors.lastname: Heusler
authors.forename: Liesbeth
authors.initials: L
authors.lastname: Bruins Slot
authors.forename: Isabelle
authors.initials: I
authors.lastname: Rauly-Lestienne
authors.forename: Christiane
authors.initials: C
authors.lastname: Palmier
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Tardif
authors.forename: Amélie
authors.initials: A
authors.lastname: Tourette
authors.forename: Marie-Christine
authors.initials: MC
authors.lastname: Ailhaud
authors.forename: Didier
authors.initials: D
authors.lastname: Cussac
mesh_terms: D000818:Animals; D014150:Antipsychotic Agents / Q000494:pharmacology; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004298:Dopamine / Q000494:pharmacology; D018491:Dopamine Agonists / Q000494:pharmacology*; D018492:Dopamine Antagonists / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004837:Epinephrine / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000819:agonists* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009638:Norepinephrine / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D011869:Radioligand Assay; D050638:Receptors, Dopamine D4 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000502:physiology*
chemical_list: D014150:Antipsychotic Agents; D018491:Dopamine Agonists; D018492:Dopamine Antagonists; D050638:Receptors, Dopamine D4; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019204:GTP-Binding Proteins; D004298:Dopamine; D009638:Norepinephrine; D004837:Epinephrine