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

• DOI: 10.1007/s00210-008-0333-4
• Publication Date: 2008-08-05T03:28:53Z
• Authors: Peter Heusler; Liesbeth Bruins Slot; Isabelle Rauly-Le...; Christiane Palmier; Stéphanie Tardif; Amélie Tourette; Marie-Christine A...; Didier Cussac
• Keywords: Mitogen-Activated Protein Kinase 1; 0303 health sciences; Mitogen-Activated Protein Kinase 3; Dose-Response Relationship, Drug; Epinephrine; Dopamine; CHO Cells; Binding, Competitive; Enzyme Activation; Norepinephrine; 03 medical and health sciences; Cricetulus; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Cricetinae; Dopamine Agonists; Animals; Dopamine Antagonists; Humans; Phosphorylation; Antipsychotic Agents; Protein Binding

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.