Activation of G proteins and extracellular signal-regulated kinase 1/2 phosphorylation via human dopamine D_4.4 receptors: differential pathway-dependent potencies of receptor agonists

摘要

Agonist activity at recombinant human dopamine D_4.4 receptors was compared in stably transfected CHO cells using two functional readouts: G protein activation by [35S]GTPγS 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]GTPγS 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]GTPγS), selected compounds were tested in a modified short-duration [35S]GTPγS 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. Keywords Dopamine D_4.4 receptor - G proteins - MAP-Kinase - Extracellular signal-regulated kinase 1/2 phosphorylation - [35S]GTPγS binding - Antipsychotics This paper is especially dedicated to the memory of our appreciated colleague and co-author, Dr. Liesbeth Bruins Slot, who left us much too soon the 4th of April, 2008.