Comparative pharmacology of human dopamine D2-like receptor stable cell lines coupled to calcium flux through Gαqo5

Abstract The goal of this study was to develop a new approach to study the pharmacology of the dopamine D 4 receptor that could be used in comparative studies with dopamine D 2 and D 3 receptors. Stable HEK-293 cell lines co-expressing recombinant human D 2L , D 3 or D 4 receptors along with Gα qo5 cDNA were prepared. Dopamine induced a robust, transient calcium signal in these cell lines with EC 50 s for D 2L , D 3 and D 4 of 18.0, 11.9 and 2.2 nM, respectively. Reported D 4 -selective agonists CP226269 and PD168077 were potent, partial D 4 agonists exhibiting 31–1700-fold selectivity for D 4 over D 3 or D 2 . Non-selective D 2 -like agonists apomorphine and quinpirole showed full efficacy but did not discriminate across the three receptors. D 3 -selective agonists 7-hydroxy-DPAT and PD128907 were potent but non-selective D 2 -like agonists. The reported D 3 partial agonist BP-897 exhibited minimal agonist activity at D 3 but was a potent D 3 antagonist and a partial D 4 agonist. Other D 2 -like antagonists, haloperidol, clozapine, and domperidone showed concentration-dependent inhibition of dopamine responses at all three receptors with K i ranging from 0.05 to 48.3 nM. The D 3 selective antagonist S33084 and D 4 -selective antagonist L-745870 were highly selective for D 3 and D 4 receptors with K b of 0.7 and 0.1 nM, respectively. Stable co-expression of D 2 -like receptors with chimeric Gα qo5 proteins in HEK-293 cells is an efficient method to study receptor activation in a common cellular background and an efficient method for direct comparison of ligand affinity and efficacy across human D 2L , D 3 and D 4 receptors.