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

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 Galpha(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 nonselective 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 Galpha(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. (C) 2004 Elsevier Inc. All rights reserved.