Comparative pharmacology of human dopamine D 2-like receptor stable cell lines coupled to calcium flux through Gα qo5

• Published in: Biochemical pharmacology Vol. 68; no. 4; pp. 761 - 772
• Main Authors: Moreland, Robert B, Nakane, Masaki, Donnelly-Roberts, Diana L, Miller, Loan N, Chang, Renjie, Uchic, Marie E, Terranova, Marc A, Gubbins, Earl J, Helfrich, Rosalind J, Namovic, Marian T, El-Kouhen, Odile F, Masters, Jeffrey N, Brioni, Jorge D
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 2004
• Subjects: A-369508; Calcium flux; Chimeric G-protein; D 4 receptor; Dopamine; Gα qo5; L-741626; PD168077; SCH23390; D 4 receptor; Ins(1,4,5)P 3; BP897; 7-hydroxy-DPAT; S33084; PTX; L-745870; RIPA buffer; SKF81297; CP226269; PD128907; PIPAT; Dopamine; Chimeric G-protein; TBST; A-369508; A23187; ABT-724; PNU95666E; FLIPR; Gα qo5; DPBS; Calcium flux
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/j.bcp.2004.05.019

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 50s 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.