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

• Published in: Biochemical pharmacology 2004-08, Vol.68 (4), p.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: Article
• Language: English
• Subjects: Animals; Biological Transport; Calcium - metabolism; Cell Line; Cells, Cultured; CHO Cells; Cricetinae; Dopamine; GTP-Binding Protein alpha Subunits - metabolism; Humans; Receptors, Dopamine D2 - metabolism; Receptors, Dopamine D3; Receptors, Dopamine D4; Recombinant Fusion Proteins - metabolism
• ISSN: 0006-2952

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 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 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.