Cloning, expression and characterization of a human dopamine D402 receptor (CHO K1 cells) and various D4.2/D2L chimeras (COS-7 cells)

Abstract 1. 1. Using the gene splicing technique a synthetic human dopamine (DA) D 4.2 gene was constructed and subsequently stably expressed in CHO K1 cells. 2. 2. Binding of [ 3 H]spiperone to membranes prepared from human DA D 4.2 CHO Kl cells was saturable with a K d of 93 ± 0.51 pM and a B max of 768 ± 22 fmol per mg protein. 3. 3. Clozapine, apomorphine, and S(+)-NPA were more selective for D 4.2 than for D 2L receptors, with c ratios of 5.7, 7.1, and 19.6, respectively. 4. 4. Functional studies indicated that DA D 4.2 receptors expressed in CHO K1 cells inhibited forskolin stimulated cAMP levels showing coupling to G-proteins. 5. 5. Two reciprocal human D 2L and D 4.2 chimeric receptors (D 2L /D 4.2 and D 4.2 /D 2L ) were constructed by exchanging the amino-terminal end to the third transmembrane (TM) of one receptor with the counter part of the other receptor and expressing them transiently into COS-7 cells. The chimeric D 2L /D 4.2 receptor displayed non-detectable specific binding of [ 3 H] spiperone and other ligands. The chimeric D 4.2 /D 2L receptor binding affinities of DA agonists were more affected than that of antagonists, suggesting that binding affinities of agonists are more sensitive to changes in receptor conformation than that of antagonists. 6. 6. This study characterized the pharmacology of a novel synthesized DA D 4.2 receptor that provides a useful model for screening of potential D 4.2 receptor agonist and antagonist .