CLONING, EXPRESSION AND CHARACTERIZATION OF A HUMAN DOPAMINE D-4.2 RECEPTOR (CHO K1 CELLS) AND VARIOUS D-4.2 D-2L CHIMERAS (COS-7 CELLS)/

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. Binding of [H-3]spiperone to membranes prepared from human D A D-4.2 CHO K1 cells was saturable with a K-d of 93 +/- 0.51 pM and a B-max of 768 +/- 22 fmol per mg protein. 3. Clozapine, apomorphine, an d S(+)-NPA were more selective for D-4.2 than for D-2L receptors, with D-2L/D-4.2 ratios of 5.7, 7.1, and 19.6, respectively. 4. Functional studies indicated that DA D-4.2 receptors expressed in CHO K1 cells in hibited forskolin stimulated cAMP levels showing coupling to G-protein s.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 en d to the third transmembrane (TM) of one receptor with the counter par t of the other receptor and expressing them transiently into COS-7 cel ls. The chimeric D-2L/D-4.2 receptor displayed non-detectable specific binding of [H-3] spiperone and other ligands. The chimeric D-4.2/D-2L receptor binding affinities of DA agonists were more affected than th at of antagonists, suggesting that binding affinities of agonists are more sensitive to changes in receptor conformation than that of antago nists. 6. This study characterized the pharmacology of a novel synthes ized DA D-4.2 receptor that provides a useful model for screening of p otential D-4.2 receptor agonist and antagonist.