Using the gene splicing technique a synthetic human dopamine (DA) D4.2gene was constructed and subsequently stably expressed in CHO K1 cells. Binding of [3H]spiperone to membranes prepared from human DA D4.2CHO K1 cells was saturable with a K(d) of 93 ± 0.51 pM and a B(max) of 768 ± 22 fmol per mg protein. Clozapine, apomorphine, and S(+)-NPA were more selective for D4.2than for D(2L) receptors, with D(2L)/D4.2ratios of 5.7, 7.1, and 19.6, respectively. Functional studies indicated that DA D4.2receptors expressed in CHO K1 cells inhibited forskolin stimulated cAMP levels showing coupling to G-proteins. Two reciprocal human D(2L) and D4.2chimeric receptors (D(2L)/D4.2and D4.2/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)/D4.2receptor displayed non-decetable specific binding of [3H] spiperone and other ligands. The chimeric D4.2/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. This study characterized the pharmacology of a novel synthesized DA D4.2receptor that provides a useful model for screening of potential D4.2receptor agonist and antagonist.
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