Functional characterization of the human dopamine D4.2 receptor using vaccinia virus as an expression system

Abstract

Recombinant vaccinia viruses harboring the human dopamine D₄ receptor cDNA containing two 48 base pair-repeats (D_4.2) or the rat dopamine D₂ short (D_2s) receptor cDNA were used to infect rat-1 fibroblasts. Heterologous expression of both dopamine receptors was demonstrated in binding assays. The affinity constants of these receptors were consistent with values previously reported, including D_4.2's higher affinity for the antipsychotic clozapine and raclopride's selectivity for D₂ receptors. In the presence of 200 μM 5′-guanylyl- imidodiphosphate (Gpp[NH]p) both receptors exhibited reduced affinities for dopamine. Furthermore, when rat-1 cells were infected with the D_2s or the D_4.2 recombinant vaccinia viruses and exposed to dopamine agonists, the inhibition of adenylyl cyclase activity was prevented in pertussis toxin-treated cells. This study demonstrates the utility of recombinant receptor-vaccinia viruses in studies of expression, pharmacology and functional coupling of inhibitory G protein-coupled receptors.