Functional characterization of a rat dopamine D-2 receptor cDNA expressed in a mammalian cell line

We recently cloned a complementary DNA for the rat dopamine D-2 receptor, making it possible to create cell lines expressing this receptor. A cell line (LZR1) was created by transfecting the D-2 cDNA (RGB-2) into mouse fibroblast Ltk- cells. LZR1 cells, previously described as L-RGB2Zem-1 cells, express a high density of D-2 receptors, whereas the wild-type cells do not. A number of agonists competitively and stereoselectively inhibited the binding of [3H]spiroperidol to the expressed D-2 receptors in a GTP-sensitive manner. The potency of dopamine was decreased by the addition of GTP. NaCl and GTP together caused a further decrease in potency and increased the Hill slope for inhibition of radioligand binding by dopamine almost to 1.0. Pretreatment of cells with pertussis toxin inhibited high affinity binding of dopamine and prevented further inhibition of binding by GTP. The NaCl-induced decrease in affinity was not prevented by pertussis toxin treatment. Dopamine reduced forskolin-stimulated adenylate cyclase activity by 27% in membranes prepared from LZR1 cells. Inhibition by dopamine was blocked by (+)-butaclamol or prior treatment of intact cells with pertussis toxin. Other dopamine receptor agonists stereoselectively inhibited adenylate cyclase activity. These data indicate that the RGB-2 cDNA directs the expression of a dopamine D-2 receptor capable of interacting with guanine nucleotide-binding proteins and inhibiting adenylate cyclase activity. Furthermore, the RGB-2 cDNA provides a means of creating many cell lines that will be useful tools for the biochemical and pharmacological characterization of dopamine D-2 receptors.