FUNCTIONAL EXPRESSION AND CHARACTERIZATION OF HUMAN D-2, AND D-3, DOPAMINE-RECEPTORS

Functional characteristics of human D-2 and D-3 receptors (DRs) were e xamined using a new bioassay suited for the study of G(i)-protein-coup led receptors (G(i)Rs). The bioassay utilizes pigment granule aggregat ion within cultured Xenopus laevis melanophores for the quantitative e valuation of ligands as agonists or antagonists upon particular G(i)Rs . Initial feasibility studies were performed by analyzing a melanocyte receptor endogenous to the melanophores. In dose-dependent manners, m elatonin inhibited melatonin-stimulating hermone-induced cAMP accumula tion and caused pigment aggregation that could be monitored over time. Next, melanophores were transiently transfected with cDNAs coding for the human D(2B)R (short form) and D(3)R. Expression of either recepto r conferred upon the cells the ability to aggregate their melanosomes in response to selective dopaminergic agonists. The same ligands also inhibited cAMP accumulation within the transfected melanophores, and t he agonist-induced pigment aggregation was shown to be sensitive to pe rtussis toxin. EC(50) and IC50 value determinations revealed that agon ists activated the D(2)R and D(3)R at similar concentrations, while ea ch of the antagonists displaying an effect was more potent upon the D( 2)R. The results reveal functional similarities and differences betwee n the D(2)R and D(3)R.