Functional expression and characterization of human D2 and D3 dopamine receptors

Functional characteristics of human D2 and D3 receptors (DRs) were examined using a new bioassay suited for the study of Gi-protein-coupled receptors (GiRs). The bioassay utilizes pigment granule aggregation within cultured Xenopus laevis melanophores for the quantitative evaluation of ligands as agonists or antagonists upon particular GiRs. Initial feasibility studies were performed by analyzing a melanocyte receptor endogenous to the melanophores. In dose-dependent manners, melatonin inhibited melatonin-stimulating hormone-induced cAMP accumulation and caused pigment aggregation that could be monitored over time. Next, melanophores were transiently transfected with cDNAs coding for the human D2BR (short form) and D3R. Expression of either receptor 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 the agonist-induced pigment aggregation was shown to be sensitive to pertussis toxin. EC50 and IC50 value determinations revealed that agonists activated the D2R and D3R at similar concentrations, while each of the antagonists displaying an effect was more potent upon the D2R. The results reveal functional similarities and differences between the D2R and D3R.