Abstract

The human dopamine D_2L receptor couples promiscuously to multiple members of the Gα_i/o subfamily. Despite the high homology of the D_2L and D₃ receptors, the G protein coupling specificity of the human D₃ receptor is less clearly characterized. The primary aim of this study, then, was the parallel characterization of the G protein coupling specificity of the D_2L and D₃ receptors. By using both receptor-G protein fusion proteins and stable cell lines in which pertussis toxin-resistant mutants of individual Gα_i-family G proteins were expressed in an inducible fashion, we demonstrated highly selective coupling of the D₃ receptor to Gα_o1. Furthermore, by using the fusion proteins to ensure identical stoichiometry of receptor to G protein for each pairing, a range of ligands displayed higher potency and, for partial agonists, higher efficacy at the D₃ receptor when coupled to Gα_o1 compared with the D_2L receptor. The second aim of this study was to investigate the molecular basis of the above differential G protein coupling specificity. The importance of a 12-amino acid sequence from the C-terminal end of the third intracellular loop of the D_2L receptor in providing promiscuity in G protein coupling was demonstrated using a chimeric D₃/D₂ receptor in which the equivalent region of the D₃ receptor was exchanged for this sequence. This chimera displayed D₃-like affinity for [³H]spiperone and potency for agonists but gained D₂-like ability to couple to each of Gα_i1–3 as well as Gα_o1.