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CELLULAR AND MOLECULAR

G Protein Coupling and Ligand Selectivity of the D_2L and D₃ Dopamine Receptors

Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, United Kingdom (J.R.L., G.M.); and Screening and Compound Profiling, GlaxoSmithKline Research and Development, New Frontiers Science Park, Harlow, Essex, United Kingdom (B.P., A.W., S.R.)

The human dopamine D_2L receptor couples promiscuously to multiple members of the G_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 G_i-family G proteins were expressed in an inducible fashion, we demonstrated highly selective coupling of the D₃ receptor to G_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 G_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 G_i1–3 as well as G_o1.

Address correspondence to: Dr. Graeme Milligan, Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK. E-mail: g.milligan{at}bio.gla.ac.uk