Activation of Multiple G Protein Pathways to Characterize the Five Dopamine Receptor Subtypes Using Bioluminescence Technology

G protein-coupled receptors show preference for G protein subtypes but can recruit multiple G proteins with various downstream signaling cascades. This functional selection can guide drug design. Dopamine receptors are both stimulatory (D₁-like) and inhibitory (D₂-like) with diffuse expression across the central nervous system. Functional selectivity of G protein subunits may help with dopamine receptor targeting and their downstream effects. Three bioluminescence-based assays were used to characterize G protein coupling and function with the five dopamine receptors. Most proximal to ligand binding was the miniG protein assay with split luciferase technology used to measure recruitment. For endogenous and selective ligands, the G-CASE bioluminescence resonance energy transfer (BRET) assay measured G protein activation and receptor selectivity. Downstream, the BRET-based CAMYEN assay quantified cyclic adenosine monophosphate (cAMP) changes. Several dopamine receptor agonists and antagonists were characterized for their G protein recruitment and cAMP effects. G protein selectivity with dopamine revealed potential G_q coupling at all five receptors, as well as the ability to activate subtypes with the “opposite” effects to canonical signaling. D₁-like receptor agonist (+)-SKF-81297 and D₂-like receptor agonist pramipexole showed selectivity at all receptors toward G_s or G_i/o/z activation, respectively. The five dopamine receptors show a wide range of potentials for G protein coupling and activation, reflected in their downstream cAMP signaling. Targeting these interactions can be achieved through drug design. This opens the door to pharmacological treatment with more selectivity options for inducing the correct physiological events.