G_o/z-biased coupling profile of the dopamine D3 receptor

Dopamine receptors are G protein coupled receptors (GPCRs) that serve as key targets for FDA-approved drugs used to treat various neuropsychiatric disorders. Notably, ~11% of all marketed GPCR-targeting drugs act on dopamine receptors. Five GPCRs mediate the effects of endogenous dopamine and compounds used to treat Parkinson's disease, schizophrenia, and other conditions. However, on-target side effects associated with these medications highlight the need to analyze dopamine receptor signaling to design safer, more effective therapeutics. We characterized the G protein coupling of dopamine D2-like receptors and observed the striking inability of D3R to engage with G_i proteins while effectively activating G_o and G_z subtypes. Applying orthogonal cell-based assays that utilize wild-type G proteins both in parental and ΔGα_i/o/z cells, we conclusively established that D3R does not activate G_i proteins. Further analysis of Gα_i2:Gα_oA and D2R:D3R chimeras revealed that this selective inability is driven by molecular determinants located within the α5 helix of Gα_i and the intracellular loop 2 (ICL2) of D3R. Guided by cryo-EM structures, we modeled the interface between these regions to better understand the structural basis of this selectivity. Finally, we treated hippocampal neurons in acute brain slices with selective agonists for D2R and D3R and observed marked differences in their ability to regulate endogenous adenylyl cyclase to produce cAMP, highlighting the neurophysiological significance of our findings.