Gα_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 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. Here, we characterize the G protein coupling of dopamine D2-like receptors and observe the striking inability of D3R to engage with Gα_i proteins while effectively activating Gα_o/z subtypes. Applying orthogonal cell-based assays with wild-type G proteins in parental and ΔGα_i/o/z cells, we conclusively establish that D3R does not activate Gα_i proteins. Further analysis of Gα_i2:Gα_oA and D2R:D3R chimeras reveals that this selective inability is driven by molecular determinants located within the α5 helix of Gα_i and ICL2 of D3R. Guided by cryo-EM structures, we model the interface between these regions to clarify the structural basis of selectivity. Finally, treating hippocampal neurons in acute slices with selective agonists and antagonists reveals marked differences in their regulation of endogenous adenylyl cyclase and cAMP, underscoring the neurophysiological significance of our findings.