Pathogenic G α o Mutants Drive Dominant GPCR Coupling in GNAO1 Encephalopathies

Heterozygous mutations in GNAO1 , which encodes the G α o subunit of heterotrimeric G proteins, cause a spectrum of neurodevelopmental disorders ranging from early‐onset epileptic encephalopathy to dystonia. Although the mechanisms underlying disease dominance remain incompletely understood, some functional disruptions in G α o mutants have been described. Intriguingly, several G α o variants have been independently reported to dominantly engage G protein‐coupled receptors (GPCRs) or to sequester G β γ—two seemingly incompatible mechanisms inferred from distinct, indirect biosensor assays. To clarify this apparent contradiction, we developed a split‐YFP‐based bimolecular fluorescence complementation (BiFC) assay to directly visualize receptor‐G _o protein complexes at the plasma membrane. Using this system, we found that severe G α o variants fail to disengage from activated Gi/o‐coupled GPCRs, thereby preventing downstream receptor phosphorylation and endocytosis. By contrast, milder dystonia‐linked mutants showed near‐normal receptor internalization and only minor phosphorylation defects. These findings establish dominant GPCR coupling as a molecular hallmark of severe GNAO1 encephalopathies and point to split‐YFP BiFC as a robust platform for probing mutant G protein behavior in genetic disease.