Pathogenic Gαo Mutants Drive Dominant GPCR Coupling in GNAO1 Encephalopathies

Heterozygous mutations in GNAO1, which encodes the Gαo 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 Gαo mutants have been described. Intriguingly, several Gαo variants have been independently reported to dominantly engage G protein-coupled receptors (GPCRs) or to sequester Gβγ-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 Gαo 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.