Visualizing the Dominant GPCR Coupling of Pathogenic Gαo Mutants in GNAO1 -Related Disorders

Heterozygous mutations in the GNAO1 gene, which encodes the Gαo subunit of heterotrimeric G proteins, cause a spectrum of rare neurodevelopmental disorders ranging from early-onset epileptic encephalopathy to milder dystonia phenotypes. Disease dominance of Gαo mutants appears to arise from multiple functional disruptions, including impaired guanine nucleotide handling, failure to adopt the active conformation, and neomorphic interactions with Ric8A/B chaperones. In parallel, several Gαo variants have been independently reported to dominantly engage G protein-coupled receptors (GPCRs) or to sequester Gβγ, two mechanistically distinct behaviors. These conclusions were inferred from different indirect biosensor assays, likely contributing to the apparent contradiction in their proposed mechanisms. To overcome this, we developed a split-YFP-based bimolecular fluorescence complementation (BiFC) assay to visualize receptor-Go protein complexes at the plasma membrane. Using this system, we found that severe Gαo variants (S47G, G203R, R209C, E246K) fail to disengage from activated Gi/o-coupled GPCRs, thereby preventing downstream receptor phosphorylation and endocytosis. By contrast, milder dystonia-linked mutants (C215Y and T241_N242insPQ) showed near-normal receptor internalization and only minor phosphorylation defects. These findings establish dominant GPCR coupling as a molecular hallmark of severe GNAO1 -related disorders and point to split-YFP BiFC as a robust platform for probing mutant G protein behavior in genetic disease.