Neomorphic Gαo mutations gain interaction with Ric8 proteins in GNAO1 encephalopathies
Gonzalo P. Solis, … , Mikhail Savitsky, Vladimir L. Katanaev
Gonzalo P. Solis, … , Mikhail Savitsky, Vladimir L. Katanaev
Published June 14, 2024
Citation Information: J Clin Invest. 2024;134(15):e172057. https://doi.org/10.1172/JCI172057.
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Research Article Cell biology Genetics

Neomorphic Gαo mutations gain interaction with Ric8 proteins in GNAO1 encephalopathies

Abstract

GNAO1 mutated in pediatric encephalopathies encodes the major neuronal G protein Gαo. Of the more than 80 pathogenic mutations, most are single amino acid substitutions spreading across the Gαo sequence. We performed extensive characterization of Gαo mutants, showing abnormal GTP uptake and hydrolysis and deficiencies in binding Gβγ and RGS19. Plasma membrane localization of Gαo was decreased for a subset of mutations that leads to epilepsy; dominant interactions with GPCRs also emerged for the more severe mutants. Pathogenic mutants massively gained interaction with Ric8A and, surprisingly, Ric8B proteins, relocalizing them from cytoplasm to Golgi. Of these 2 mandatory Gα-subunit chaperones, Ric8A is normally responsible for the Gαi/Gαo, Gαq, and Gα12/Gα13 subfamilies, and Ric8B solely responsible for Gαs/Gαolf. Ric8 mediates the disease dominance when engaging in neomorphic interactions with pathogenic Gαo through imbalance of the neuronal G protein signaling networks. As the strength of Gαo-Ric8B interactions correlates with disease severity, our study further identifies an efficient biomarker and predictor for clinical manifestations in GNAO1 encephalopathies. Our work uncovers the neomorphic molecular mechanism of mutations underlying pediatric encephalopathies and offers insights into other maladies caused by G protein malfunctioning and further genetic diseases.

Authors

Gonzalo P. Solis, Alexey Koval, Jana Valnohova, Arghavan Kazemzadeh, Mikhail Savitsky, Vladimir L. Katanaev

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Figure 3

Differential Gβγ binding induced by GNAO1 mutations.

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Differential Gβγ binding induced by GNAO1 mutations. (A–C) The interacti...
(A–C) The interaction of Gαo-GFP variants with mRFP-Gβ1 and mRFP-Gγ3 was analyzed by immunoprecipitation (IP) from N2a cells using a nanobody against GFP. (A) Immunodetection was done by Western blot using anti-GFP and anti-RFP antibodies. (B) Quantification of the Gαo-Gβ1γ3 interaction for individual Gαo variants (n = 4–6). Bars are color-coded according to the involvement of Gαo mutants in the pathology developmental and epileptic encephalopathy-17 (DEE17; red) or neurodevelopmental disorder with involuntary movements (NEDIM; blue). (C) The combined Gβ1γ3 interaction of Gαo variants grouped in the DEE17 or NEDIM categories. (D–F) A scheme of the Gβ3γ9 displacement assay by BRET (D). Wild-type Gαo internally tagged with nano-luciferase (Gαo-NLuc) excites cpVenus (Ven) fused to Gγ9 in the Gβ3γ9 heterodimer. The ability of nontagged Gαo to displace Gβ3γ9 from Gαo-NLuc (reduction in the BRET signal) was quantified for wild-type Gαo, the encephalopathy mutants, and the GTPase-dead Q205L as control (n = 4–9) (E). The combined effect of the Gαo variants on Gβ3γ9 displacement sorted in the DEE17 or NEDIM group (F). (G and H) Scatterplots illustrating a strong positive correlation between Gβ3γ9 displacement and PM localization (G), and between disease onset and Gβ1γ3 co-IP (H) of Gαo mutants. Note the log scale in the y axis of H. Data represent mean ± SEM. Data in B and E were analyzed by 1-way ANOVA followed by Dunnett’s multiple-comparison test, in C and F by 2-tailed Mann-Whitney test, and in G and H by 2-tailed Spearman’s correlation test; rank correlation coefficients (rs) and P values are indicated. NS, not significant. *P < 0.05; **P < 0.01; ****P < 0.0001.