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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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 2

Subcellular localization variations linked to GNAO1 mutations.

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Subcellular localization variations linked to GNAO1 mutations. (A) N2a c...
(A) N2a cells expressing wild-type Gαo-GFP or the encephalopathy mutants G40R and E237K were immunostained against GM130 to visualize the Golgi apparatus and stained with DAPI in blue for nuclei. Scale bar: 10 μm. (B and C) Mean fluorescence intensity ratios of Gαo-GFP variants at the plasma membrane (PM; B) or Golgi (C) versus total cell (n = 9–10). 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). (D and E) The combined localization at the PM (D) or Golgi (E) of the variants connected to DEE17 or NEDIM. (F) A scatterplot showing a significant positive correlation between disease onset and PM localization of Gαo mutants. Note the log scale in the y axis. (G) N2a cells were cotransfected with His6-tagged RGS19 and Gαo-GFP variants, and immunoprecipitation (IP) was done with a nanobody against GFP. Co-IP of RGS19 was analyzed by Western blot using antibodies against GFP for Gαo and against the His6 tag for RGS19. (H and I) Quantification of the co-IP of RGS19 by individual Gαo mutants (n = 3) (H) and pooled in the DEE17 and NEDIM classes (I). Data represent mean ± SEM. Data in B, C, and H were analyzed by 1-way ANOVA followed by Dunnett’s multiple-comparison test, in D, E, and I by 2-tailed Mann-Whitney test, and in F by 2-tailed Spearman’s correlation test; rank correlation coefficients (rs) and P value are indicated. NS, not significant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.