Neomorphic Gαo mutations gain interaction with Ric8 proteins in GNAO1 encephalopathies.

GNAO1 mutated in pediatric encephalopathies encodes the major neuronal G-protein Gαo. Of >80 pathogenic mutations, most are single amino acid substitutions spreading across Gαo sequence. We perform extensive characterization of Gαo mutants showing abnormal GTP uptake and hydrolysis, and deficiencies to bind Gßγ and RGS19. Plasma membrane localization of Gαo is decreased for a subset of mutations that leads to epilepsy; dominant interactions with GPCRs also emerge for the more severe mutants. Pathogenic mutants massively gain interaction with Ric8A and, surprisingly, Ric8B proteins, delocalizing them from cytoplasm to Golgi. Of these two mandatory Gα-subunit chaperones, Ric8A is normally responsible for the Gαi/o, Gαq, and Gα12/13 subfamilies, and Ric8B solely for Gαs/olf. Ric8A/B mediate the disease dominance when engaging in neomorphic interactions with pathogenic Gαo through disbalancing 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 discovers the neomorphic molecular mechanism of mutations underlying pediatric encephalopathies and offers insights to other maladies caused by G protein misfunctioning and further genetic diseases.

Restoration of the GTPase activity and cellular interactions of Gαo mutants by Zn2+ in GNAO1 encephalopathy models.

De novo point mutations in GNAO1, gene encoding the major neuronal G protein Gαo, have recently emerged in patients with pediatric encephalopathy having motor, developmental, and epileptic dysfunctions. Half of clinical cases affect codons Gly203, Arg209, or Glu246; we show that these mutations accelerate GTP uptake and inactivate GTP hydrolysis through displacement Gln205 critical for GTP hydrolysis, resulting in constitutive GTP binding by Gαo. However, the mutants fail to adopt the activated conformation and display aberrant interactions with signaling partners. Through high-throughput screening of approved drugs, we identify zinc pyrithione and Zn2+ as agents restoring active conformation, GTPase activity, and cellular interactions of the encephalopathy mutants, with negligible effects on wild-type Gαo. We describe a Drosophila model of GNAO1 encephalopathy where dietary zinc restores the motor function and longevity of the mutant flies. Zinc supplements are approved for diverse human neurological conditions. Our work provides insights into the molecular etiology of GNAO1 encephalopathy and defines a potential therapy for the patients.

Local and substrate-specific S-palmitoylation determines subcellular localization of Gαo.

Peripheral membrane proteins (PMPs) associate with cellular membranes through post-translational modifications like S-palmitoylation. The Golgi apparatus is generally viewed as the transitory station where palmitoyl acyltransferases (PATs) modify PMPs, which are then transported to their ultimate destinations such as the plasma membrane (PM). However, little substrate specificity among the many PATs has been determined. Here we describe the inherent partitioning of Gαo - α-subunit of heterotrimeric Go proteins - to PM and Golgi, independent from Golgi-to-PM transport. A minimal code within Gαo N-terminus governs its compartmentalization and re-coding produces G protein versions with shifted localization. We establish the S-palmitoylation at the outer nuclear membrane assay ("SwissKASH") to probe substrate specificity of PATs in intact cells. With this assay, we show that PATs localizing to different membrane compartments display remarkable substrate selectivity, which is the basis for PMP compartmentalization. Our findings uncover a mechanism governing protein localization and establish the basis for innovative drug discovery.

Structural insight into small molecule action on Frizzleds.

WNT-Frizzled (FZD) signaling plays a critical role in embryonic development, stem cell regulation and tissue homeostasis. FZDs are linked to severe human pathology and are seen as a promising target for therapy. Despite intense efforts, no small molecule drugs with distinct efficacy have emerged. Here, we identify the Smoothened agonist SAG1.3 as a partial agonist of FZD6 with limited subtype selectivity. Employing extensive in silico analysis, resonance energy transfer- and luciferase-based assays we describe the mode of action of SAG1.3. We define the ability of SAG1.3 to bind to FZD6 and to induce conformational changes in the receptor, recruitment and activation of G proteins and dynamics in FZD-Dishevelled interaction. Our results provide the proof-of-principle that FZDs are targetable by small molecules acting on their seven transmembrane spanning core. Thus, we provide a starting point for a structure-guided and mechanism-based drug discovery process to exploit the potential of FZDs as therapeutic targets.

A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection.

Class F receptors are considered valuable therapeutic targets due to their role in human disease, but structural changes accompanying receptor activation remain unexplored. Employing population and cancer genomics data, structural analyses, molecular dynamics simulations, resonance energy transfer-based approaches and mutagenesis, we identify a conserved basic amino acid in TM6 in Class F receptors that acts as a molecular switch to mediate receptor activation. Across all tested Class F receptors (FZD4,5,6,7, SMO), mutation of the molecular switch confers an increased potency of agonists by stabilizing an active conformation as assessed by engineered mini G proteins as conformational sensors. Disruption of the switch abrogates the functional interaction between FZDs and the phosphoprotein Dishevelled, supporting conformational selection as a prerequisite for functional selectivity. Our studies reveal the molecular basis of a common activation mechanism conserved in all Class F receptors, which facilitates assay development and future discovery of Class F receptor-targeting drugs.

Dishevelled enables casein kinase 1-mediated phosphorylation of Frizzled 6 required for cell membrane localization.

Frizzleds (FZDs) are receptors for secreted lipoglycoproteins of the Wingless/Int-1 (WNT) family, initiating an important signal transduction network in multicellular organisms. FZDs are G protein-coupled receptors (GPCRs), which are well known to be regulated by phosphorylation, leading to specific downstream signaling or receptor desensitization. The role and underlying mechanisms of FZD phosphorylation remain largely unexplored. Here, we investigated the phosphorylation of human FZD6 Using MS analysis and a phospho-state- and -site-specific antibody, we found that Ser-648, located in the FZD6 C terminus, is efficiently phosphorylated by casein kinase 1 ϵ (CK1ϵ) and that this phosphorylation requires the scaffolding protein Dishevelled (DVL). In an overexpression system, DVL1, -2, and -3 promoted CK1ϵ-mediated FZD6 phosphorylation on Ser-648. This DVL activity required an intact DEP domain and FZD-mediated recruitment of this domain to the cell membrane. Substitution of the CK1ϵ-targeted phosphomotif reduced FZD6 surface expression, suggesting that Ser-648 phosphorylation controls membrane trafficking of FZD6 Phospho-Ser-648 FZD6 immunoreactivity in human fallopian tube epithelium was predominantly apical, associated with cilia in a subset of epithelial cells, compared with the total FZD6 protein expression, suggesting that FZD6 phosphorylation contributes to asymmetric localization of receptor function within the cell and to epithelial polarity. Given the key role of FZD6 in planar cell polarity, our results raise the possibility that asymmetric phosphorylation of FZD6 rather than asymmetric protein distribution accounts for polarized receptor signaling.

Functional dissection of the N-terminal extracellular domains of Frizzled 6 reveals their roles for receptor localization and Dishevelled recruitment.

The Frizzled (FZD) proteins belong to class F of G protein-coupled receptors (GPCRs) and are essential for various pathways involving the secreted lipoglycoproteins of the wingless/int-1 (WNT) family. A WNT-binding cysteine-rich domain (CRD) in FZDs is N-terminally located and connected to the seven transmembrane domain-spanning receptor core by a linker domain that has a variable length in different FZD homologs. However, the function and importance of this linker domain are poorly understood. Here we used systematic mutagenesis of FZD6 to define the minimal N-terminal domain sufficient for receptor surface expression and recruitment of the intracellular scaffold protein Dishevelled (DVL). Further, we identified a triad of evolutionarily conserved cysteines in the FZD linker domain that is crucial for receptor membrane expression and recruitment of DVL. Our results are in agreement with the concept that the conserved cysteines in the linker domain of FZDs assist with the formation of a common secondary structure in this region. We propose that this structure could be involved in agonist binding and receptor activation mechanisms that are similar to the binding and activation mechanisms known for other GPCRs.

FZD10-Gα13 signalling axis points to a role of FZD10 in CNS angiogenesis.

Among the 10 Frizzled (FZD) isoforms belonging to the Class F of G protein-coupled receptors (GPCRs), FZD10 remains the most enigmatic. FZD10 shows homology to FZD4 and FZD9 and was previously implicated in both ß-catenin-dependent and -independent signalling. In normal tissue, FZD10 levels are generally very low; however, its upregulation in synovial carcinoma has attracted some attention for therapy. Our findings identify FZD10 as a receptor interacting with and signalling through the heterotrimeric G protein Gα13 but not Gα12, Gαi1, GαoA, Gαs, or Gαq. Stimulation with the FZD agonist WNT induced the dissociation of the Gα13 protein from FZD10, and led to global Gα12/13-dependent cell changes assessed by dynamic mass redistribution measurements. Furthermore, we show that FZD10 mediates Gα12/13 activation-dependent induction of YAP/TAZ transcriptional activity. In addition, we show a distinct expression of FZD10 in embryonic CNS endothelial cells at E11.5-E14.5. Given the well-known importance of Gα13 signalling for the development of the vascular system, the selective expression of FZD10 in brain vascular endothelial cells points at a potential role of FZD10-Gα13 signalling in CNS angiogenesis.

Dishevelled is a NEK2 kinase substrate controlling dynamics of centrosomal linker proteins.

Dishevelled (DVL) is a key scaffolding protein and a branching point in Wnt signaling pathways. Here, we present conclusive evidence that DVL regulates the centrosomal cycle. We demonstrate that DVL dishevelled and axin (DIX) domain, but not DIX domain-mediated multimerization, is essential for DVL's centrosomal localization. DVL accumulates during the cell cycle and associates with NIMA-related kinase 2 (NEK2), which is able to phosphorylate DVL at a multitude of residues, as detected by a set of novel phospho-specific antibodies. This creates interfaces for efficient binding to CDK5 regulatory subunit-associated protein 2 (CDK5RAP2) and centrosomal Nek2-associated protein 1 (C-NAP1), two proteins of the centrosomal linker. Displacement of DVL from the centrosome and its release into the cytoplasm on NEK2 phosphorylation is coupled to the removal of linker proteins, an event necessary for centrosomal separation and proper formation of the mitotic spindle. Lack of DVL prevents NEK2-controlled dissolution of loose centrosomal linker and subsequent centrosomal separation. Increased DVL levels, in contrast, sequester centrosomal NEK2 and mimic monopolar spindle defects induced by a dominant negative version of this kinase. Our study thus uncovers molecular crosstalk between centrosome and Wnt signaling.