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Golgi-Resident Gαo Promotes Protrusive Membrane Dynamics
olegchagin
G protein-coupled receptors (GPCRs) form the biggest receptor family in animals. Main intracellular GPCR effectors are heterotrimeric G proteins composed of α, β, and γ subunits, of which the α-subunit binds guanine nucleotides. Four main subgroups of Gα-subunits exist: Gαs, Gαq, Gαi/o, and Gα12/Gα13 (Milligan and Kostenis, 2006). When bound to guanosine diphosphate (GDP), heterotrimeric G protein is competent to interact with the cognate GPCR. The activated receptor acts as a guanine nucleotide exchange factor (GEF), catalyzing exchange of GDP for GTP on the Gα. This triggers dissociation of the G protein into Gα-GTP and the βγ-heterodimer, which can bind and activate downstream transducer proteins. When GTP on Gα is hydrolyzed, the inactive Gαβγ heterotrimer re-associates for a new cycle of activation. Alternatively, the Gα-subunit can be reloaded with GTP and continue its signaling activity (Lin et al., 2014).

As Gα-subunits provide the main specificity in GPCR-initiated signaling cascades (Milligan and Kostenis, 2006), identification of the Gα targets is crucial to understand this type of signaling. Gαo was among the first α-subunits discovered and is the major Gα-subunit of the nervous system across the animal kingdom (Sternweis and Robishaw, 1984, Wolfgang et al., 1990), controlling both development and adult physiology of the brain (Bromberg et al., 2008). Gαo is also expressed in other tissues and is a transducer of the developmentally and medically important Wnt signaling pathway (Egger-Adam and Katanaev, 2008, Koval et al., 2011).

Despite this importance, the list of known molecular targets of Gαo has been remarkably short. To uncover Gαo interactors, we performed several whole genome/proteome screenings resulting in > 250 candidate targets, most of which are not previously known to be regulated by Gα proteins. These Gαo targets can be clustered into functional modules, identifying several basic cellular activities, conserved from insects to humans, as being under regulation of Gαo-mediated GPCR signaling. We focus on vesicular trafficking as one of these functional modules and show that Gαo controls multiple steps within it. From Drosophila epithelia to mammalian neuronal cells, Gαo controls outgrowth formation through coordinated activities from the plasma membrane (PM) and the Golgi apparatus, the latter involving the KDEL receptor and small GTPases Rab1 and Rab3.

With only a few Gαo effectors previously known, we performed several screenings to massively identify Gαo partners. Our primary screenings were: yeast two-hybrid (1), proteomic (2) and genetic suppressor-enhancer screens in Drosophila using Gαo overexpression (3), and RNAi-mediated downregulation (4). In a complementary manner, we expected to cover the complete Gαo interactome with these screenings. We then complemented these screens with an extensive scrutiny of the literature data (5) and bioinformatics analysis of the resulting network and translation of this network into proteins orthologous between Drosophila and humans. This type of interactome identification has not been performed for any Gα protein and produced an impressive list of 254 proteins being candidate Gαo partners (Table S1; STAR Methods).

Next, we aimed at functional clusterization of the Gαo targets, performing the gene ontology enrichment analysis of the Gαo interactome. This analysis identifies several functional modules, such as cytoskeleton organization, cell division, cell adhesion, etc., within the Gαo interaction network (Figures 1A and S1A; Table S2). These modules may represent key cellular activities being directly controlled by Gαo-mediated GPCR signaling. As opposed to analysis of isolated individual targets, we decided to select a functional module from this network and to holistically investigate the role of Gαo in the regulation of this module. For this purpose, we selected the vesicular trafficking group of Gαo targets.

http://www.cell.com/cell/fulltext/S0092-8674(17)30822-X

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