Wnt signaling is essential for tissue homeostasis and its dysregulation causes cancer. Wnt ligands trigger signaling by activating Frizzled receptors (FZDRs), which belong to the G-protein coupled receptor superfamily. However, the mechanisms of G protein activation in Wnt signaling remain controversial. In this study, we demonstrate that FZDRs activate G proteins and trigger non-canonical Wnt signaling via the Dishevelled-binding protein, Daple. Daple contains a Gα-binding and activating (GBA) motif, which activates Gαi proteins and an adjacent domain that directly binds FZDRs, thereby linking Wnt stimulation to G protein activation. This triggers non-canonical Wnt responses, that is, suppresses the β-catenin/TCF/LEF pathway and tumorigenesis, but enhances PI3K-Akt and Rac1 signals and tumor cell invasiveness. In colorectal cancers, Daple is suppressed during adenoma-to-carcinoma transformation and expressed later in metastasized tumor cells. Thus, Daple activates Gαi and enhances non-canonical Wnt signaling by FZDRs, and its dysregulation can impact both tumor initiation and progression to metastasis.DOI: http://dx.doi.org/10.7554/eLife.07091.001
GIV/Girdin, a nonreceptor GEF, directly links integrins to activation of trimeric G proteins to promote the acquisition of proinvasive traits in cancer cells.
Highlights d Optical biosensors directly detect active G-proteins in real time in live cells d Unimolecular sensors report activity of natively expressed GPCRs/G-proteins d Versatile modular design allows customization for multiple G-protein specificities d Proof-of-principle implementation for cancer and neurobiology applications
Heterotrimeric G proteins are quintessential signalling switches activated by nucleotide exchange on Gα. Although activation is predominantly carried out by G-protein-coupled receptors (GPCRs), non-receptor guanine-nucleotide exchange factors (GEFs) have emerged as critical signalling molecules and therapeutic targets. Here we characterize the molecular mechanism of G-protein activation by a family of non-receptor GEFs containing a Gα-binding and -activating (GBA) motif. We combine NMR spectroscopy, computational modelling and biochemistry to map changes in Gα caused by binding of GBA proteins with residue-level resolution. We find that the GBA motif binds to the SwitchII/α3 cleft of Gα and induces changes in the G-1/P-loop and G-2 boxes (involved in phosphate binding), but not in the G-4/G-5 boxes (guanine binding). Our findings reveal that G-protein-binding and activation mechanisms are fundamentally different between GBA proteins and GPCRs, and that GEF-mediated perturbation of nucleotide phosphate binding is sufficient for Gα activation.
Trimeric G protein signaling is a fundamental mechanism of cellular communication in eukaryotes. The core of this mechanism consists of activation of G proteins by the guanine-nucleotide exchange factor (GEF) activity of G protein coupled receptors. However, the duration and amplitude of G protein-mediated signaling are controlled by a complex network of accessory proteins that appeared and diversified during evolution. Among them, nonreceptor proteins with GEF activity are the least characterized. We recently found that proteins of the ccdc88 family possess a Gα-binding and activating (GBA) motif that confers GEF activity and regulates mammalian cell behavior. A sequence similarity-based search revealed that ccdc88 genes are highly conserved across metazoa but the GBA motif is absent in most invertebrates. This prompted us to investigate whether the GBA motif is present in other nonreceptor proteins in invertebrates. An unbiased bioinformatics search in Caenorhabditis elegans identified GBAS-1 (GBA and SPK domain containing-1) as a GBA motif-containing protein with homologs only in closely related worm species. We demonstrate that GBAS-1 has GEF activity for the nematode G protein GOA-1 and that the two proteins are coexpressed in many cells of living worms. Furthermore, we show that GBAS-1 can activate mammalian Gα-subunits and provide structural insights into the evolutionarily conserved determinants of the GBA–G protein interface. These results demonstrate that the GBA motif is a functional GEF module conserved among highly divergent proteins across evolution, indicating that the GBA-Gα binding mode is strongly constrained under selective pressure to mediate receptor-independent G protein activation in metazoans.
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