Abstract
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.
Highlights
Heterotrimeric G proteins are quintessential signalling switches activated by nucleotide exchange on Ga
We focused our initial efforts on GIV, the first-identified and best-characterized member of the Ga-binding and -activating (GBA) motif-containing family of non-receptor guanine-nucleotide exchange factors (GEFs)
Full-length GIV co-immunoprecipitated with a G-protein mutant unable to bind nucleotides, mimicking Gai3-[ ]37, as efficiently as with the wild-type (WT) protein (Supplementary Fig. 1). These results indicate that the association of GIV with G proteins along the activation pathway differs from that of G-protein-coupled receptors (GPCRs) (Fig. 1c): GIV binds with high affinity to monomeric Gai-[GDP], remains bound to Gai with similar affinity on nucleotide release and eventually dissociates on GTP binding to release the active G protein
Summary
Heterotrimeric G proteins are quintessential signalling switches activated by nucleotide exchange on Ga. Despite the moderate GEF activity of GIV and DAPLE in vitro, it has been shown that their GBA motif leads to increased G-protein activation in cells, as determined by read-outs that monitor either the formation of Gai-GTP (for example, conformation-specific antibodies or cAMP)[20,26,27,28] or free Gbg (for example, PI3K-Akt or resonance energy transfer-based biosensors)[18,20,27,29,30] As a consequence, they impact cell behaviour, and their dysregulation is associated with human diseases like cancer, liver fibrosis, nephrotic syndrome, insulin resistance and pathologic neovascularization[16,20,31]. Our results provide the first detailed view of the molecular mechanism by which proteins with a GBA motif regulate Gai[3], revealing fundamental differences with GPCR-mediated activation of G proteins
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