Abstract
GIV (aka Girdin) is a guanine nucleotide exchange factor that activates heterotrimeric G protein signaling downstream of RTKs and integrins, thereby serving as a platform for signaling cascade cross-talk. GIV is recruited to the cytoplasmic tail of receptors upon stimulation, but the mechanism of activation of its G protein regulatory function is not well understood. Here we used assays in humanized yeast models and G protein activity biosensors in mammalian cells to investigate the role of GIV subcellular compartmentalization in regulating its ability to promote G protein signaling. We found that in unstimulated cells GIV does not co-fractionate with its substrate G protein Gαi3 on cell membranes and that constitutive membrane anchoring of GIV in yeast cells or rapid membrane translocation in mammalian cells via chemically induced dimerization leads to robust G protein activation. We show that membrane recruitment of the GIV "Gα binding and activating" motif alone is sufficient for G protein activation and that it does not require phosphomodification. Furthermore, we engineered a synthetic protein to show that recruitment of the GIV "Gα binding and activating" motif to membranes via association with active RTKs, instead of via chemically induced dimerization, is also sufficient for G protein activation. These results reveal that recruitment of GIV to membranes in close proximity to its substrate G protein is a major mechanism responsible for the activation of its G protein regulatory function.
Highlights
Under resting conditions the nucleotide-binding ␣ subunit (G␣) is loaded with GDP and forms a complex with G␥
The main finding of this work is the identification of GIV recruitment to membranes as a major mechanism responsible for the activation of its G protein regulatory function
This mechanism operates in the context of G protein-coupled receptors (GPCRs)-independent G protein activation and provides novel insights into a long standing gap in the understanding of signaling cross-talk and G protein transactivation by receptor tyrosine kinases (RTKs)
Summary
Tide exchange [17, 23], and it promotes G protein activation in cells, as determined by readouts for either G␣i-GTP (e.g. conformation-specific antibodies, cAMP dampening) [25,26,27] or free G␥ (e.g. PI3K-Akt signaling, resonance energy transferbased biosensors) [17, 26, 28]. Stimulation of EGFR, a prototypical RTK, has been recently shown to lead to the phosphorylation of GIV at serine 1674, a residue adjacent to its GBA motif, which enhances the GEF activity [47]. This enhancement is unlikely to fully account for all GIV-mediated G protein activation because it is modest (ϳ1.5-fold enhancement of GEF activity in vitro) and dependent on a particular kinase (cyclin-dependent kinase 5), which might not be relevant for all RTKs or other receptor types, like integrins, that rely on GIV for signaling. By using synthetic biology approaches and G protein activity biosensors, here we provide evidence that the spatial relocalization of GIV to membranes is a mayor mechanism responsible for the activation of its G protein regulatory function
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