DAPLE protein inhibits nucleotide exchange on Gαs and Gαq via the same motif that activates Gαi

Abstract

Besides being regulated by G-protein–coupled receptors, the activity of heterotrimeric G proteins is modulated by many cytoplasmic proteins. GIV/Girdin and DAPLE (Dvl-associating protein with a high frequency of leucine) are the best-characterized members of a group of cytoplasmic regulators that contain a Gα-binding and -activating (GBA) motif and whose dysregulation underlies human diseases, including cancer and birth defects. GBA motif–containing proteins were originally reported to modulate G proteins by binding Gα subunits of the Gi/o family (Gαi) over other families (such as Gs, Gq/11, or G12/13), and promoting nucleotide exchange in vitro. However, some evidence suggests that this is not always the case, as phosphorylation of the GBA motif of GIV promotes its binding to Gαs and inhibits nucleotide exchange. The G-protein specificity of DAPLE and how it might affect nucleotide exchange on G proteins besides Gαi remain to be investigated. Here, we show that DAPLE's GBA motif, in addition to Gαi, binds efficiently to members of the Gs and Gq/11 families (Gαs and Gαq, respectively), but not of the G12/13 family (Gα12) in the absence of post-translational phosphorylation. We pinpointed Met-1669 as the residue in the GBA motif of DAPLE that diverges from that in GIV and enables better binding to Gαs and Gαq. Unlike the nucleotide-exchange acceleration observed for Gαi, DAPLE inhibited nucleotide exchange on Gαs and Gαq. These findings indicate that GBA motifs have versatility in their G-protein–modulating effect, i.e. they can bind to Gα subunits of different classes and either stimulate or inhibit nucleotide exchange depending on the G-protein subtype.

Planar cell polarity induces local microtubule bundling for coordinated ciliary beating

“…Daple has been reported to both trigger Gα i protein activation through the Gα binding and activating (GBA) motif ( Aznar et al, 2015 ; Leyme et al, 2017 ; Aznar et al, 2018 ; Marivin et al, 2019 ; Marivin and Garcia-Marcos, 2019 ; Marivin et al, 2020 ) and interact with Dvl and PARD3 through a PDZ-binding motif (PBM; Fig. 5 A ; Oshita et al, 2003 ; Ishida-Takagishi et al, 2012 ; Ear et al, 2020 ).…”

Optogenetic activation of heterotrimeric Gi proteins by LOV2GIVe— a rationally engineered modular protein

“…We also found that LOV2GIVe retains the same G-protein specificity as GIV. Much like GIV (Garcia-Marcos et al, 2010;Marivin et al, 2020), LOV2GIVe bound robustly only to members of the G i/o family among the 4 families of Gα proteins (G i/o , G s , G q/11 and G 12/13 ), and could discriminate within the G i/o family by binding to Gαi1, Gαi2 and Gαi3 but not to Gαo (Fig. 2B).…”

Optogenetic activation of heterotrimeric G-proteins by LOV2GIVe, a rationally engineered modular protein

“…We also found that LOV2GIVe retains the same G-protein specificity as GIV. Much like GIV ( Garcia-Marcos et al, 2010 ; Marivin et al, 2020 ), LOV2GIVe bound robustly only to members of the G i/o family among the four families of Gα proteins (G i/o , G s , G q/11, and G 12/13 ), and could discriminate within the G i/o family by binding to Gαi1, Gαi2 and Gαi3 but not to Gαo ( Figure 2B ). Next, we assessed if LOV2GIVe can activate G-proteins in vitro, that is whether it retains GIV’s GEF activity.…”