Specific inhibition of GPCR-independent G protein signaling by a rationally engineered protein

Abstract

Activation of heterotrimeric G proteins by cytoplasmic nonreceptor proteins is an alternative to the classical mechanism via G protein-coupled receptors (GPCRs). A subset of nonreceptor G protein activators is characterized by a conserved sequence named the Gα-binding and activating (GBA) motif, which confers guanine nucleotide exchange factor (GEF) activity in vitro and promotes G protein-dependent signaling in cells. GBA proteins have important roles in physiology and disease but remain greatly understudied. This is due, in part, to the lack of efficient tools that specifically disrupt GBA motif function in the context of the large multifunctional proteins in which they are embedded. This hindrance to the study of alternative mechanisms of G protein activation contrasts with the wealth of convenient chemical and genetic tools to manipulate GPCR-dependent activation. Here, we describe the rational design and implementation of a genetically encoded protein that specifically inhibits GBA motifs: GBA inhibitor (GBAi). GBAi was engineered by introducing modifications in Gαi that preclude coupling to every known major binding partner [GPCRs, Gβγ, effectors, guanine nucleotide dissociation inhibitors (GDIs), GTPase-activating proteins (GAPs), or the chaperone/GEF Ric-8A], while favoring high-affinity binding to all known GBA motifs. We demonstrate that GBAi does not interfere with canonical GPCR-G protein signaling but blocks GBA-dependent signaling in cancer cells. Furthermore, by implementing GBAi in vivo, we show that GBA-dependent signaling modulates phenotypes during embryonic development. In summary, GBAi is a selective, efficient, and convenient tool to dissect the biological processes controlled by a GPCR-independent mechanism of G protein activation mediated by cytoplasmic factors.

A biochemical and genetic discovery pipeline identifies PLCδ4b as a nonreceptor activator of heterotrimeric G-proteins

“…6B), which is even lower than that of the GBA motif of GIV or of DAPLE. Because GIV and DAPLE have been shown to regulate G-protein signaling in cells via GBA-dependent binding (23,26,31,36,37,51), this suggests that the GBA motif of PLCD4 might also be bioactive. In contrast, NOC2L binding was so weak that the FP signal did not reach saturation even at the highest concentration of G-protein tested (32 M) ( Fig.…”

“…This assay was performed using the previously described BRET-based biosensor NLuc-EPAC-VV (51,57). HEK293T cells were seeded, transfected, and harvested as described above (under "BRET-based G-protein activation assays") except that the plasmids used were as follows (quantities in parentheses): Nluc-EPAC-VV (0.2 g); Lyn11-FRB (3 g); and FKBP-GBA constructs (0.1 g).…”

GPCR-independent activation of G proteins promotes apical cell constriction in vivo

“…The long-held tenet that activation of heterotrimeric G proteins is a function performed exclusively by GPCRs has been challenged recently. For example, some cytoplasmic factors bearing the so-called Gα-binding-and-activating (GBA) motif (de Opakua et al, 2017; DiGiacomo et al, 2018) possess GEF activity in vitro for Gα proteins of the Gi subfamily (Gαi1, Gαi2, or Gαi3; Garcia-Marcos et al, 2009, 2011; Aznar et al, 2015) and promote G protein signaling in cells, as determined by readouts for the generation of either GTP-bound Gαi (Lin et al, 2014; Lopez-Sanchez et al, 2014; Aznar et al, 2015; Midde et al, 2015) or free Gβγ (Garcia-Marcos et al, 2009; Aznar et al, 2015; Leyme et al, 2015, 2017; Midde et al, 2015; Parag-Sharma et al, 2016; Maziarz et al, 2018). Despite abundant information on the biochemical and signaling mechanisms of this growing family of regulators, direct in vivo evidence for a physiological process controlled by them is still lacking.…”

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

“…This assay was performed using the previously described BRET-based biosensor NLuc-EPAC-VV ( Leyme et al, 2017 ; Masuho et al, 2015 ). HEK293T cells were seeded, transfected, and harvested as described above (‘ BRET-based G-protein activation assay ’) except that the plasmids used were (quantities in parenthesis): Nluc-EPAC-VV (0.05 μg), Gαi3 (1 μg) Gβ1 (0.5 μg), Gγ2 (0.5 μg) and the mLOV2GIVe constructs indicated in the figures (2 µg).…”