Optimization Of A Synthetic Protein Inhibitor Of GPCR-Independent G Protein SignalingIn Vivo

Abstract ID 91447 Poster Board 345 Activation of heterotrimeric G-proteins is viewed as a task exclusively carried out by GPCRs. However, this canonical idea has been challenged by the identification of non-receptor proteins that also activate heterotrimeric G-protein signaling. While these non-receptor activators were originally believed to be a heterogeneous group of proteins, a subclass of them is defined by the presence of a conserved stretch of ∼20-30 amino acids named the Gα-binding-and-activating (GBA) motif. While GBA motifs promote G-protein signaling in various cellular contexts, the physiological functions of this atypical mechanism of G protein regulation and the complement proteins that mediate it remain unknown. Here, we set out to develop a transgenic mouse that conditionally expresses a synthetic construct that specifically binds GBA motifs with high affinity to block their actions in cells. For this, we leveraged a previously described construct named GBA inhibitor (GBAi). Briefly, GBAi is an engineered Gα subunit that binds to GBA motifs but not any other known G-protein regulator or effector. We rationally modified GBAi to improve its affinity for GBA motif. The optimized construct, which we named GBAi 2.0, improved GBA motif binding affinity up to 10 times, while lacking binding to other G-protein interactors. We have generated a transgenic mouse line to conditionally express GBAi 2.0 in Cre-dependent manner. We foresee that this model will become useful to define redundant and/or tissue specific physiological functions of GBA motif-mediated signaling in vivo, and to identify new members of this class of regulators using GBAi 2.0 as an affinity probe.