GoLoco/GPR motif-dependent regulation of Rap1GAP1 by Gαo is disrupted by Gαo encephalopathy variants

This is the authors' abstract. We don't add key points for this paper.

G protein-coupled receptors (GPCRs) that couple to Gα i/o family members are major therapeutic targets. Among heterotrimeric G proteins, Gα o is the most abundant Gα subunit in the brain but the mechanistic pathways controlled by Gα o have not been thoroughly established. Understanding Gα o -mediated signalling pathways is especially critical given recent reports of a neurodevelopmental disorder ( GNAO1 encephalopathy) associated with mutations in the Gα o -encoding gene. To address this gap, we sought to uncover novel Gα o effectors using a proximity-based proteomics screen in differentiated PC12 cells. Our analysis revealed a diverse set of potential Gα o -GTP effector proteins including a Rap1 GTPase activating protein, Rap1GAP1. Regulation of Rap1GAP1 by G protein α subunits is controversial, with Rap1GAP1 reported to bind preferentially to Gα o -GDP via a GoLoco/G protein regulatory (GPR) motif. We establish that Gα o -GTP binds and regulates Rap1GAP1 activity and reveal a novel mechanism for Gα subunit recognition by Rap1GAP1 where the presence or absence of key contact residues in the GoLoco/GPR motif confer differential recognition of Gα o guanine nucleotide binding status. We also show that pathologic GNAO1 mutations disrupt this functional relationship by preventing the activated Gα subunit from attaining a conformation required for effector binding. These data resolve controversies in the literature regarding activation-dependent binding and regulation of Rap1GAP by Gα o and help establish Rap1GAP1a as a bone fide G protein regulated effector. Furthermore, our study finds that multiple mutants in Gα o associated with GNAO1 encephalopathy have defects in downstream effector interactions, which could underly some of the manifestations of this disease.