GoLoco/GPR Motif-Dependent Regulation of Rap1GAP1 by Gα is Disrupted by Gα Encephalopathy Variants.

G protein-coupled receptors (GPCRs) that couple to Gα family members are major therapeutic targets. Among heterotrimeric G proteins, Gα is the most abundant Gα subunit in the brain but the mechanistic pathways controlled by Gα have not been thoroughly established. Understanding Gα-mediated signalling pathways is especially critical given recent reports of a neurodevelopmental disorder (GNAO1 encephalopathy) associated with mutations in the Gα-encoding gene. To address this gap, we sought to uncover novel Gα effectors using a proximity-based proteomics screen in differentiated PC12 cells. Our analysis revealed a diverse set of potential Gα-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-GDP via a GoLoco/G protein regulatory (GPR) motif. We establish that Gα-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 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 and help establish Rap1GAP1a as a bone fide G protein regulated effector. Furthermore, our study finds that multiple mutants in Gα associated with GNAO1 encephalopathy have defects in downstream effector interactions, which could underly some of the manifestations of this disease.