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

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 signaling 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 bona 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 underlie some of the manifestations of this disease.