G protein-coupled receptors (GPCRs) are ubiquitously expressed in mammalian tissue and regulate a wide variety of physiological and pathological processes. Functionally, GPCRs exert diverse intracellular outputs through coupling to four distinct families of heterotrimeric G proteins comprised of the a and bg subunits. Upon activation, GPCRs promote guanine nucleotide exchange on the Ga subunit, facilitating guanosine-5’-diphosphate(GDP) release and guanosine-5’-triphosphate (GTP) binding to the Ga subunit. This nucleotide exchange triggers the dissociation of the GTP-Ga subunit from both the receptor and the Gbg dimer. This dissertation primarily focuses on the Gi/o family of G proteins. Canonically, Gai inhibits adenylyl cyclase activity. However, although Gao is a member of the Gi family, it has not been shown to modulate adenylyl cyclase in vitro. In comparison to Gai/o subunits, Gbg effectors are relatively well characterized and serve as primary signaling drivers downstream of activated Gi/o-coupled receptors. As such, intracellular effector proteins that directly interact with Gai are often overlooked, and those specific to Gao remain poorly defined. To address these gaps, we aimed to uncover novel effectors of Gai/o subunits using proximity-dependent proteomic approaches. Our strategy to identify Gai1-GTP signaling interaction partners was by comparing proteins enriched by the constitutively active Gai1 mutant (Gai1(Q204L)) relative to Gai1(WT) via proximitylabeling. This initial proteomic screen detected several reported Gai1 interactors. Among the novel candidates identified was the brain abundant activity-dependent neuroprotective protein (ADNP), which showed a strong enrichment by Gai1(Q204L). Interaction between ADNP and active Gai1 was demonstrated through orthogonal luciferase complementation assays. Further characterization revealed a 46 amino-acid stretch sequence in ADNP containing the putative nuclear localization signal necessary for its interaction with Gai1-GTP, laying the groundwork for future studies on the functional relevance of this novel protein-protein interaction. The Gai proteomic-based approach helped overcome many of the limitations associated with studying Gai signaling, identifying not only known Gai interactors but also novel Gai effectors. The success of this approach led us to adopt similar methodology to identify Gao-GTP effectors in neuronally differentiated PC12 cells. This screen identified several known interactors, including G protein signaling modulator 1 and 2 (GPSM1/2), Gbg isoforms, and zDHHC palmitoyl transferases. Rap1 GTPase activating protein 1 (Rap1GAP1) was the most enriched protein by the constitutively active Gao subunit (Gao(Q205L)), prompting us to investigate its controversial interaction with Gao. Using a series of biochemical approaches, we identified a stretch of amino acids within the N-terminal helix of the GoLoco motif in Rap1GAP1 that modulate its nucleotide-dependent interaction with Gao at distinct cellular compartments. Our findings further establish the Rap1GAP1a splice variant as a bona fide Gao-GTP effector, while Rap1GAP1b selectively engages Gao-GDP. The study of Gao-effector interactions is particularly important due to disease-associated mutations in the GNAO1 gene that encodes Gao. Our work reveals that a subset of these Gao mutants fail to interact with Rap1GAP1a and are unable to modulate its activity following receptor stimulation. Together, our findings expand the Gai/o interactome and also resolve existing ambiguities regarding Gao-effector interactions. Moreover, they uncover several Golgi-localized proteins enriched by active Gao and provide major insights to understand mechanisms for differential engagement of Rap1GAP1 GoLoco motif variants with Gao-GDP and Gao-GTP subunits. Ultimately, this work emphasizes the continued importance of characterizing Gai/o effectors and highlights defects in Ga effector engagement in the pathology of neurodevelopmental disorders