Architecture of inhibitory G protein signaling in the hippocampus

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SUMMARY Dysregulation of inhibitory G protein-dependent signaling, including signaling controlled by the GABAB receptor (GABABR), is implicated in many neurological disorders and diseases. In the hippocampus (HPC), GABABR exerts much of its inhibitory influence by activating G protein-gated Inwardly Rectifying K+ (GIRK/Kir3) channels and by inhibiting Adenylyl Cyclase (AC). We have shown that GABABR-GIRK signaling in the HPC is modulated by Regulator of G protein Signaling (RGS) proteins of the R7 sub-family, which sharpen the timing and dampen the sensitivity of this signaling pathway. The R7 RGS sub-family includes RGS6 and RGS7, which form stable complexes with G5. Genetic ablation of RGS7/G5 complexes in mice profoundly alters GABABR-GIRK signaling, disrupting synaptic plasticity and HPC-dependent behaviors. R7 RGS/G5 complexes can also associate with adaptor proteins, including the small palmitoylated protein R7BP and an orphan Class C GPCR (GPR158). Our recent work suggests the intriguing prospect that discrete R7 RGS/G5 complexes, together with R7BP and GPR158, play an essential role in the selective routing of GABABR signals to GIRK channels or AC. These findings fuel our central hypothesis that R7 RGS/G5 complexes and their adaptors orchestrate the assembly of inhibitory "signalosomes” – distinct physical and/or functional arrays of receptors, G proteins, RGS and related proteins, and effectors – to ensure the dedicated and selective regulation of effector enzymes and ion channels by GABABR. To test this innovative hypothesis, we propose a multi-disciplinary program with two AIMs: 1) Identify mechanisms mediating GABABR-effector signaling dynamics and compartmentalization. We will employ loss- and gain-of-function/rescue genetic manipulations in HPC pyramidal neurons, along with electrophysiological and real-time optical assessments of GABABR-effector signaling, to test the prospect that discrete R7 RGS/G5 complexes and their adaptors, working in concert with their inhibitory G protein substrates, orchestrate the assembly of distinct and dedicated GABABR-GIRK and GABABR-AC signalosomes. 2) Interrogate the organization of the GABABR-GIRK signalosome. Leveraging insights derived from our recent crystal structure of RGS7/G5 and associated predictive modeling, we will use cell-based biochemical and optical reporter assays, as well as orthogonal biochemical reconstitution approaches, to test hypotheses related to interactions and interfaces among GABABR-GIRK signalosome elements. Structural insights into GABABR- GIRK signalosome assembly will then be exploited to probe the functional relevance of specific interactions. Summary: Our efforts will reveal whether R7 RGS/G5 complexes and adaptors orchestrate selective signaling between GABABR and its effectors. This project will yield new mechanistic insights into the functional and physical compartmentalization of GABABR-effector signaling, knowledge that can be used to design novel therapeutic interventions for neurological disorders and diseases characterized by aberrant GABABR-dependent signaling.