Architecture of inhibitory G protein signaling in the hippocampus

摘要 抑制性G蛋白依赖信号的失调，包括由GABAB受体控制的信号 (GABR)，与许多神经疾病和疾病有关。在海马区(HPC)，GABABR 通过激活G蛋白门控的内向整流钾(GIRK/Kir3)通道来发挥其抑制作用 并通过抑制腺酰环化酶(AC)。我们已经证明了GABABR-GIRK信令在HPC中是被调制的 由R7亚家族的G蛋白信号转导(RGS)蛋白调节，从而缩短计时和抑制 这一信号通路的敏感性。R7 RGS亚家族包括RGS6和RGS7，它们形成稳定的 G--GIRK复合体-GABABR--GIRK基因突变 信号，破坏突触的可塑性和HPC依赖的行为。R7 RGS/G5复合体也可以 与接头蛋白相关，包括小棕榈酰化蛋白R7BP和孤儿C类GPCR (GPR158)。我们最近的工作提出了一个有趣的前景，即离散的R7RGS/G5复合体与 R7BP和GPR158在GABABR信号到GIRK通道或AC的选择性路由中起着至关重要的作用。 这些发现支持了我们的中心假设，即R7RGS/G5复合体及其适配器协调 抑制性“信号小体”的组装--受体、G蛋白、RGS的不同物理和/或功能阵列 以及相关的蛋白质和效应器--以确保效应器酶和效应器的专用和选择性调节 离子通道由GABABR提供。为了检验这一创新假设，我们提出了一个由两个学科组成的多学科项目 目的：1)明确介导GABABR-效应信号动力学和区划的机制。我们 将在HPC锥体神经元中使用功能丧失和功能获得/挽救的遗传操作，以及 GABABR效应器信号的电生理和实时光学评估，以测试 离散的R7RGS/G5复合体及其接头，与其抑制性G蛋白底物协同工作， 协调不同和专用的GABABR-GIRK和GABABR-AC信号体的组装。2) 询问GABABR-GIRK信号体的组织。利用从我们最近的 RGS7/G5的晶体结构和相关的预测模型，我们将使用基于细胞的生化和 光学报告分析，以及正交生化重建方法，以检验相关假设 GABABR-GIRK信号体元件之间的相互作用和界面。对GABABR的结构性见解- 然后将利用GIRK信号体组装来探索特定相互作用的功能相关性。 摘要：我们的努力将揭示R7RGS/G5复合体和适配器是否协调选择性信号 GABR和它的效应器之间。该项目将产生新的机械洞察功能和 GABABR效应信号的物理划分，可用于设计新的知识 神经疾病和以异常GABABR依赖为特征的疾病的治疗干预 发信号。

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.