Momplaisir Nathalie L, Chandan Naincy R, Wang Beiyun, Qu Elaine, Smrcka Alan V
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI.
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI.
J Biol Chem. 2025 Jul 2:110446. doi: 10.1016/j.jbc.2025.110446.
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.
与Gα家族成员偶联的G蛋白偶联受体（GPCRs）是主要的治疗靶点。在异源三聚体G蛋白中，Gα是大脑中最丰富的Gα亚基，但由Gα控制的机制途径尚未完全确立。鉴于最近有报道称一种神经发育障碍（GNAO1脑病）与Gα编码基因突变有关，了解Gα介导的信号通路尤为关键。为了填补这一空白，我们试图在分化的PC12细胞中使用基于邻近性的蛋白质组学筛选来发现新的Gα效应器。我们的分析揭示了一系列潜在的Gα-GTP效应蛋白，包括一种Rap1 GTP酶激活蛋白Rap1GAP1。G蛋白α亚基对Rap1GAP1的调节存在争议，据报道Rap1GAP1通过GoLoco/G蛋白调节（GPR）基序优先与Gα-GDP结合。我们证实Gα-GTP结合并调节Rap1GAP1的活性，并揭示了Rap1GAP1识别Gα亚基的一种新机制，其中GoLoco/GPR基序中关键接触残基的存在与否赋予了对Gα鸟嘌呤核苷酸结合状态的差异识别。我们还表明，病理性GNAO1突变通过阻止活化的Gα亚基获得效应器结合所需的构象来破坏这种功能关系。这些数据解决了文献中关于Gα对Rap1GAP的激活依赖性结合和调节的争议，并有助于将Rap1GAP1确立为一种真正的G蛋白调节效应器。此外，我们的研究发现，与GNAO1脑病相关的Gα中的多个突变体在下游效应器相互作用方面存在缺陷，这可能是该疾病某些表现的基础。
