Probing the molecular mechanisms that regulate key steps in the GPCR-sensory response pathway responsible for vision in dim light

探索调节负责弱光视觉的 GPCR 感觉反应通路关键步骤的分子机制

• 批准号: 10635707
• 负责人: RICHARD A. CERIONE
• 金额: $ 37.92万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10635707
• 关键词: 3-Dimensional; Address; Antibodies; Binding; Biochemical; Biological; Biological Assay; C-terminal; Catalysis; Catalytic Domain; Complex; Coupled; Coupling; Cryoelectron Microscopy; Cyclic GMP; Disease; Electron Spin Resonance Spectroscopy; Environment; Enzymes; Eye diseases; Fluorescence; Fluorescence Resonance Energy Transfer; G-Protein Signaling Pathway; G-Protein-Coupled Receptors; GTP Binding; GTP-Binding Proteins; Goals; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Ion Channel Gating; Label; Laboratories; Learning; Light; Link; Liposomes; Membrane; Membrane Lipids; Modeling; Molecular Probes; Monitor; Noise; Optic Nerve; Output; Pathway interactions; Photons; Photoreceptors; Phototransduction; Physiologic pulse; Physiological; Play; Proteins; Regulation; Retina; Retinal Degeneration; Rhodopsin; Rod Outer Segments; Role; Sensory; Sensory Receptors; Signal Pathway; Signal Transduction; Site; Structure; System; Testing; Time; Transducin; Vision; Vision Disorders; Work; absorption; alpha Subunit Transducin; clinically relevant; design; insight; nanodisk; novel; novel therapeutic intervention; phosphodiesterase 6; phosphoric diester hydrolase; reconstitution; response; retinal rods; three dimensional structure

Project Abstract Our laboratory has used the phototransduction pathway in retinal rods, a beautifully designed sensory response system, to study how G protein coupled receptors (GPCRs) propagate highly amplified signals. This pathway starts with the absorption of a photon by the GPCR rhodopsin, resulting in its activation of the heterotrimeric G protein transducin by catalyzing GDP-GTP exchange on the transducin-alpha subunit (GT). GTP-bound GT subunits then interact with their effector protein, the cyclic GMP (cGMP) phosphodiesterase-6 (PDE6), a tetrameric enzyme with two catalytic subunits (PDE, PDE) and two subunits (PDE) that bind GT. Binding of GTP-bound GT subunits to PDE6 activates its ability to hydrolyze cGMP to GMP, thus closing cGMP-gated ion channels in retinal rod membranes and sending a signal to the optic nerve. We determined structures for the rhodopsin-transducin complex by cryo-electron microscopy (cryoEM), which together with efforts from other laboratories, led to a detailed picture of how GPCRs activate their G protein partners. However, there is still a great deal to learn about how activated G proteins execute a precise regulation of their effector proteins. Recently, we solved a cryoEM structure for a complex in solution that contains two GTP-bound GT subunits and PDE6, leading to a model describing how transducin activates its biological effector. We will now test important aspects of this model through two broad experimental aims, each comprised of a number of sub-aims: 1) Determine how activated G subunits of the retinal G protein transducin exert a highly tuned regulation of their biological effector PDE6. We will perform: (i) fluorescence read-outs we developed to monitor GT-PDE6 interactions, (ii) studies with a bivalent GT antibody that enables us to form different asymmetric configurations of GT-PDE6 complexes and (iii) site-directed spin probe labeling with electron spin resonance spectroscopy, to test our model for how two GT subunits activate PDE6, as well as (iv) determine if the model is consistent with how RGS9 deactivates signal propagation. 2) Establish a mechanistic basis for how a membrane environment influences the ability of the retinal G protein to activate its biological effector. We will use: (i) fluorescence read-outs to monitor GT-PDE6 interactions to determine how membranes facilitate PDE6 activation by GT, and (ii) FRET to examine the orientation of the PDE subunits on PDE6 in the presence and absence of GTP-bound GT in a membrane environment. We will also: (iii) reconstitute GT- stimulated PDE6 activity in nanodiscs, and (iv) undertake structure determinations of PDE6 alone and bound to GT, to test our model for PDE6 activation in a more physiological setting. The results of these studies will enable us to further develop a comprehensive mechanistic picture for how an activated G protein regulates its biological effector in phototransduction, and how this signal is rapidly terminated when its stimulation has ceased, as well as provide fundamentally important insights into key steps essential for other GPCR-sensory responses.

项目摘要 我们的实验室使用了视网膜杆中的光转导通路，这是一种设计精美的感官反应 系统，研究 G 蛋白偶联受体 (GPCR) 如何传播高度放大的信号。 首先，GPCR 视紫红质吸收光子，从而激活异三聚体 G 通过催化转导蛋白-α 亚基 (GT) 上的 GDP-GTP 交换来实现蛋白质转导蛋白。 然后，亚基与其效应蛋白、环 GMP (cGMP) 磷酸二酯酶 6 (PDE6) 相互作用， 具有两个催化亚基（PDE、PDE）和两个与 GT 结合的亚基（PDE）的四聚酶。 GTP 与 PDE6 结合的 GT 亚基激活其将 cGMP 水解为 GMP 的能力，从而关闭 cGMP 门控离子 我们确定了视网膜杆膜中的通道并向视神经发送信号。 通过冷冻电子显微镜（cryoEM）观察视紫红质转导蛋白复合物，与其他人的努力一起 实验室详细了解了 GPCR 如何激活其 G 蛋白伴侣。 了解激活的 G 蛋白如何对其效应蛋白进行精确调节有很多意义。 最近，我们解决了溶液中复合物的冷冻电镜结构，该复合物包含两个 GTP 结合的 GT 亚基 和 PDE6，产生一个描述转导蛋白如何激活其生物效应器的模型，我们现在将进行测试。 该模型的重要方面通过两个广泛的实验目标来实现，每个目标都包含许多子目标： 1) 确定视网膜 G 蛋白转导蛋白的活化 Gα 亚基如何发挥高度调节作用 我们将执行：（i）我们开发的荧光读数。 监测 GαT-PDE6 相互作用，(ii) 使用二价 GαT 抗体进行研究，该抗体使我们能够形成不同的 GT-PDE6 复合物的不对称构型和 (iii) 使用电子自旋进行定点自旋探针标记 共振光谱，测试我们的模型，了解两个 GT 亚基如何激活 PDE6，以及 (iv) 确定是否 该模型与 RGS9 停用信号传播的方式一致 2) 建立机制基础。 膜环境如何影响视网膜 G 蛋白激活其生物活性的能力 我们将使用：(i) 荧光读数来监测 GT-PDE6 相互作用，以确定膜如何变化。 促进 GαT 激活 PDE6，以及 (ii) FRET 以检查 PDE6 上 PDEα 亚基的方向 膜环境中存在和不存在 GTP 结合的 GT 我们还将： (iii) 重建 GT-。 刺激纳米圆盘中的 PDE6 活性，并且 (iv) 单独测定 PDE6 并与 GT，在更生理的环境中测试我们的 PDE6 激活模型。这些研究的结果将。 使我们能够进一步开发一个全面的机制图，了解激活的 G 蛋白如何调节其 光转导中的生物效应器，以及当其刺激停止时该信号如何快速终止， 以及为其他 GPCR 感官反应所必需的关键步骤提供根本性的重要见解。