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

项目摘要 我们的实验室已经在视网膜视杆中使用了光传导通路，这是一种设计精美的感官反应 系统，研究G蛋白偶联受体(GPCRs)如何传播高度放大的信号。这条路 从GPCR视紫质对光子的吸收开始，导致它激活异三聚体G 蛋白转导蛋白通过催化转导蛋白-α亚基(G-T)上的Gdp-GTP交换。Gtp结合的GT 然后，亚基与它们的效应蛋白-环鸟苷酸(CGMP)磷酸二酯酶-6(PDE6)相互作用，a 具有两个催化亚基(pde、pde)和两个亚基(pde)与GT结合的四聚酶。 GT亚基与PDE6结合可激活其将cGMP分解为GMP的能力，从而关闭cGMP门控离子 视网膜视杆细胞膜上的通道并向视神经发送信号。我们确定了 视紫红质-转导蛋白复合体的冷冻电子显微镜(CryoEM)，这与其他人的努力一起 导致了GPCRs如何激活它们的G蛋白伙伴的详细图景。然而，仍然有一个 要了解激活的G蛋白是如何执行其效应蛋白的精确调控的，还有很多事情要做。 最近，我们解决了包含两个gtp结合的GT亚基的溶液中的络合物的低温EM结构 和PDE6，导致了一个描述转导蛋白如何激活其生物效应器的模型。我们现在要测试一下 这一模式的重要方面通过两个广泛的实验目标实现，每个目标都由若干子目标组成： 1)确定激活的视网膜G蛋白转导蛋白的G亚单位如何发挥高度调节的 它们的生物效应因子PDE6的调节。我们将执行：(I)我们开发的荧光读出 监测GT与PDE6的相互作用，(Ii)用二价GT抗体进行研究，使我们能够形成不同的 G-T-PdE_6络合物的不对称构型及(III)电子自旋标记定点自旋探针 以测试我们的模型，了解两个GT亚基如何激活PDE6，以及(Iv)确定 该模型与RGS9如何停用信号传播是一致的。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 效应器。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 通过GT促进PDE6的激活，以及(Ii)FRET检测PDE6上的PDE亚基在 膜环境中GTP结合的G-T的存在和缺失。我们还将：(Iii)重组GT- 刺激纳米盘中的PDE6活性，以及(Iv)单独进行PDE6的结构测定并与其结合 GT，以在更具生理学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张全面的机制图，说明激活的G蛋白是如何调节其 光传导中的生物效应器，以及当刺激停止时该信号如何迅速终止， 以及对其他GPCR-感官反应所必需的关键步骤提供根本上重要的见解。

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.