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Molecular Mechanism of Photoreceptor G Protein Signaling

光感受器G蛋白信号转导的分子机制

基本信息

批准号：
10330548
负责人：
Nikolai O Artemyev
金额：
$ 36.98万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-05-01 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10330548
关键词：
Affect Biochemical Biology Blindness Cells Cholinesterase Inhibitors Complex Data Defect Electrophysiology (science)Enzymes Family GTP-Binding Protein alpha Subunits GTP-Binding Proteins Generations Glean Goals Homologous Gene Human In Vitro Knock-out Knockout Mice Light Mediating Medical Modeling Molecular Molecular Chaperones Morphology Mutation Analysis Nature Outcome Study Output Photoreceptors Phototransduction Point Mutation Property Proteins Regulation Research Resistance Resolution Retina Retinal Cone Retinal Degeneration Retinal Diseases Rhodopsin Rod Roentgen Rays Role SNAP receptor Signal Pathway Signal Transduction Signaling Protein Structure Synapses Synaptic Transmission System Testing Transducin Vision Disorders Visual X-Ray Crystallography alpha Subunit Transducin base chaperone machinery crosslink experimental study in vivo insight mouse model phosphodiesterase 6 photoreceptor degeneration presynaptic programs protein folding response retinal rods transmission process

项目摘要

The long-term goal of this research program is to elucidate the molecular mechanisms underlying transducin signaling in rod and cone photoreceptors. Although a remarkable level of understanding of how transducin functions in the phototransduction cascade has been achieved, the mechanisms underlying the folding of transducin-α (Gαt1) in rod photoreceptors (RPs) remains poorly understood. Defects in protein folding are a common cause of retinal degeneration and blindness; this underscores the need to investigate the folding mechanisms of key photoreceptor proteins including transducin. Evidence has emerged that the protein known as resistance to inhibitors of cholinesterase 8 homolog A (Ric8A) is a chaperone of G-protein α-subunits of the Gαi/o family, to which transducin belongs. Based on our finding that Ric8A is expressed in RPs, we hypothesize that Ric8A is a chaperone for newly synthesized and/or light-translocated Gαt1. In order to elucidate the mechanism of Ric8A chaperone activity, we will investigate the structure and properties of the complex between Gαt1 and Ric8A. The structure of the Gαt1-Ric8A complex in solution will be determined by small angle X-ray scattering (SAXS), using atomic models of Gαt1 and Ric8A as a framework, and distance constraints derived from cross-linking experiments. In parallel, X-ray crystallography will be used to determine high-resolution structures of Ric8A, alone and in complex with Gαt1. Structural information on the Gαt1-Ric8A complex will serve as a starting point for mutational and biochemical analyses of both the protein interface and the chaperone activity of Ric8A. We developed a mouse model in which Ric8A is knocked out specifically in RPs (Ric8AF/FCre+), and our preliminarily data support a role for Ric8A as a Gαt1 chaperone. In parallel with the structural studies, we will investigate the functional significance of Ric8A in RPs by conducting comprehensive examination of this mouse model. In addition, we will examine the potential role of Ric8A as a chaperone of Gαo in rod bipolar cells (RBCs). Elucidation of molecular details of Gαt1 folding by Ric8A is expected to have important implications for retinal diseases and to deepen our understanding of the G-protein chaperone machinery more generally. A second major focus of the proposed research is on the mechanisms whereby synaptic transmission between RPs and RBCs is modulated by light- translocated transducin. Based on our initial finding of Cav1.4 Ca2+ channel activation by transducin- (Gβ1γ1) in HEK293T cells, we hypothesize that Gβ1γ1 modulates signaling at the RP-RBC synapse. The mechanisms underlying the modulation of RP output by Gβ1γ1 will be investigated in biochemical and electrophysiological experiments using in vitro and in vivo approaches. The proposed analysis of synapse modulation by transducin is expected to cause a profound paradigm shift, expanding the role of transducin from the generation of visual signals to the modulation of the RP output.

这项研究计划的长期目标是阐明潜在的分子机制视杆和视锥光感受器中的转导蛋白信号传导。尽管对人类的理解如何转导功能的光转导级联已经实现，机制在视杆细胞感光器（RPs）中转导素-α（Gαt1）的折叠机制仍知之甚少。蛋白质折叠缺陷是视网膜变性和失明的常见原因;这强调了需要研究包括transducin在内的关键感光蛋白的折叠机制。有证据表明，已知的抗胆碱酯酶抑制剂的蛋白质8同源物A （Ric 8A）是Gαi/o家族的G蛋白α亚基的伴侣蛋白，转导素属于该家族。基于我们发现Ric 8A在RPs中表达，我们假设Ric 8A是新的RPs的伴侣。合成和/或光易位的Gαt1。为了阐明Ric 8A分子伴侣的作用机制，活性的基础上，研究了Gαt1与Ric 8A形成的复合物的结构和性质。的溶液中Gαt1-Ric 8A复合物的结构将通过小角X射线散射测定（SAXS），使用Gαt1和Ric 8A的原子模型作为框架，以及从交联实验同时，X射线晶体学将用于确定高分辨率 Ric 8A的结构，单独和与Gαt1的复合物。Gαt1-Ric 8A复合物的结构信息将作为蛋白质界面和蛋白质结构的突变和生化分析的起点。 Ric 8A的分子伴侣活性。我们开发了一种小鼠模型，其中Ric 8A被特异性敲除， RPs（Ric 8AF/FCre+），我们的初步数据支持Ric 8A作为Gαt1分子伴侣的作用。并行在结构研究的基础上，我们将通过对Ric 8A的结构和功能的研究，对该小鼠模型的全面检查。此外，我们将研究Ric 8A的潜在作用在视杆双极细胞（RBC）中作为Gαo的伴侣。Gαt1折叠的分子细节解析 Ric 8A有望对视网膜疾病有重要意义，并加深我们对视网膜疾病的理解。 G蛋白分子伴侣机制。拟议研究的第二个主要重点是关于RP和RBC之间的突触传递受光调制的机制- 易位转导素基于我们最初发现的Cav1.4钙通道通过转导蛋白激活，在HEK 293 T细胞中，我们假设Gβ1γ1调节RP-RBC突触处的信号传导。的 Gβ1γ1调节RP输出的潜在机制将在生化和生物化学方面进行研究。使用体外和体内方法进行电生理学实验。拟议的分析预计转导素的突触调节将引起深刻的范式转变，扩大神经元的作用。从视觉信号的产生到RP输出的调制的转导。

项目成果

期刊论文数量（30）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Unique transducins expressed in long and short photoreceptors of lamprey Petromyzon marinus.

DOI：
10.1016/j.visres.2008.07.006
发表时间：
2008-09
期刊：
VISION RESEARCH
影响因子：
1.8
作者：
Muradov, Hakim;Kerov, Vasily;Boyd, Kimberly K.;Artemyev, Nikolai O.
通讯作者：
Artemyev, Nikolai O.

A point mutation uncouples transducin-alpha from the photoreceptor RGS and effector proteins.

点突变使转导蛋白-α 与光感受器 RGS 和效应蛋白解偶联。

DOI：
10.1046/j.1471-4159.2003.02103.x
发表时间：
2003
期刊：
Journal of neurochemistry
影响因子：
4.7
作者：
Natochin,Michael;Artemyev,NikolaiO
通讯作者：
Artemyev,NikolaiO

Transducin Partners Outside the Phototransduction Pathway.

跨核蛋白在光转导途径之外的合作伙伴。

DOI：
10.3389/fncel.2020.589494
发表时间：
2020
期刊：
Frontiers in cellular neuroscience
影响因子：
5.3
作者：
Srivastava D;Yadav RP;Inamdar SM;Huang Z;Sokolov M;Boyd K;Artemyev NO
通讯作者：
Artemyev NO

Dominant negative mutants of transducin-alpha that block activated receptor.

阻断激活受体的转导蛋白-α 的显性负突变体。

DOI：
10.1021/bi060381e
发表时间：
2006
期刊：
Biochemistry
影响因子：
2.9
作者：
Natochin,Michael;Barren,Brandy;Artemyev,NikolaiO
通讯作者：
Artemyev,NikolaiO

Expression and subcellular distribution of UNC119a, a protein partner of transducin α subunit in rod photoreceptors.

UNC119a 的表达和亚细胞分布，UNC119a 是视杆光感受器中转导蛋白 α 亚基的蛋白质伴侣。

DOI：
10.1016/j.cellsig.2012.10.005
发表时间：
2013
期刊：
Cellular signalling
影响因子：
4.8
作者：
Sinha,Satyabrata;Majumder,Anurima;Belcastro,Marycharmain;Sokolov,Maxim;Artemyev,NikolaiO
通讯作者：
Artemyev,NikolaiO