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.

该研究计划的长期目标是阐明基础机制杆和锥形感受器中的透明蛋白信号传导。虽然对跨多霉素如何在光转导级联反应中的功能，机制杆光感受器（RPS）中透明蛋白-α（GαT1）的折叠的基础知识仍然很差。蛋白质折叠的缺陷是永久性变性和失明的常见原因。这突显了需要研究关键感光蛋白在内的折叠机制，包括转霉素。有证据表明，被称为抗胆碱酯酶抑制剂的蛋白质8同源物A （RIC8A）是跨核素所属的GαI/O家族G蛋白α-亚基的伴侣。基于我们发现RIC8A以RPS表示，我们假设RIC8A是新的伴侣合成和/或光转分配的GαT1。为了阐明RIC8A链酮的机制活性，我们将研究GαT1和RIC8A之间复合物的结构和特性。这溶液中GαT1-RIC8A复合物的结构将由小角度X射线散射确定（SAXS），使用GαT1和RIC8A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 RIC8A的结构，单独使用GαT1复合体。 GαT1-RIC8A复合物的结构信息将作为蛋白质界面和生化分析的突变和生化分析的起点 RIC8A的伴侣活动。我们开发了一个鼠标模型，其中RIC8A专门在 RPS（RIC8AF/FCRE+），我们的初步数据支持RIC8A作为GαT1伴侣的作用。并联通过结构研究，我们将通过进行RIC8A的功能意义来通过进行RIC8A的功能意义全面检查该鼠标模型。此外，我们将研究RIC8A的潜在作用作为杆双极细胞（RBC）中GαO的伴侣。阐明GαT1折叠的分子细节 RIC8A有望对残余疾病具有重要影响，并加深我们对 G蛋白伴侣机械更普遍。拟议研究的第二个主要重点是关于RPS和RBC之间突触传播的机制，光转透明素。基于我们最初发现CAV1.4 Ca2+通道激活的基于转霉素- （Gβ1γ1）在HEK293T细胞中，我们假设Gβ1γ1调节RP-RBC突触处的信号传导。这 Gβ1γ1调节RP输出的基础机制将在生化和使用体外和体内方法的电生理实验。提出的分析预期通过转霉素调制突触会导致深远的范式转移，从而扩大了从视觉信号的产生到RP输出的调制。

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

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

Transducin Partners Outside the Phototransduction Pathway.

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

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