Molecular Mechanism of Photoreceptor G Protein Signaling

该研究计划的长期目标是阐明潜在的分子机制。 视杆细胞和视锥细胞中的转导信号。尽管人们对这一问题的理解 转导蛋白在光信号转导级联反应中的作用机制 杆状光感受器中的转导蛋白-α(GαT1)的折叠机制目前仍知之甚少。 蛋白质折叠缺陷是视网膜退化和失明的常见原因；这强调了 有必要研究包括转导蛋白在内的关键光感受器蛋白的折叠机制。 有证据表明，被称为对胆碱酯酶8抑制剂具有抵抗力的蛋白质同系物A (RIC8A)是转导蛋白所属的G蛋白α-亚基的伴侣，属于G-αI/O家族。基于 我们发现Ric8A在RPS中表达，我们假设Ric8A是新的伴侣 合成和/或光转位GαT1。为了阐明Ric8A分子伴侣的作用机制 我们将研究GαT1与RIC8A之间的络合物的结构和性质。这个 G-αT1-Ric8A络合物在溶液中的结构将通过小角X射线散射确定 (SAXS)，以GαT1和RIC8A的原子模型为框架，并从 交联性实验。同时，X射线结晶学将用于确定高分辨率 RIC8A的结构，单独和与GαT1的复合体。GαT1-RIC8A复合体的结构信息 将作为突变和生化分析的起点，对蛋白质界面和 Ric8A的伴侣活性。我们开发了一种小鼠模型，在这种模型中，Ric8A被特别地敲除了 RPS(RIC8AF/FCRE+)，我们的初步数据支持RIC8A作为GαT1伴侣的作用。并行的 在结构研究的基础上，我们将通过对RPS中RIC8A的功能研究来探讨其在RPS中的作用。 全面检查这一小鼠模型。此外，我们还将研究Ric8A的潜在作用 作为视杆双极细胞(RBC)中Gαo的伴侣。G-αT1折叠的分子细节 RIC8A有望对视网膜疾病有重要影响，并加深我们对 更广泛地说，G蛋白的伴侣机制。拟议研究的第二个主要重点是 关于RPS和RBC之间的突触传递受光调制的机制-- 转导蛋白移位。基于转导蛋白-激活Cav1.4钙通道的初步发现 (Gβ1γ1)在HEK293T细胞中，我们假设Gβ1γ1调节RP-红细胞突触的信号转导。这个 G-β-1-γ-1调节RP输出的潜在机制将在生化和 电生理实验采用体外和体内两种方法。建议的分析 转导蛋白对突触的调制有望导致一种深刻的范式转变，扩大 从视觉信号的产生到RP输出的调制的转换。

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.