Molecular Mechanism of Photoreceptor G Protein Signaling

描述(申请人提供)：该计划的长期目标是研究脊椎动物感光细胞中转导蛋白(GT)信号的分子机制。对杆状转导蛋白GT1在光传导过程中的性质和调节的了解远远超过对锥形转导蛋白GT2的了解。目前尚不清楚视杆细胞和视锥细胞转导蛋白α(GAT1)亚单位的保守序列差异是否转化为导致视杆细胞和视锥细胞明显不同的生理学的功能差异。我们推测，由于Gat N端和结构域间界面的保守差异，锥体GT2被光激色素激活的效率低于杆状GT1，并且Gat2介导的cGMP-光二酯酶6的激活作用弱于杆状体内的效应器酶激活。为了验证这一假设，我们将对Gat2和异三聚体锥体转导蛋白Gat2？3？8进行详细的鉴定和突变分析。该分析将基于一种新的细菌表达程序。这些研究将促进我们对区别视锥细胞和视杆细胞中光传导的分子机制的理解。转导蛋白生物学和信号转导的另一个模糊方面涉及转导蛋白在杆状体内和外节段之间的双向运输机制，光依赖的区隔的决定因素，以及光感受器膜上的迁移率。我们将探索转导蛋白/视紫红质相互作用和脂质修饰在转导蛋白靶向、膜迁移率和盘间转移中的作用，这些作用是通过在杆状感光器中表达突变的绿色荧光蛋白融合的Gat1亚基的转基因非洲爪蛙实现的。突变的Gat1模型将通过EGFP成像、免疫荧光和光漂白后荧光恢复(FRAP)横向和纵向扩散分析进行检查。这项拟议的研究将为光感受器细胞中外周膜蛋白的运输和迁移提供重要的见解。与公共健康相关：光感受器GTP结合蛋白，转导蛋白，是视觉中的关键信号分子。锥体转导蛋白的功能特性以及视锥转导蛋白和视杆转导蛋白信号的差异在很大程度上是未知的。这些研究将使我们对视锥转导蛋白的功能和调控有一个新的认识，并促进我们对视锥转导蛋白和视锥转导蛋白的分子机制的理解。这项研究还将为光感受器细胞中转导蛋白的运输和迁移提供重要的见解。

DESCRIPTION (provided by applicant): The long-term objective of this program is to investigate molecular mechanisms of transducin (Gt) signaling in the vertebrate photoreceptor cells. The properties and regulation of rod transducin Gt1 during phototransduction are understood to a far greater extent than those of cone transducin Gt2. It remains unknown whether the conserved sequence differences in rod and cone transducin-a (Gat1) subunits translate into the functional differences that contribute the remarkably distinct physiology of rods and cones. We hypothesize that because of conserved differences within the Gat N-termini and the interdomain interfaces, cone Gt2 is activated by photoexcited pigment with a lower efficiency than rod Gt1,and the Gat2-mediated stimulation of cGMP-phoshodiesterase 6 is less potent in comparison to the effector enzyme activation in rods. In order to test this hypothesis, we will carry out detailed characterization and mutational analysis of Gat2 and heterotrimeric cone transducin Gat2¿3?8. The proposed analysis will be based on a newly established procedure for bacterial expression of human Gat2. These studies will advance our understanding of the molecular mechanisms that distinguish phototransduction in cones and rods. Another obscure aspect of transducin biology and signaling involves the mechanisms of its bi-directional transport between the inner and outer segments in rods, the determinants of light-dependent compartmentalization, and mobility on photoreceptor membranes. We will explore the roles of transducin/rhodopsin interactions and lipid modifications in transducin targeting, membrane mobility and interdisc transfer using transgenic Xenopus laevis expressing mutant EGFP-fused Gat1 subunits in rod photoreceptors. The mutant Gat1 models will be examined with EGFP imaging, immunofluorescence, and Fluorescence Recovery After Photobleaching (FRAP) analysis of lateral and longitudinal diffusion. The proposed research will provide important insights into transport and mobility of peripheral membrane proteins in photoreceptor cells. PUBLIC HEALTH RELEVANCE: Photoreceptor GTP-binding proteins, transducins, are the key signaling molecules in vision. Functional properties of cone transducin and the differences in signaling of cone and rod transducins are largely unknown. The proposed studies will yield a new level of understanding the function and regulation of cone transducin and advance our understanding of the molecular mechanisms responsible for the markedly distinct physiology of cones and rods. This research will also provide important insights into the transport and mobility of transducin in photoreceptor cells.