Molecular Mechanism of Photoreceptor G Protein Signaling

描述（由申请人提供）：该项目的长期目标是研究脊椎动物光感受器细胞中转导素（Gt）信号传导的分子机制。杆状转导蛋白Gt1在光导过程中的特性和调控被了解的程度远远大于锥状转导蛋白Gt2。杆状细胞和锥体细胞转导素-a （Gat1）亚基的保守序列差异是否转化为导致杆状细胞和锥体细胞显著不同生理的功能差异，目前尚不清楚。我们推测，由于Gat n端和结构域间界面的保守差异，锥状体Gt2被光激发色素激活的效率低于杆状体Gt1，并且与杆状体中的效应酶激活相比，gat2介导的cgmp -磷酸二酯酶6的刺激更弱。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进行详细的表征和突变分析。提出的分析将基于新建立的细菌表达人Gat2的程序。这些研究将促进我们对区分视锥细胞和视杆细胞光转导的分子机制的理解。转导素生物学和信号传导的另一个鲜为人知的方面涉及其在杆细胞内外段之间的双向运输机制，光依赖性区隔化的决定因素和光感受器膜上的迁移。我们将利用在杆状光感受器中表达egfp融合Gat1亚基突变体的转基因非洲脚，探讨转导蛋白/视紫红质相互作用和脂质修饰在转导蛋白靶向、膜迁移和盘间转移中的作用。突变体Gat1模型将通过EGFP成像、免疫荧光和光漂白后荧光恢复（FRAP）分析进行横向和纵向扩散检测。该研究将为光感受器细胞外周膜蛋白的转运和迁移提供重要的见解。公共卫生相关性：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锥体和杆状转导蛋白的信号传导差异在很大程度上是未知的。这些研究将使我们对视锥细胞转导素的功能和调控的理解达到一个新的水平，并促进我们对视锥细胞和视杆细胞生理差异的分子机制的理解。这项研究也将为光感受器细胞中转导素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.