Molecular Mechanism of Photoreceptor G Protein Signaling

光感受器G蛋白信号转导的分子机制

• 批准号: 8117514
• 负责人: Nikolai O Artemyev
• 金额: $ 34.83万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8117514
• 关键词: Biochemical; Biology; Conserved Sequence; Cyclic GMP; Diffusion; Dissociation; Enzyme Activation; Fluorescence Recovery After Photobleaching; GTP-Binding Proteins; Goals; Health; Heterotrimeric GTP-Binding Proteins; Human; Image; Immunofluorescence Immunologic; Investigation; Lateral; Light; Lipids; Mediating; Membrane; Membrane Proteins; Modeling; Modification; Molecular; Peripheral; Photoreceptors; Phototransduction; Physiology; Pigments; Procedures; Property; Protein Family; Recombinants; Regulation; Research; Retinal Cone; Rhodopsin; Role; Signal Transduction; Signaling Molecule; System; Testing; Transducin; Transgenic Organisms; Translating; Vertebrate Photoreceptors; Vision; Xenopus; Xenopus laevis; base; insight; member; model design; mutant; programs; protein transport; reconstitution; retinal rods

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.

描述（由申请人提供）：本项目的长期目标是研究脊椎动物感光细胞中转导素（Gt）信号传导的分子机制。视杆细胞转导素Gt 1在光转导过程中的特性和调节比视锥细胞转导素Gt 2的特性和调节了解的程度要大得多。视杆细胞和视锥细胞转导蛋白-a（Gat 1）亚基的保守序列差异是否转化为功能差异仍然未知，这些差异导致视杆细胞和视锥细胞的显著不同的生理学。我们假设，由于保守的差异内的Gat N-末端和域间接口，锥Gt 2被激活的光激发色素的效率低于杆Gt 1，和Gat 2介导的刺激cGMP-磷酸二酯酶6是不太有效的相比，在杆中的效应酶激活。为了验证这一假设，我们将进行详细的表征和突变分析的Gat 2和异源三聚体锥转导Gat 2 <$3？8.拟议的分析将基于新建立的人类Gat 2细菌表达程序。这些研究将推进我们对区分视锥细胞和视杆细胞中光转导的分子机制的理解。转导蛋白生物学和信号传导的另一个模糊方面涉及其在视杆细胞内外节之间的双向运输机制、光依赖性区室化的决定因素以及感光膜上的流动性。我们将探讨转导蛋白/视紫红质的相互作用和脂质修饰的转导蛋白靶向，膜流动性和interdisc转移使用转基因非洲爪蟾表达突变的EGFP融合Gat 1亚基在杆光感受器的作用。突变体Gat 1模型将用EGFP成像、免疫荧光和横向和纵向扩散的光漂白后荧光恢复（FRAP）分析进行检查。该研究将为感光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：感光器GTP结合蛋白，转导蛋白，是视觉的关键信号分子。视锥细胞转导蛋白的功能特性以及视锥细胞和视杆细胞转导蛋白信号转导的差异在很大程度上是未知的。拟议的研究将产生一个新的水平的理解锥转导蛋白的功能和调节，并推进我们的理解的分子机制，负责显着不同的生理锥和杆。这项研究也将提供重要的见解，转运和感光细胞中的运动。