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: 8317673
• 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/8317673
• 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（由适用提供）：该程序的长期目的是研究脊椎动物感光细胞中跨核素（GT）信号传导的分子机制。在光转导过程中，杆状蛋白GT1的特性和调节比锥体透明蛋白GT2的程度要大得多。尚不清楚杆和锥体透射蛋白A（GAT1）亚基的保守序列差异是否转化为功能差异，这些功能差异有助于杆和锥体的显着不同生理学。我们假设，由于GAT N-末端和域间界面内的差异，锥体GT2被光激活的色素激活，其效率低于ROD GT1，并且GAT2介导的刺激CGMP-磷酸二酯酶6的刺激与效应的效应相比较小。为了检验这一假设，我们将对GAT2和异源三蛋白锥蛋白GAT2¿3？8进行详细的表征和突变分析。拟议的分析将基于一种新建立的人类GAT2的细菌表达的程序。这些研究将促进我们对区分锥体和棒中光转导的分子机制的理解。透明蛋白生物学和信号传导的另一个晦涩的方面涉及杆中内部和外部段之间其双向转运的机制，光依赖性分隔的决定剂以及对光感受器膜的迁移率。我们将使用转基因Xenopus laevis表达突变体EGFP融合的GAT1亚基在棒杆型中，探索透明蛋白/视紫红蛋白相互作用和脂质修饰在跨核素靶向，膜迁移率和间盘转移中的作用。将使用EGFP成像，免疫荧光和光漂白后的荧光恢复（FRAP）分析侧面和纵向差异后，将检查突变的GAT1模型。拟议的研究将为光感受器细胞中周围膜蛋白的运输和迁移率提供重要的见解。公共卫生相关性：光感受器GTP结合蛋白，透明蛋白是视觉中的关键信号分子。锥体透明蛋白的功能特性以及锥体和杆状蛋白信号传导的差异在很大程度上尚不清楚。拟议的研究将产生新的理解锥体转丁蛋白的功能和调节水平，并促进我们对负责锥和杆明显不同生理的分子机制的理解。这项研究还将提供有关跨受体细胞中透明蛋白的运输和迁移率的重要见解。