Mechanisms of Cone Pigment Deactivation

锥体颜料失活的机制

• 批准号: 7033295
• 负责人: CHERYL Mae CRAFT
• 金额: $ 36.66万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7033295
• 关键词: arrestins; biochemistry; cell surface receptors; cone cell; electrophysiology; gene deletion mutation; gene targeting; genetically modified animals; high performance liquid chromatography; immunoprecipitation; laboratory mouse; phenotype; phosphorylation; protein binding; protein isoforms; protein protein interaction; protein structure function; retinoids; rhodopsin; synapses; visual phototransduction; visual pigments; yeast two hybrid system

DESCRIPTION (provided by applicant): Both rod and cone photoreceptors absorb light, triggering an amplification cascade, which produces membrane hyperpolarization through closure of selective ion channels. Radiating from the light-initiated event is a G-protein-coupled response of secondary activities, including rhodopsin or cone opsin receptor shut-off through a GRK1 phosphorylation and subsequent binding of either rod or cone arrestin. Cone photoreceptors are distinct from rods in morphology, light sensitivity, recovery rate, thermal stability, timing of outer segment shedding and resistance to programmed cell death by apoptosis. Characterization of the dynamic interactions and functions of these cone gene and their gene products may provide a basis for diagnosis, treatment or prevention of age related macular degeneration and other retinal rod and cone degenerations, thus preserving vision for currently untreatable forms of blindness. To address the distinct aspects inherent to the cone photo-transduction pathway and to accomplish our goals, experiments are designed to explore the function(s) of cone arrestin (CAR), its targeted G protein-coupled receptors (S and M opsin pigments) and other potential relevant partners in the cone synapse. Our working hypothesis, based in part on our ongoing biochemical and electrophysiological studies, support a role for CAR in regulating cone photo-transduction through binding to light-activated, GRK1 phosphorylated S and M opsins. We propose that when this X-chromosomal gene encoding CAR is genetically deleted with mouse knockout (KO) technology, a defective receptor shut-off will lead to a delayed recovery of cone photoresponses. To test this hypothesis, the specific aims and experimental design include 1) characterization of the morphological, biochemical and electrophysiological retinal phenotypes of the newly generated Car KO. Further experiments will explore these parameters in Grk1/Car double KO mice on two genetic backgrounds (transducin alpha -/- with normal rod morphology but no rod response and Nrl -/- with pure cone retina) compared to wildtype; 2) examine the effects of Grk1 S and M opsin phosphorylation and CAR binding on the cone visual retinoid cycle pathway; and 3) identification of other potential interacting cone synaptic partners for CAR and its alternatively spliced isoforms. Studies of the photo-transduction cascade and the molecular triggers for initiation and termination of high acuity vision are vital for sustaining lifelong vision.

描述(申请人提供)：杆状和锥状感光器都吸收光线，触发放大级联，通过关闭选择性离子通道产生膜超极化。从光引发的事件中辐射的是G蛋白偶联的二级活性反应，包括视紫红质或视锥视蛋白受体通过GRK1磷酸化和随后与视杆或视锥arrestin的结合而关闭。锥状光感受器在形态、光敏感度、恢复率、热稳定性、外段脱落的时间和抵抗细胞程序性死亡等方面与杆状感受器不同。鉴定这些视锥基因及其基因产物的动态相互作用和功能，可能为诊断、治疗或预防老年性黄斑变性和其他视网膜视杆细胞和视锥细胞变性提供基础，从而为目前无法治疗的失明保留视力。为了解决视锥细胞光转导通路中固有的不同方面，并实现我们的目标，我们设计了实验来探索视锥细胞突触中视锥蛋白(CAR)及其靶向G蛋白偶联受体(S和M视蛋白色素)以及其他潜在的相关合作伙伴的功能(S)。我们的工作假说部分基于我们正在进行的生化和电生理研究，支持CAR通过与光激活的GRK1磷酸化的S和M opsins结合来调节锥体光传导。我们提出，当这个编码CAR的X染色体基因用小鼠敲除(KO)技术被遗传缺失时，一个有缺陷的受体关闭将导致锥体光反应的延迟恢复。为了验证这一假设，具体的目标和实验设计包括：1)表征新生成的CAR KO的视网膜形态、生化和电生理表型。进一步的实验将探索GRK1/CAR双KO小鼠在两种遗传背景下(转导蛋白α-/-具有正常的视杆细胞形态但没有视杆反应，以及纯视锥视网膜的NRL-/-)与野生型相比的这些参数；2)检测Grk1、S和Mopsin的磷酸化以及CAR结合对视锥视黄醇周期通路的影响；3)鉴定CAR及其替代剪接异构体的其他潜在相互作用的视锥突触伙伴。研究高锐度视觉的光转导级联反应和分子触发机制对维持终身视力至关重要。