Rhodopsin endocytic trafficking and Drosophila visual sensitivity

视紫红质内吞运输和果蝇视觉敏感性

• 批准号: 7895552
• 负责人: HONG-SHENG LI
• 金额: $ 41.13万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7895552
• 关键词: ARRB1 gene; Back; Biochemical; Cell membrane; Cell surface; Clinical; Cultured Cells; Development; Disease; Drosophila eye; Drosophila genus; Drug Design; Drug Tolerance; Endocytosis; Failure; Family; Feedback; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; Genes; Genetic; Goals; Growth Factor; Hormones; Ligand Binding; Light; Lysosomes; Mediating; Membrane; Modeling; Molecular Genetics; Morphine; Neuropeptides; Neurotransmitters; Opioid; Organism; Pathway interactions; Pharmacologic Substance; Photophobia; Photoreceptors; Physiological; Process; Proteins; Receptor Signaling; Recycling; Regulation; Research; Retinal Degeneration; Rhodopsin; Sensory; Signal Transduction; Sorting - Cell Movement; Stimulus; Tertiary Protein Structure; Testing; Visual; Work; cytokine; fly; in vivo; mutant; neuronal cell body; public health relevance; receptor; receptor recycling; response; sensory stimulus; trafficking

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to reveal the physiological functions and in vivo mechanisms of G protein-coupled receptor (GPCR) endocytosis and postendocytic trafficking. GPCRs are the largest family of membrane receptors that receive sensory stimuli and mediate responses to neurotransmitters, neuropeptides, hormones, cytokines and growth factors. Activity-dependent endocytosis of GPCR reduces receptor numbers on the cell surface and is an important feedback regulation on the receptor signaling. In addition to being sorted into lysosome for degradation, endocytosed GPCRs are more frequently recycled back to the plasma membrane. The process of endocytosis and recycling is required for many receptors to dissociate from the binding ligand so that they can receive new stimuli. Failure of this process has been implicated in drug tolerances such as that to morphine. Although a large body of works has elicited various mechanisms of receptor endocytosis in cultured cells, the studies on GPCR endocytosis in intact organisms are still limited. More importantly, it is unclear how the endocytosed receptors are recycled back to the cell surface. In addition, the specific physiological functions of endocytosis and recycling have yet to identify for each GPCR. The major light receptor Rh1 rhodopsin in Drosophila eye is a model molecule for genetic characterization of GPCR signaling and regulation. Recently we identified a null mutant of a gene that encodes a CUB- and LDLa-domain protein (CULD), and found that a large amount of endocytosed Rh1 protein was retained in the cell body of the mutant photoreceptor. Our preliminary studies suggest that this is due to a failure of Rh1 recycling. We propose to take advantage of this culd mutant and several additional new mutant flies to study the mechanisms and the regulations of Rh1 endocytosis and recycling, and to characterize their impacts on the visual sensory function. Using a combination of molecular genetic, biochemical and electrophysiological approaches, we will 1. Confirm that the CULD protein is required for the recycling of Rh1 in photoreceptor 2. Test the hypothesis that CULD interacts with Arr1 for the localization of Rh1 in the rhabdomere 3. Test the hypothesis that loss of CULD impairs the development of light sensitivity in photoreceptors 4. Test the hypothesis that LAP is involved in the Rh1 endocytosis 5. Test the hypothesis that the deglycosylation of Rh1 restricts its endocytosis 6 Screen for additional molecules involved in the recycling of Rh1. PUBLIC HEALTH RELEVANCE: G protein-coupled receptor (GPCR) proteins on the cell membrane mediate >80% of transmembrane signaling activities, and are the major targets for pharmaceutical drug designs. In this proposal we plan to use Drosophila rhodopsin Rh1, a light-stimulated GPCR, as a model to genetically characterize the mechanisms underlying the receptor endocytosis and recycling. These processes regulate the intensity of GPCR signaling, and have been implicated in clinical disorders such as retinal degenerations and opioid tolerance.

描述（由申请人提供）：拟议的研究的长期目标是揭示G蛋白偶联受体（GPCR）内吞作用和后内细胞运输的生理功能和体内机制。 GPCR是最大的膜受体家族，可接受感觉刺激，并介导对神经递质，神经肽，激素，细胞因子和生长因子的反应。 GPCR的活性依赖性内吞作用会减少细胞表面的受体数量，并且是受体信号传导的重要反馈调节。除了被分类为溶酶体降解外，内吞GPCR还更常见回到质膜。许多受体需要与结合配体分离的内吞作用和回收的过程，以便它们可以接受新的刺激。该过程的失败与吗啡这样的药物耐受性有关。尽管大量作品引起了培养细胞中受体内吞作用的各种机制，但对完整生物的GPCR内吞作用的研究仍然受到限制。更重要的是，尚不清楚内吞受体如何回收回细胞表面。此外，内吞作用和回收利用的特定生理功能尚未确定每个GPCR。果蝇眼中的主要光受体RH1视紫红质是GPCR信号传导和调节遗传表征的模型分子。最近，我们鉴定了一个编码CUB和LDLA粘蛋白蛋白（CULD）的基因的零突变体，发现大量内吞Rh1蛋白保留在突变体光感受器的细胞体中。我们的初步研究表明，这是由于RH1回收的失败。我们建议利用这种Culd突变体和几个新的新突变蝇，以研究RH1内吞和回收的机制和法规，并表征它们对视觉感觉功能的影响。 Using a combination of molecular genetic, biochemical and electrophysiological approaches, we will 1. Confirm that the CULD protein is required for the recycling of Rh1 in photoreceptor 2. Test the hypothesis that CULD interacts with Arr1 for the localization of Rh1 in the rhabdomere 3. Test the hypothesis that loss of CULD impairs the development of light sensitivity in photoreceptors 4. Test the hypothesis该圈涉及RH1内吞作用5。检验假设RH1的退化限制了其内吞作用6筛选6筛查，以便与RH1回收的其他分子有关。公共卫生相关性：细胞膜介导> 80％的跨膜信号传导活性的G蛋白偶联受体（GPCR）蛋白，并且是药物设计的主要靶标。在此提案中，我们计划使用果蝇Rhodopsin RH1（一种光刺激的GPCR）作为一种模型，以遗传表征受体内吞作用和回收利用的机制。这些过程调节了GPCR信号的强度，并与临床疾病有关，例如视网膜变性和阿片类药物耐受性。