Rhodopsin endocytic trafficking and Drosophila visual sensitivity

描述（由申请人提供）：拟议研究的长期目标是揭示G蛋白偶联受体（GPCR）内吞和胞内后转运的生理功能和体内机制。GPCR是最大的膜受体家族，其接收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减少了细胞表面受体的数量，是对受体信号传导的重要反馈调节。除了被分选到溶酶体中进行降解外，内吞的GPCR更频繁地再循环回到质膜。许多受体需要内吞和再循环的过程才能从结合配体解离，以便它们能够接受新的刺激。这一过程的失败与药物耐受性有关，如吗啡。虽然大量的工作已经引出了培养细胞中受体内吞的各种机制，但在完整生物体中对GPCR内吞的研究仍然有限。更重要的是，目前还不清楚内吞的受体如何再循环回到细胞表面。此外，内吞作用和再循环的具体生理功能尚未确定每一个GPCR。果蝇眼睛中的主要光受体Rh1视紫红质是GPCR信号转导和调控的遗传表征的模型分子。最近，我们确定了编码CUB和LDLa结构域蛋白（CULD）的基因的无效突变体，并发现大量的内吞Rh1蛋白保留在突变光感受器的细胞体中。我们的初步研究表明，这是由于Rh1回收失败。我们建议利用这个culd突变体和其他几个新的突变苍蝇研究的机制和调节Rh1的内吞和回收，并表征其对视觉感觉功能的影响。使用分子遗传学，生物化学和电生理学方法的组合，我们将1。确认CULD蛋白是Rh1在光感受器2中再循环所需的。检验CULD与Arr 1相互作用以定位Rh1在横纹肌3中的假设。检验CULD的缺失损害光感受器中光敏感性的发展的假设4。检验Rh1内吞作用中涉及到Rh2的假设5。检验Rh1的去糖基化限制其内吞作用的假设6筛选参与Rh1再循环的其他分子。公共卫生相关性：细胞膜上的G蛋白偶联受体（GPCR）蛋白介导>80%的跨膜信号传导活性，并且是药物设计的主要靶标。在这个建议中，我们计划使用果蝇视紫红质Rh1，光刺激的GPCR，作为一个模型，从遗传学上表征的受体内吞和回收的机制。这些过程调节GPCR信号传导的强度，并且已经涉及临床病症，例如视网膜变性和阿片耐受性。

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.