Rhodopsin endocytic trafficking and Drosophila visual sensitivity

描述（由申请人提供）：拟建研究的长期目标是揭示G蛋白偶联受体（GPCR）内吞作用和内吞后转运的生理功能和体内机制。GPCRs是最大的膜受体家族，它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减少了细胞表面的受体数量，是对受体信号传导的重要反馈调节。除了被分类到溶酶体中进行降解外，内吞的gpcr更频繁地循环回到质膜中。许多受体需要内吞作用和循环过程才能与结合配体分离，从而接受新的刺激。这一过程的失败与药物耐受性有关，例如对吗啡的耐受性。虽然大量的工作已经引出了培养细胞中受体内吞作用的各种机制，但对完整生物体中GPCR内吞作用的研究仍然有限。更重要的是，目前还不清楚内吞受体是如何循环回到细胞表面的。此外，每种GPCR的内吞作用和再循环的具体生理功能尚未确定。果蝇眼的主要光受体Rh1视紫红质是GPCR信号传导和调控的遗传表征模型分子。最近，我们发现了一个编码CUB-和ldla -结构域蛋白（CULD）的基因的零突变，并发现在突变的光感受器的细胞体中保留了大量内吞噬的Rh1蛋白。我们的初步研究表明，这是由于Rh1循环失败造成的。我们拟利用这一突变体和另外几个新的突变体蝇来研究Rh1内吞和再循环的机制和调控，并表征它们对视觉感觉功能的影响。结合分子遗传学，生物化学和电生理学的方法，我们将1。确认CULD蛋白是光感受器2中Rh1循环所必需的。验证CULD与Arr1相互作用导致Rh1定位于横纹肌3的假设。验证CULD的丧失会损害光感受器的光敏发育的假设。验证LAP参与Rh1胞吞作用的假设5。验证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%的跨膜信号活动，是药物设计的主要靶点。在这个提议中，我们计划使用果蝇视紫红质Rh1，一个光刺激的GPCR，作为一个模型来遗传表征受体内吞和循环的机制。这些过程调节GPCR信号的强度，并涉及临床疾病，如视网膜变性和阿片类药物耐受性。

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.