Rhodopsins: from biosynthesis and degradation to unconventional functions

描述(申请人提供)：建议的研究目标是使用果蝇作为动物模型，以揭示视紫红质的生物合成、周转和非经典功能的分子机制。视紫红质由视蛋白和维生素A衍生的发色团组成，后者能感知光线。最常见的视网膜变性形式是由视觉周期(类视黄素周期)缺陷引起的--这是生色团再生所需的一种酶途径。直到最近，人们还认为苍蝇并不使用视觉周期，因为发色团通常不会从光激活的视紫红质中释放出来。然而，一些视紫红质被内化，视蛋白被降解，从而释放发色团。在上一次资助期间，我们发现苍蝇使用视觉周期来再生释放的发色团。目标1中提出的实验旨在区分相互竞争的假说，以解释由这一酶途径缺陷导致的视网膜退化的基础。我们还提出了实验来检验新形成的无脊椎动物周期模型所预测的假说。由于哺乳动物的视蛋白(黑素蛋白)在固有的感光视网膜神经节细胞中的功能似乎更类似于果蝇的视紫红质，而不是杆状和锥形光色素，这些研究还表明，视觉周期可能起到再生黑素所使用的发色团的作用。虽然一些视紫红质正常情况下是内化的，但视紫红质的过度内化和降解发生在各种突变的果蝇身上，这些突变过度激活了光转导级联反应。这似乎是一种限制过度信号传递的反馈机制。第二个目标解决了一个新的假说，该假说将解释异源三聚体G蛋白的不受控制的活性如何导致视紫红质的过度周转。尽管在视网膜中发挥作用的视紫红质是特征最好的受体蛋白之一，但在过去的几年中，一些视黄素在视网膜外表达已经变得明显。然而，人们对它们在视网膜外的作用知之甚少。拟议研究的目标3和4将描述在嗅觉系统和中央大脑的神经元中表达的两种视蛋白的作用，这两种视蛋白与光反应无关。我们建议测试这些视紫红质对动物行为的贡献。拟议中的实验提出了有趣的可能性，即哺乳动物视网膜外Opsins的潜在作用，如Opn3和OPN5，这些蛋白尚未进行遗传分析。为了实现我们的目标，我们建议采用一种结合了遗传学、细胞生物学、电生理、分子和生化技术的多学科方法。这些研究的长期目标是1)揭示由视觉周期缺陷引起的视网膜退行性变的潜在机制，最终目标是发现新的治疗方法，2)揭示神秘的视网膜外视蛋白的作用。 与公共卫生相关：视紫红质是对检测光线至关重要的受体，影响视紫红质的突变会导致常见的视网膜变性。这项拟议工作的重点是利用果蝇作为动物模型的巨大技术优势来揭示视紫红质受损导致视网膜退化的潜在机制，并确定在未知受光功能的细胞中表达的视蛋白的作用。

DESCRIPTION (provided by applicant): The goal of the proposed research is to use the fruit fly, Drosophila melanogaster, as an animal model to unravel the molecular mechanisms underlying the biosynthesis, turnover and non-classical functions of rhodopsins. Rhodopsin is comprised of an opsin protein and a vitamin A-derived chromophore, which senses light. Among the most common forms of retinal degeneration are those that result from defects in the visual cycle (retinoid cycle)-an enzymatic pathway required for regeneration of the chromophore. Until recently it was thought that flies do not employ a visual cycle, since the chromophore does not normally release from photoactivated rhodopsin. However, some rhodopsin is internalized and the opsin gets degraded, thereby releasing the chromophore. During the last funding period, we made the discovery that flies use a visual cycle to regenerate the released chromophore. The experiments proposed in aim 1 are designed to differentiate between competing hypotheses to explain the basis for the retinal degeneration that results from defects in this enzymatic pathway. We also propose experiments to test hypotheses predicted by the newly formulated model for the invertebrate cycle. Since the mammalian opsin (melanopsin) that functions in the intrinsically photosensitive retinal ganglion cells appears to be more akin to Drosophila rhodopsins than to rod and cone photopigments, these studies also suggest that a visual cycle might function to regenerate the chromophore used by melanopsin. Although some rhodopsin is normally internalized, excessive internalization and degradation of rhodopsin occurs in a variety of flies with mutations that hyperactivate the phototransduction cascade. This appears to be a feedback mechanism to limit excessive signaling. The second aim addresses a new hypothesis that would explain how uncontrolled activity of the heterotrimeric G-protein leads to excessive turnover of rhodopsin. Although rhodopsins that function in the retina are among the best-characterized receptor proteins, in the past few years it has become clear that some opsins are expressed outside the retina. However, their extra-retinal roles are understood poorly. Aims 3 and 4 of the proposed research will characterize the roles of two opsins that are expressed in neurons in the olfactory system and central brain and that have not been associated with a light response. We propose to test the contributions of these rhodopsins to animal behaviors. The proposed experiments raise intriguing possibilities as to the potential roles for mammalian extra-retinal opsins, such as Opn3 and Opn5, which have not been subjected to genetic analysis. To accomplish our goals, we propose to employ a multidisciplinary approach using a combination of genetic, cell biological, electrophysiological, molecular and biochemical techniques. The long-term goals of these studies are to 1) uncover mechanisms underlying the retinal degenerations that result from defects in the visual cycle with the ultimate goal of discovering new therapeutic approaches, and 2) uncover the roles of the enigmatic extra-retinal opsins. PUBLIC HEALTH RELEVANCE: Rhodopsin is the receptor that is critically important for detecting light, and mutations affecting rhodopsin lead to common forms of retinal degeneration. The focus of the proposed work is to exploit the great technical advantages of the fruit fly as an animal model to uncover mechanisms underlying the retinal degeneration resulting from impairments in rhodopsins, and to identify roles for opsins that are expressed in cells that are not known to function in light reception.