MOLECULAR GENETICS OF RHODOPSIN SENSITIVITY

本申请中提出的研究的目的是获得洞察力 关于视觉信号转导的分子机制 系统。预计这些研究将有助于更好地 了解受体结构、功能、 以及生物系统中的信息处理。 编码四种果蝇视蛋白的基因已经被分离出来并 特征的(Rh1-4)。其中的每一个都在不同的类中表示 并显示了光感受器细胞的特征图案 对光的敏感性。Rh1视蛋白是一种蓝敏感色素 (lambda/max 480 nm)，它在六个外光感受器中表达 细胞(R1-R6)。RH2的光敏性为波长/最大420 nm， 在枕骨细胞中表达。Rh3和Rh4对紫外线敏感， 在非重叠的R7细胞组中表达。 在以前的研究中，我们产生了一系列嵌合的果蝇视蛋白 基因，以确定视蛋白的区域是 参与调节视紫红质敏感性和后视紫红质 吸收。这些嵌合蛋白来自于 蓝色和紫色敏感颜料。表达了嵌合基因 在缺乏NINA E突变动物的R1-R6光感受器细胞中 野生型Rh1基因产物。在这些苍蝇中，嵌合的眼球是 只有光色素在R1-R6感光细胞中表达。我们 检测了表达这些修饰的动物的光谱敏感性 在体内利用生理学技术，并研究了 In对活化形式的色素(变视紫质)的吸收 活体显微分光光度测定法。我们发现视神经的多个区域 蛋白质参与调节视紫红质光谱敏感性和 天然的和活化的色素可以被调谐 独立的。 在本申请中，我们建议1)识别视神经的区域 导致光谱敏感性差异的蛋白质 果蝇Rh1和Rh2的视紫红质。2)确定残留物 视蛋白的第二跨膜片段负责 关于活化形式(变视紫质)吸收的差异 果蝇Rh1和Rh2的视紫红质。3)确定更多地点 在视紫红质中起重要作用的视蛋白 敏感度和功能。4)分离和鉴定编码基因 在果蝇和其他无脊椎动物中发现的新型感光色素。5) 产生并鉴定异位表达的转基因果蝇 来自其他无脊椎动物和脊椎动物的视蛋白。 这些研究将提供对潜在机制的洞察 视紫红质光谱敏感性的调节及其相互关系 视紫红质的结构和光激活过程。这些问题是 是视觉研究的中心，并将提供一个独特的模型系统 了解G蛋白偶联受体是如何发挥作用的。

The aim of the research proposed in this application is to gain insight into the molecular mechanisms of signal transduction in the visual system. It is expected that these studies will contribute to a better understanding of the relationship between receptor structure, function, and information processing in biological systems. The genes encoding four Drosophila opsins have been isolated and characterized (Rh1-4). Each of these is expressed in distinct classes of photoreceptor cells and displays characteristic patterns of sensitivity to light. The Rh1 opsin is a blue sensitive pigment (lambda/max 480nm) which is expressed in the six outer photoreceptor cells (R1-R6). Rh2 displays light sensitivity with lambda/max 420nm and is expressed in the occelli. Rh3 and Rh4 are UV sensitive and are expressed in non-overlapping sets of R7 cells. In previous studies, we generated a series of chimeric Drosophila opsin genes, in order to identify regions of the opsin protein that are involved in regulating rhodopsin sensitivity and metarhodopsin absorption. These chimeric proteins were derived from regions of the blue and violet sensitive pigments. The chimeric genes were expressed in the R1-R6 photoreceptor cells of ninaE mutant animals which lack the wild type Rh1 gene product. In these flies, the chimeric opsins are the only photopigments expressed in the R1-R6 photoreceptor cells. We examined the spectral sensitivity of animals expressing these modified pigments in vivo using physiological techniques, and studied the absorption of the activated form of the pigment (metarhodopsin) by in vivo microspectrophotometry. We found that multiple regions of the opsin protein are involved in regulating rhodopsin spectral sensitivity and that the native and activated forms of the pigment can be tuned independently. In the present application we propose to 1) identify regions of the opsin protein that are responsible for differences in the spectral sensitivity of the Drosophila Rh1 and Rh2 rhodopsins. 2) Identify residues in the second transmembrane segment of the opsin protein that are responsible for differences in the absorption of the activated form (metarhodopsin) of the Drosophila Rh1 and Rh2 rhodopsins. 3) Identify additional sites within the opsin protein that play an important role in rhodopsin sensitivity and function. 4) Isolate and characterize the genes encoding novel photopigments found in Drosophila and other invertebrates. 5) Generate and characterize transgenic flies that ectopically express opsins from other invertebrate and vertebrate organisms. These studies will provide insight into the mechanisms underlying the regulation of rhodopsin spectral sensitivity and the relationship between rhodopsin structure and the process of photoactivation. These issues are central to the study of vision, and will provide a unique model system for understanding how G-protein coupled receptors function.