Comparative Study on Molecular Mechanism of Photoreception in the Retina and Pineal Gland

许多脊椎动物有几种类型的感光细胞：视网膜中的视杆和视锥细胞，以及松果体中的松果体细胞。重要的是要了解导致细胞功能分化的分子机制的不同；即视杆细胞用于暗视，视锥细胞用于色觉，松果体细胞用于生物钟的昼夜调节。本项目旨在比较研究三种类型的感光细胞中的光信号转导过程，特别是细胞中感光色素和信号转导G蛋白的结构和功能。[Rod]为了确定转导蛋白α和β亚基(Talpha和Tbetagamma)的脂质修饰的生理作用，我们研究了亚单位相互作用和膜结合的机制。这清楚地表明，Tγ的异戊二烯和甲基通过影响1)Talpha-Tbetagamma偶联Th…来调节光子信号转导的效率2)转导蛋白与细胞膜的相互作用。识别Talpha N-末端脂肪酸结构的单抗对牛视网膜中几乎所有的杆状外节进行了染色，这表明一个杆状细胞含有四种脂肪酸修饰的Talpha的四种异构体。恢复素的靶点是视紫红质激酶。[CONE]锥体和杆状感光色素的低温光谱、激光光解和圆二色谱对比研究表明，锥体色素活性中间体的寿命比视紫红质快得多。这清楚地表明，锥体色素的分子性质是锥体生理光响应的主要决定因素之一。由杆状和锥形色素组成的嵌合突变体的光化学反应表明，螺旋1到3的结构决定了感光色素活性中间体的寿命。[松果体细胞]对光敏松果体中的光子信号转导过程知之甚少。免疫组织化学染色发现腺体内有视蛋白样免疫反应。然后我们分离了编码松果体特异性视蛋白样蛋白的cDNA.11顺式视网膜重建实验显示其对蓝光敏感，因此我们根据松果体视蛋白将其命名为Pinopsin。这是首次证明视蛋白在非视网膜组织中表达。较少

Many vertebrates have several types of photoreceptive cells ; rods and cones in the retina, and pinealocytes in the pineal gland. It is important to understand the difference in molecular mechanism responsible for the diverged cellular functions ; that is, rods for scotopic vision, cones for color vision, and pinealocytes for circadian regulation of biological clock. This project aimed at comparative studies on the photon-signal transduction processes in the three types of photoreceptive cells, particularly at the structures and functions of photoreceptive pigments and signal-transducing G-proteins in the cells.[Rod] To identify the physiological roles of lipid modifications of transducin alpha-and betagamma-subunits (Talpha and Tbetagamma), we studied the mechanism of the subunit interaction and the membrane association. It ws clearly shown that the isoprenyl and methyl groups of Tgamma regulate the efficiency of photon-signal transduction by affecting 1) Talpha-Tbetagamma coupling th … More rough direct contact with alpha-subunit, and 2) the interaction between transducin and cellular membranes. Monoclonal antibodies recognizing the structure of the N-terminal fatty acid of Talpha stained almost all of the rod outer segments in a bovine retina, suggesting that a single rod cell contains the four isoforms of Talpha modified with four kinds of fatty acids. The target of recoverin was shown to be rhodopsin kinase. The complex of calcium-recoverin-rhodopsin kinase was stabilized by the membrane association, by which the kinase activity was effectively inhibited.[Cone] Comparative studies on cone and rod photoreceptive pigments by low temperature spectroscopy, laser flash photolysis, and circular dichroism showed that the life times of the active intermediates of cone pigments are much faster than that of rhodopsin. This clearly indicates that the molecular property of cone pigment is one of the major determinants for physiological photoresponse of cones. Photochemical reactions of chimeric mutants composed of rod-and cone-type pigments demonstrated that the structures of helices 1 through 3 determines the life time of the active intermediate of photoreceptive pigments.[Pinealocytes] Little is known about the photon-signal transduction process in a photosensitive pineal gland. We found opsin-like immunoreaction in the gland by immunohistochemistry. Then we isolated the cDNA encoding a pineal specific opsin-like protein. Reconstitution experiment with 11-cis-retinal revealed its blue-light sensitivity, and thus we named it "pinopsin" after pineal opsin. This is the first demonstration of an opsin expressed in non-retinal tissues. Less