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；视网膜中的视杆细胞和视锥细胞，松果体中的松果体细胞。了解导致细胞功能分化的分子机制差异具有重要意义；也就是说，视杆细胞负责暗视，视锥细胞负责色觉，松果体细胞负责生物钟的昼夜调节。本项目旨在比较研究三种类型的光敏细胞的光子信号转导过程，特别是光敏色素和信号转导g蛋白在细胞中的结构和功能。[Rod]为了确定转导素α和β - γ亚基（Talpha和Tbetagamma）的脂质修饰的生理作用，我们研究了亚基相互作用和膜关联的机制。结果表明，γ的异戊烯基和甲基通过影响α -亚基与α - γ的偶联以及α -亚基与α - γ的直接接触和α - γ与细胞膜的相互作用来调节光子信号转导效率。识别Talpha n端脂肪酸结构的单克隆抗体染色了牛视网膜中几乎所有的杆状细胞外段，表明单个杆状细胞含有四种被四种脂肪酸修饰的Talpha的四种同工型。恢复蛋白的靶点是视紫红质激酶。钙-恢复-视紫红质激酶复合体通过膜结合得到稳定，从而有效地抑制了激酶活性。[锥]通过低温光谱、激光闪光光解和圆二色性对锥状和杆状感光色素进行对比研究，发现锥状色素活性中间体的寿命要比视紫红质快得多。这清楚地表明，锥体色素的分子特性是决定锥体生理光响应的主要因素之一。由棒状和锥状色素组成的嵌合突变体的光化学反应表明，螺旋1至3的结构决定了光敏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。我们通过免疫组化发现腺体内有视蛋白样免疫反应。然后我们分离出编码松果体特异性视蛋白样蛋白的cDNA。对11-顺式视网膜进行重组实验，发现其具有蓝光敏感性，因此我们将其命名为pinopsin，以松果体视蛋白命名。这是首次证明视蛋白在非视网膜组织中表达。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