Making a model system for signal transduction and modulation, and study on photosignal transduction with the system.

光信号转导系统是目前研究最多的G蛋白偶联信号转导系统之一，它由感光细胞视紫红质、信号转导蛋白G蛋白以及视紫红质激酶、抑制蛋白、我们首先计划用来自无脊椎动物的视紫红质和来自脊椎动物的G蛋白建立模型系统，由于无脊椎动物视紫红质的光可逆性和与无脊椎动物视紫红质的重构模型系统可以重复使用，我们使用章鱼视紫红质来构建模型系统，在研究过程中，发现章鱼视紫红质与G蛋白参与脊椎动物光信号转导系统transducin中的另一类G蛋白偶联，因此对章鱼光信号转导系统的阐明和表征是研究的重点 关于我们 本课题的主要研究成果如下：1）在章鱼视紫红质的光反应过程中发现了一个新的长寿命光中间体，该中间体是章鱼光信号转导系统中G蛋白的候选偶联物; 2）对章鱼视紫红质的一个N-连接糖链的分子结构进行了解析，发现了一个新的N-连接糖链的分子结构; 3）在章鱼视紫红质的光反应过程中发现了四种不同类型的G-连接糖链的α-亚基，并对它们的结构进行了初步的研究。4）对章鱼G蛋白α亚基的北方印迹分析表明，Gq类蛋白主要在章鱼眼中表达。只有一类来自章鱼眼睛和视叶的G蛋白β亚基。我们成功地用Vaccuro病毒和Sf 9昆虫培养细胞表达了视蛋白。7）我们成功地漂白和重组了章鱼视紫红质，这一技术为提供视紫红质类似物提供了可能。使用章鱼视紫红质重构的囊泡，我们发现两类G蛋白，Gqp和Gip，与光活化的章鱼视紫红质结合。少

The aim of this project research was to elucidate an molecular mechanism of signal transduction and that of modulation of the system.The system for photasignal transduction is one of the most elucidated G-protein coupled signal transduction system, which is composed of photoreceptor rhodopsin, signal transducer G-protein, and several modulator of the system such as rhodopsin kinase, arrestin, and S-modulin.We first planned to make a model system with rhodopsin from invertebrate and G-protein from vertebrate, because an invertebrate rhodopsin in photoreversible and a reconstituted model system with an invertebrate rhodopsin can be used repeatedly.We used an octopus rhodopsin for making the model system.On the course of the study we found that octopus rhodopsin couples with a different class of G-protein from transducin, a G-protein participating vertebrate photosignal transduction system.Thus the study to elucidate and to characterize the octopus photosignal transduction system was main … More ly progressed.The achievements with this project were listed below.1) A new long-lived photointermediate was found in the photoreaction process of octopus rhodopsin.This intermediate is a candidate couples with G-protein on Octopus photosignal transduction system.2) The molecular structure of a N-linked sugar chain of octopus rhodopsin was elucidated and found to be a new one.3) We found four different classed of alpha subunit of G-protein were expressed in octopus eye.An alpha subunit of G-protein in the same class as transducin was not found.4) Northern blot analysis of alpha subunits of octopus G-protein elucidates that Gq class was predominately expressed in the eye of octopus.5) We found, although, only one class of beta subunit of G-protein from octopus eye and optic lobe.6) We succeeded to express opsin protein with vaccuro virus and Sf9 insect cultured cell.7) We succeeded to bleach and to reconstitute octopus rhodopsin.This technique makes it possible to supply an analog pigment of rhodopsin.8) Using a vesicle reconstituted with octopus rhodopsin, we found that two classes of G-protein, Gqp and Gip, coupled with photo-activated octopus rhodopsin. Less