Influence Of Protein/lipid Interactions On Signal Transd

蛋白质/脂质相互作用对信号转导的影响

• 批准号: 6676956
• 负责人: Burton J Litman
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6676956
• 关键词: G protein; biological signal transduction; enzyme activity; ethanol; fluorescence spectrometry; intermolecular interaction; lipid bilayer membrane; lipid structure; membrane structure; phosphodiesterases; phospholipids; physical chemical interaction; posttranslational modifications; protein structure function; receptor coupling; rhodopsin; rod cell; time resolved data; unsaturated fatty acids

G protein-coupled receptors (GPCR) are ubiquitous components of signal transduction pathways, including taste, smell, vision, and many neurotransmitter systems. GPCRs are also targets of a great many pharmaceutical drugs. This project is designed to assess the role of membrane lipid composition, especially polyunsaturated phospholipids, in modulating GPCR signal transduction and to elucidate the mechanism of action of ethanol in these systems. The visual transduction pathway of the retinal rod photoreceptor is the best characterized member of this receptor superfamily and is being used as a model system in these studies. System properties under study include: 1. the kinetics and extent of formation of metarhodopsin II (MII), the G protein activating form of activated rhodopsin; 2. MII/G protein complex formation; 3. the rate of G protein activation; 4. cGMP phosphodiesterase (PDE) activation; and 5. the GTPase activity of the G protein. Both functional measures in the transduction pathway and lipid bilayer physical properties are being investigated. Current studies demonstrate that the kinetics and extent of formation of the MII-G protein complex are dependent on both acyl chain formation and cholesterol content. In particular the kinetic of MII-G protein formation is slowed by 50% in 18:0,18:1PC relative to 18:0,22:6PC. The addition of cholesterol doubles the lag time in complex formation in 18:0,18:1PC, whereas the lag time is essentially unchanged upon the addition of cholesterol to 18:0,22:6PC. In other experiments, it is shown that the PDE activity, a measure of the integrated visual pathway function, is also dependent upon acyl chain composition. Here again, 22:6n-3 phospholipids yield the highest levels of activity. A novel method of varying ROS disk membrane cholesterol content was developed which allowed studying the initial steps in the visual signaling pathway over the range of 5 to 45 mole percent cholesterol. Here again, both the kinetics and level of MII and MII-G complex formation were found to be dependent on cholesterol content. Taken together, these studies indicate that 22:6n-3 containing phospholipids enhance the efficiency of a G protein-coupled signaling system. In a recent collaboration, various steps in the visual signaling pathway were studied in ROS from rats raised on n-3 adequate and deficient diets. Under these conditions, 22:6n-3 is replaced by 22:5n-6. We have found that the ROS from n-3 deficient rats were less sensitive to light stimulation than were the ROS from n-3 adequate rats. These studies provide a basis for understanding the visual and cognitive deficits associated with a nutritional deficiency of n-3 fatty acids.

G蛋白偶联受体（GPCR）是信号转导途径中普遍存在的成分，包括味觉、嗅觉、视觉和许多神经递质系统。gpcr也是许多药物的靶标。本项目旨在评估膜脂组成，特别是多不饱和磷脂在调节GPCR信号转导中的作用，并阐明乙醇在这些系统中的作用机制。视网膜杆状光感受器的视觉转导通路是该受体超家族中最具特征的成员，并且在这些研究中被用作模型系统。所研究的系统特性包括：1。后视紫红质II （metarhodopsin II，活化视紫红质的G蛋白激活形式）的形成动力学和程度；2. MII/G蛋白复合物形成；3. G蛋白活化率；4. cGMP磷酸二酯酶（PDE）活化；和5。G蛋白的GTPase活性。在转导途径中的功能测量和脂质双分子层的物理性质都在研究中。目前的研究表明，MII-G蛋白复合物的形成动力学和程度取决于酰基链的形成和胆固醇含量。特别是在18:0,18:1PC时，与18:0,22:6PC相比，MII-G蛋白形成的动力学减慢了50%。在18:0,18:1PC时，胆固醇的加入使复合地层的滞后时间增加了一倍，而在18:0,22:6PC时，胆固醇的加入使滞后时间基本保持不变。在其他实验中，显示PDE活性，综合视觉通路功能的量度，也依赖于酰基链组成。这里，22:6n-3磷脂产生最高水平的活性。开发了一种改变ROS盘膜胆固醇含量的新方法，可以研究5%至45%摩尔胆固醇范围内视觉信号通路的初始步骤。再一次，MII和MII- g复合物形成的动力学和水平都被发现依赖于胆固醇含量。综上所述，这些研究表明含有磷脂的22:6n-3可提高G蛋白偶联信号系统的效率。在最近的一项合作中，研究人员研究了n-3充足和缺乏饮食的大鼠ROS中视觉信号通路的各个步骤。在这些条件下，22:6n-3被22:5n-6取代。我们发现n-3缺乏大鼠的ROS对光刺激的敏感性低于n-3充足大鼠的ROS。这些研究为理解与n-3脂肪酸营养缺乏相关的视觉和认知缺陷提供了基础。