Influence Of Protein/lipid Interactions On Signal Transd

G蛋白偶联受体是信号转导通路中普遍存在的成分，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成分，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。视网膜视杆感受器的视觉传导通路是该受体超家族中最具特征的成员，并被用作这些研究的模型系统。所研究的系统性质包括：1.活化视紫红质的G蛋白激活形式--后紫红质II(MII)的形成动力学和形成程度；2.MII/G蛋白复合体的形成；3.G蛋白的激活速率；4.cGMP磷酸二酯酶(PDE)的激活；以及5.G蛋白的GTP酶活性。转导途径的功能措施和脂质双分子层的物理性质正在研究中。目前的研究表明，MII-G蛋白复合体的形成动力学和形成程度取决于酰链的形成和胆固醇含量。特别是在18：0、18：1pC时，MII-G蛋白的形成速度比18：0、22：6pC慢了50%。在18：0，18：1的情况下，胆固醇的加入使形成复合体的滞后时间增加了一倍，而在加入胆固醇到18：0，22：6的情况下，滞后时间基本不变。在其他实验中，研究表明，PDE活性也依赖于酰链组成，PDE活性是综合视觉通路功能的衡量标准。在这里，22：6N-3磷脂产生最高水平的活性。发展了一种改变ROS盘膜胆固醇含量的新方法，该方法可以在5-45摩尔百分比的胆固醇范围内研究视觉信号通路的初始步骤。这里再次发现，MII和MII-G复合体的形成动力学和水平都取决于胆固醇含量。综上所述，这些研究表明，含有22：6N-3的磷脂提高了G蛋白偶联信号系统的效率。在最近的一次合作中，研究了在以n-3充足和不足的饮食饲养的大鼠的ROS中视觉信号通路的各个步骤。在这种情况下，22：6N-3将被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脂肪酸营养缺陷相关的视觉和认知缺陷提供了基础。

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.