Influence Of Protein/lipid Interactions On Signal Transd

G蛋白偶联受体（GPCR）是信号转导途径中普遍存在的组分，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。该项目旨在评估膜脂质组成，特别是多不饱和磷脂，在调节GPCR信号转导的作用，并阐明乙醇在这些系统中的作用机制。视网膜视杆细胞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。研究中的系统特性包括：1。活化视紫红质的G蛋白活化形式的后视紫红质II（MII）的形成的动力学和程度; 2. MII/G蛋白复合物形成; 3. G蛋白激活率; 4. cGMP磷酸二酯酶（PDE）活化;和5. G蛋白的GTP酶活性。这两个功能的措施，在转导途径和脂质双层的物理性质正在调查。目前的研究表明，MII-G蛋白复合物形成的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、18：1 PC中MII-G蛋白形成的动力学比18：0、22：6PC减慢了50%。胆固醇的加入使18：0、18：1 PC中复合物形成的滞后时间增加一倍，而胆固醇的加入使18：0、22：6PC中复合物形成的滞后时间基本不变。在其他实验中，它表明，PDE活性，综合视觉通路功能的量度，也取决于酰基链的组成。在此，22：6 n-3磷脂再次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，该方法允许在5至45摩尔%胆固醇的范围内研究视觉信号通路中的初始步骤。在此，再次发现MII和MII-G复合物形成的动力学和水平均依赖于胆固醇含量。总之，这些研究表明，22：6 n-3含有磷脂增强G蛋白偶联信号系统的效率。在最近的一项合作中，研究了在n-3充足和缺乏饮食的大鼠中ROS的视觉信号通路的各个步骤。在这些条件下，22：6 n-3被22：5 n-6取代。我们已经发现，从n-3缺乏大鼠的ROS是不太敏感的光刺激比从n-3充足的大鼠的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。

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.