INFLUENCE OF PROTEIN/LIPID INTERACTIONS ON SIGNAL TRANSD

G蛋白偶联受体是信号转导途径中普遍存在的成分，包括许多神经递质系统。本项目旨在评估多不饱和磷脂在调节G蛋白偶联信号转导中的作用，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被用作模型系统。我们正在研究的系统属性包括：1。后视紫红质II （metarhodopsin II，即视紫红质的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。磷脂双分子层的转导途径和酰基链填充特性的功能措施正在研究中。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与G蛋白的动力学偶联减慢了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。利用酰基链特异性荧光探针和荧光能量转移技术研究了信号系统中横向结构域形成的作用。这些研究表明，相对于更饱和的磷脂环境，视紫红质更喜欢富含多不饱和酰基链脂质的环境。综上所述，这些研究表明含有磷脂的22:6n-3提高了G蛋白偶联信号系统的效率，并似乎以侧结构域的形式赋予了双分子层独特的结构特性。

G protein-coupled receptors are ubiquitous components of signal transduction pathways, including many neurotransmitter systems. This project is designed to assess the role of polyunsaturated phospholipids in modulating G protein-coupled signal transduction and to elucidate of the mechanism of action of ethanol in these systems. The visual transduction pathway of the retinal rod photoreceptor is being used as a model system. System properties we are studying include: 1. the kinetics and extent of formation of metarhodopsin II (MII), the G protein activating form of 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 acyl chain packing properties of the phospholipid bilayer 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 coupling of MII to G protein 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. The role of lateral domain formation in signaling systems was investigated using acyl chain specific fluorescence probes and fluorescence energy transfer techniques. These studies demonstrate that rhodopsin prefers an environment enriched in polyunsaturated acyl chain lipids relative to a more saturated phospholipid environment. Taken together, these studies indicate that 22:6n-3 containing phospholipids enhance the efficiency of a G protein-coupled signaling system and appear to impart unique structural properties to bilayers in the form of lateral domains.