Protein/lipid Interactions & Signal Transduction

G蛋白偶联受体是包括许多神经递质系统在内的信号转导通路的普遍存在的组分。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。视网膜视杆细胞的视觉信号转导通路被用作模型系统。我们正在研究的系统特性包括：1。视紫红质的G蛋白活化形式，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。正在研究磷脂双层的转导途径和酰基链包装特性的功能措施。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与G蛋白的偶联动力学比18：0、22：6PC慢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。利用酰基链特异性荧光探针和荧光能量转移技术研究了侧结构域形成在信号传导系统中的作用。这些研究表明，视紫红质更喜欢的环境中富含多不饱和酰基链脂质相对于一个更饱和的磷脂环境。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，并似乎赋予独特的结构特性的双层的形式的横向域。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 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 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. These studies provide a basis for understanding the visual and cognitive deficits associated with a nutritional deficiency of n-3 fatty acids.