Protein/lipid Interactions and Signal Transduction

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。在最近的一次合作中，研究了以n-3充足和不足的饮食饲养的大鼠视网膜视杆外段(ROS)视觉信号通路的各个步骤。在此条件下，膜磷脂中的n-3酰基链22：6n-3被n-6酰基链22：5n-6取代。我们发现，n-3缺陷大鼠的ROS降低了视紫红质的激活水平，减缓了受体G蛋白复合体的形成，并通过PDE活性衡量，信号通路的敏感性降低了3倍。此外，我们还证明了22：5N-6酰链的排列比22：6N-3的排列更有序。22：5N-6和22：6N-3在碳19位只有一个双键的差异证明了膜物理性质和膜蛋白功能的精细耦合。观察到的差异解释了在n-3缺陷动物和非人类灵长类动物的视网膜电信号中观察到的功能变化。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。利用原子力显微镜(AFM)的合作努力已经导致在重组的脂质-视紫红质双层中观察到横向结构域。利用原子力显微镜，单个视紫红质分子在天然盘膜中被分解。在这项研究中，正在研究视觉信号通路中侧域的存在和作用。这些结构域对胆固醇含量的依赖性也在研究中。

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. In a recent collaboration, various steps in the visual signaling pathway were studied in retinal rod outer segment (ROS) from rats raised on n-3 adequate and deficient diets. Under these conditions, the n-3 acyl chain, 22:6n-3, is replaced by n-6 acyl chain, 22:5n-6, in membrane phospholipids. We have found that the ROS from n-3 deficient rats have reduced levels of rhodopsin activation, slower receptor G protein complex formation and a 3-fold reduction in sensitivity of the signaling pathway, as measured by the PDE activity. In addition we have demonstrated that the 22:5n-6 acyl chains are packed in a more ordered array than those of 22:6n-3. The exquisite coupling of membrane physical properties and membrane protein function is demonstrated by the fact that 22:5n-6 and 22:6n-3 differ by only one double bond at carbon nineteen. The observed differences provide an explanation of the functional changes observed in the electroretinograms of n-3 deficient animals and non-human primates. These studies provide a basis for understanding the visual and cognitive deficits associated with a nutritional deficiency of n-3 fatty acids. Collaborative efforts employing atomic force microscopy (AFM) have led to the observation of lateral domains in reconstituted lipid-rhodopsin bilayers. Using AFM, individual rhodopsin molecules were resolved in native disk membranes. In this study, the presence and role of lateral domains in the visual signaling pathway is being investigated. The dependence of these domains on cholesterol content is also being investigated.