Protein/lipid Interactions and Signal Transduction

蛋白质/脂质相互作用和信号转导

• 批准号: 6818448
• 负责人: Burton J Litman
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6818448
• 关键词: G protein; atomic force microscopy; biological signal transduction; cell surface receptors; cholesterol; dietary lipid; electroretinography; enzyme activity; ethanol; guanosinetriphosphatases; intermolecular interaction; laboratory rat; lipid bilayer membrane; lipid structure; malnutrition; membrane lipids; membrane structure; nutrition related tag; phosphodiesterases; phospholipids; physical chemical interaction; posttranslational modifications; protein structure function; receptor coupling; rhodopsin; rod cell; unsaturated fatty acids; visual phototransduction

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.

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。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视网膜杆外段（ROS）中视觉信号通路的各个步骤。在这些条件下，膜磷脂中的n-3酰基链22：6 n-3被n-6酰基链22：5 n-6取代。我们已经发现，从n-3缺陷大鼠的ROS具有降低的视紫红质活化水平，更慢的受体G蛋白复合物的形成和3倍的信号传导途径的敏感性降低，如通过PDE活性所测量的。此外，我们已经证明，22：5 n-6酰基链包装在一个更有序的阵列比22：6 n-3。22：5 n-6和22：6 n-3在碳19上只有一个双键不同，这一事实证明了膜物理性质和膜蛋白功能的精密耦合。观察到的差异提供了一个解释的n-3缺陷的动物和非人灵长类动物的视网膜电图中观察到的功能变化。这些研究为理解与n-3脂肪酸营养缺乏相关的视觉和认知缺陷提供了基础。采用原子力显微镜（AFM）的合作努力已经观察到了重组脂质-视紫红质双层中的侧向结构域。使用原子力显微镜，单独的视紫红质分子被解决在本机磁盘膜。在这项研究中，侧域的存在和视觉信号通路中的作用正在调查。这些结构域对胆固醇含量的依赖性也正在研究中。