INFLUENCE OF PROTEIN/LIPID INTERACTIONS ON SIGNAL TRANSD

G蛋白偶联受体是普遍存在的信号成分 转导系统 该项目旨在评估 多不饱和磷脂在调节G蛋白偶联信号中的作用 转导，并阐明乙醇的作用机制， 这些系统。 视杆细胞中的视觉传导通路， 一个典型的G蛋白偶联系统，被用作模型系统。 醇类和脂质成分对：动力学和程度 后视紫红质II（Metarhodopsin II，MII）是一种G蛋白活化形式， 视紫红质; MII/G蛋白复合物形成; G蛋白速率 激活; cGMP磷酸二酯酶激活;和GT3活性 G蛋白的研究。 沿着功能措施 在转导途径中， 磷脂双分子层是通过使用时间分辨 荧光光谱法 视紫红质的分离与重组 进入确定的脂质组成的双层允许评估 磷脂酰基链组成的作用。短链醇， 例如乙醇，促进MII形成，而较长链醇，例如 如癸醇，是抑制性的。 中等长度的醇显示出平滑的 从兴奋性到抑制性的转变。 磷脂双层 含有22：6 n3酰基链的化合物对 醇比更饱和的酰基链磷脂。 的影响 脂肪醇、酰基链组成和胆固醇的含量具有很好的相关性 随着磷脂酰基链堆积自由体积的变化， 其特征在于时间分辨荧光各向异性行为， 膜探针，二苯基己三烯（DPH）。 这些结果证明 含22：6 n3的磷脂是最好的视紫红质促进剂 活化，并提供独特的结构特征，以双层在 横向域形成的形式。 此外，还发现了富含22：6 n3的双层膜 受胆固醇的酰基链排序效应的影响最小， 发现其对视紫红质活化具有抑制作用。 这里描述的研究强烈支持脂质介导的成分 在酒精调节激活的作用机制中， G蛋白偶联受体 我们的观察最好用一个 本实验室开发的新型磷脂酰基链堆积模型， 其中磷脂的存在类似于在视网膜中发现的那些， 突触体（例如，多不饱和酰基链，在混合 饱和-不饱和和二多不饱和酰基链磷脂） 导致在表面中形成横向域或簇 膜。 这些结构特征似乎起到了 在介导膜中受体功能的重要作用， 长链多不饱和磷脂。

G protein-coupled receptors are ubiquitous components of signal transduction 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 in the rod photoreceptor, a prototypical G protein-coupled system, is being used as a model system. The effect of alcohols and lipid composition on: the kinetics and extent of formation of metarhodopsin II (MII), the G protein activating form of rhodopsin; MII/G protein complex formation; the rate of G protein activation; cGMP phosphodiesterase activation; and the GTPase activity of the G protein are being studied. Along with the functional measures in the transduction pathway, acyl chain packing properties of the phospholipid bilayer are being determined by use of time-resolved fluorescence spectroscopy. The isolation and reconstitution of rhodopsin into bilayers of defined lipid composition has allowed an evaluation of the role of phospholipid acyl chain composition. Short chain alcohols, such as ethanol, promote MII formation, while longer chain alcohols, such as decanol, are inhibitory. Intermediate length alcohols show a smooth transition from excitatory to inhibitory. Phospholipid bilayers containing 22:6n3 acyl chains are more sensitive to the effect of alcohols than are more saturated acyl chain phospholipids. The effects of ethanol, acyl chain composition and cholesterol are well correlated with changes in phospholipid acyl chain packing free volume, as characterized by the time-resolved fluorescence anisotropy behavior of the membrane probe, diphenylhexatriene (DPH). These results demonstrate that 22:6n3 containing phosphoplids are the best promoters of rhodopsin activation and provide unique structural features to the bilayer in the form of lateral domain formation. In addition, bilayers rich in 22:6n3 are least effected by the acyl chain ordering effects of cholesterol, which are found to be inhibitory with respect to rhodopsin activation. The studies described here strongly support a lipid-mediated component in the mechanism of action for alcohols in modulating the activation of a G protein-coupled receptor. Our observations are best explained by a novel phospholipid acyl chain packing model developed in this laboratory, in which the presence of phospholipids like those found in the retina and synaptosomes (e.g., polyunsaturated acyl chains, in mixed saturated-unsaturated and dipolyunsaturated acyl chain phospholipids) leads to the formation of lateral domains or clusters in the surface of the membrane. It would appear that these structural features play an important role in mediating receptor function in membranes containing long chain polyunsaturated phospholipids.