Structural Characterization of Muscarinic and Alpha Adrenergic Receptors

这些研究的目的是了解两个重要的膜信号受体的结构和功能：毒蕈碱乙酰胆碱（亚型M1）受体和α 2B肾上腺素能受体。 两者都是结构相关的G蛋白偶联受体大家族的成员。 实验方法是使用一种新的蛋白质表达系统，该系统具有产生每种受体的潜力，其数量足以允许许多尚不可能的标准结构和功能分析。 该方法需要构建编码受体的基因和细菌视紫红质基因的嵌合体，以指导嗜盐古菌盐杆菌中的高水平表达。 筛选具有细菌视紫红质基因的不同部分的嵌合体将确定受体过表达的最佳策略。 此外，构建体也将在先前采用的真核细胞系统中表达，作为体内测试受体功能的手段。 该方法将确保成功表达的嵌合体保留其与内源性下游信号传导组分偶联的能力。 这些研究有很高的潜力，以产生重要的新信息的分子机制的信号通过这些膜蛋白。

The objective of these studies is to understand the structure and function of two important membrane signaling receptors: the muscarinic acetylcholine (subtype M1) receptor and the alpha 2B adrenergic receptor. Both are members of a large family of structurally related G protein-coupled receptors. The experimental approach is to use a novel protein expression system with the potential of generating each receptor in the quantities large enough to permit many standard structural and functional analyses that have not yet been possible. The approach entails the construction of chimeras of the genes encoding the receptors and the bacteriorhodopsin gene to direct high level expression in the halophilic archaeon Halobacterium salinarum. Screening of chimeras with different portions of the bacteriorhodopsin gene will define an optimal strategy for receptor over-expression. In addition, constructs will also be expressed in previously employed eukaryotic cell systems as a means to test receptor function in vivo. The method will ensure that the successfully expressed chimeras retain their ability to couple to endogenous downstream signaling components. The studies have a high potential to yield significant new information about the molecular mechanisms of signaling by these membrane proteins.