INTERHELICAL INTERACTIONS IN THE CANNABINOID RECEPTOR

描述：(申请人摘要) 这些建议的总体目标是获得以下结构性知识 大麻素受体的跨膜区。了解 大麻素作用的基础以及潜在疗法的设计 需要有关螺旋间相互作用的详细信息 蛋白质内的跨膜片段。 具体目的是展示细胞的跨膜片段。 受体形成特定的螺旋-螺旋相互作用，以表征性质 以生成结构信息，并 从片段中重组活性受体以证明 特异性跨膜片段与受体功能的相互作用。 跨膜片段将以融合蛋白的形式在大肠杆菌和 检测它们的二聚化能力。突变将被设计和制造 以表征二聚化所需的相互作用。生成的数据 将用于构建CB1跨膜的三维模型 域。为了重组活性受体，CB1将表达为 一对不连续的片段，它们一起构成全长 蛋白。CB1重组可提供跨膜区的证据 CB1作为组织中心进行适当的组装。 所采用的方法有可能适用于其他 膜蛋白，从而提供必要的工具来研究 其他药物受体和膜蛋白的结构。

DESCRIPTION: (Applicant's Abstract) The overall objective of the proposals is to gain structural knowledge of the transmembrane domain of the cannabinoid receptor. Understanding the basis of cannabinoid action as well as design of potential therapeutics requires detailed information about interhelical interactions of transmembrane segments within the protein. The specific aims are to demonstrate the transmembrane segments of the receptor form specific helix-helix interactions, to characterize the nature of those interactions in order to generate structural information, and to reassemble active receptor from fragments to demonstrate the necessity of specific transmembrane segment interactions to receptor function. Transmembrane segments will be expressed as fusion proteins in E. Coli and assayed for their ability to dimerize. Mutations will be designed and made to characterize interactions necessary for dimerization. The data generated will be used to construct a 3-dimensional model of the CB1 transmembrane domain. In order to reassemble active receptor, CB1 will be expressed as pairs of discontinuous fragments that together comprise the full length protein. CB1 reassembly can provide evidence that transmembrane domain of CB1 acts as an organizing center for proper assembly. It is possible that the methods to be employed will be applicable to other membrane proteins, thereby providing the tools necessary to study the structures of other drugs receptors and membrane proteins in general.