Structural Studies on the Cannabinoid Receptor

描述(申请人提供)：拟议研究的长期目标是从机制上了解神经元大麻素受体(CB1)是如何被激活的。我们将探讨这个问题的几个方面。以下是我的具体目标的简要描述。目的1)目前被广泛接受的G蛋白偶联受体激活模型是二态模型，但最近的证据表明，该模型过于简单，不能描述G蛋白偶联受体的激活。CB1激活的两态模型的有效性将通过荧光光谱学来监测激活受体的构象状态，以确定是否存在一个激活状态构象或多个不同的构象状态。(目的2)与对接的G蛋白的核苷酸结合已被证明影响GPCR与其配体的亲和力。通过探索CB1受体是否如此，我将确定配体结合的核苷酸变构调节是否通过诱导受体的构象变化而起作用。(3)通过尝试使受体发生突变来降低或取消CB1的构成活性，从而检验CB1的构成活性的机制。然后将对这些突变体进行结构研究，以检查任何构象变化是否以及如何与构成活性联系在一起。由于大麻是一种广泛使用的街头药物，而大麻素受体是G蛋白偶联受体家族(最大的一类药物发现靶点)的成员，了解CB1受体的调节机制具有重要的临床和科学意义。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to understand mechanistically how the neuronal cannabinoid receptor (CB1) becomes activated. Several aspects of this question will be explored. Below is a brief description of my specific aims. (Aim 1) The most widely accepted model for G-protein coupled receptor (GPCR) activation is the two-state model yet recent evidence suggests that this model is too simple to describe GPCR activation. The validity of the two-state model for CB1 activation will be assessed using fluorescence spectroscopy to monitor conformational states of the activated receptor to determine if there is one active state conformation or multiple, distinct conformational states. (Aim 2) Nucleotide binding to docked G-proteins has been shown to affect a GPCR's affinity for its ligands. By exploring whether this is true for the CB1 receptor, I will determine if nucleotide allosteric regulation of ligand binding works through inducing conformational changes in the receptor. (3) The mechanisms by which CB1 is constitutively active will be examined by attempting to make mutations in the receptor that reduce or abolish the constitutive activity. These mutants will then be studied structurally to examine if and how any conformational changes are linked to constitutive activity. Because marijuana is a widely used street drug, and the cannabinoid receptor is a member of the G-protein coupled receptor family (the largest class of drug discovery targets), understanding the mechanism by which the CB1 receptor is modulated is important both clinically and scientifically.