Deciphering the complex pharmacology of CB1: towards the understanding of a third signaling pathway

摘要 了解gpr信号转导的机制对于充分理解它们在 生理学和病理生理学。除了规范的第二信使(cAMP、cGMP和IP3)和β- Arrestin信号、小分子GTP酶蛋白，如Rho GTP酶，主要参与GPCR介导的信号转导 转导。鸟嘌呤核苷酸交换因子(GEF)将Rho GTP酶从失活的(GDP结合的) 状态设置为活动状态(GTP绑定)。GQ、G12/13和GS蛋白均可激活Rho-GEF。然而， 目前还没有证据表明GI/0-WT可以直接激活RhoGEF。PDZ结构域是一种结构蛋白 识别简单线性氨基酸基序的结构域，通常位于蛋白质的C-末端(C-基序)。RhoA，激活 通过PDZ-RhoGEFs，通过激活磷脂酶D(PLD)和转录因子，具有重要的信号转导作用。 大麻素受体Cb1是一种主要与GI/O偶联的GPCRa，其表达水平较高，具有EAL C基序(LAST 三个氨基酸)，与PDZ III类蛋白结合，包括PDZ-Rhogef。我们的中心假设是CB1 受体激活，除了参与cAMP抑制和β-arrestin通路外，还启动了另一种 PDZ-Rhogef下游的信号机制导致RhoA的激活和随后的激活 可编程逻辑器件。PLD的激活产生了两个不同的第二信使，磷脂酸，它激活了mTOR 途径和胆碱，激活Sigma1受体。我们将使用多学科方法，结合 全面研究信号转导的最新分子和药理学方法 CB1受体C基序激活引发的通路及通路选择性配体的鉴定我们 将使用受体和PDZ-Rhogef突变，测量第二信使水平(cAMP，胆碱和 磷脂酸)以及PLD激活的实时成像。我们提供了坚实的初步结果来支持 项目的可行性和我们团队完成拟议工作的能力。该项目有两个目标： 目的1.研究PDZ结合域在CB1诱导的信号转导中的作用；实验设计旨在 鉴定由CB1基序(CB1-PDZ结合域)激活的细胞内级联。目标2.调查 PDZ-Rhogef/RhoA通路在原代培养神经元和体内CB1受体信号转导中的作用这个 该项目的成功完成将增加目前对GPCR信令的了解，并将作为 为进一步开发选择性靶向该途径的配体奠定了基础。

ABSTRACT Understanding the mechanisms underlying GPCR signaling is crucial in order to fully comprehend their role in physiology and pathophysiology. In addition to canonical second messengers (cAMP, cGMP and IP3) and β- arrestin signaling, small GTPase proteins, such as Rho GTPases are largely involved in GPCR-mediated signal transduction. Guanine nucleotide exchange factors (GEFs) convert Rho GTPases from an inactive (GDP-bound) state to an active state (GTP-bound). Rho-GEFs can be activated by Gq, G12/13 and Gs proteins. However, currently there is no evidence that Gi/0-WT can directly activate RhoGEFs. PDZ domains are structural protein domains that recognize simple linear amino acid motifs often at the protein C-terminal (C-motif). RhoA, activated by PDZ-RhoGEFs, has important signaling roles, by activating phospholipase D (PLD) and transcription factors. Cannabinoid receptor CB1, an abundantly expressed GPCR that mainly couples to Gi/o, has a EAL C-motif (last three amino acids) that binds PDZ class III proteins, including PDZ-RhoGEF. Our central hypothesis is that CB1 receptor activation, in addition to engaging cAMP inhibition and β-arrestin pathways, initiates an additional signaling mechanism downstream to PDZ-RhoGEF leading to activation of RhoA and subsequent activation of PLD. Activation of PLD generates two distinct second messengers, phosphatidic acid, which activates the mTOR pathway, and choline, which activates Sigma1 receptors. We will use a multidisciplinary approach, combining state-of-the-art molecular and pharmacological approaches for a comprehensive investigation of the signaling pathways elicited by activation of C-motifs of CB1 receptor and identification of pathways-selective ligands. We will use receptor and PDZ-RhoGEF mutations, measurements of second messengers levels (cAMP, choline and phosphatidic acid) as well as live imaging of PLD activation. We provide solid preliminary results supporting the feasibility of the project and the ability of our team to complete the work proposed. The project has two aims: Aim 1. Investigate the role of PDZ-binding domain in CB1-induced signaling; experiments are designed to characterize intracellular cascades activated by CB1-motifs (CB1-PDZ binding domain. Aim 2. Investigate the role of PDZ-RhoGEF/RhoA pathway in CB1 receptor signaling in cultured primary neurons and in vivo. The successful completion of this project will increase the current knowledge of GPCR signaling and will serve as a basis for further development of ligands selectively targeting this pathway.