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

解读 CB1 的复杂药理学：了解第三条信号通路

• 批准号: 10667865
• 负责人: EUGEN BRAILOIU
• 金额: $ 23.78万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10667865
• 关键词: Agonist; Amino Acid Motifs; Amino Acids; Analgesics; Arrestins; Behavioral; Binding; Biological Assay; Body Temperature; C-terminal; C57BL/6 Mouse; CNR1 gene; Cannabinoids; Catalepsy; Cells; Choline; Complex; Consensus; Couples; Cues; Cyclic AMP; Cyclic GMP; Data; Development; Experimental Designs; FRAP1 gene; Female; Functional disorder; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP Binding; Generations; Genomics; Goals; Guanine Nucleotide Exchange Factors; Hydrophobicity; Image; Investigation; Knowledge; Ligands; Measurement; Measures; Mediating; Molecular; Monomeric GTP-Binding Proteins; Motor Activity; Mutate; Mutation; Neurons; Pathway interactions; Pertussis Toxin; Pharmacology; Phosphatidic Acid; Phospholipase D; Physiology; Protein C; Proteins; Receptor Activation; Receptor Mediated Signal Transduction; Receptor Signaling; Role; Second Messenger Systems; Sequence Alignment; Signal Induction; Signal Pathway; Signal Transduction; Solid; Specificity; Stimulus; Structural Protein; Tail; Tertiary Protein Structure; Testing; Transfection; Work; antinociception; beta-arrestin; drug development; experimental study; extracellular; in vivo; inhibitor; interdisciplinary approach; male; mutant; pharmacologic; receptor; response; rho; rho GTP-Binding Proteins; sex; therapeutic development; transcription factor

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.

抽象的 了解GPCR信号的基础机制对于完全理解其在 生理学和病理生理学。除了规范的第二使者（CAMP，CGMP和IP3）和β- 逮捕蛋白信号传导，小的GTPase蛋白，例如Rho GTPases，很大程度上参与了GPCR介导的信号 转导。鸟嘌呤核苷酸交换因子（GEFS）从非活性（GDP结合）转换Rho GTPase 状态至活动状态（GTP结合）。 Rho-GEF可以被GQ，G12/13和GS蛋白激活。然而， 当前没有证据表明GI/0-WT可以直接激活Rhogefs。 PDZ结构域是结构蛋白 经常在蛋白质C末端（C-MOTIF）上识别简单线性氨基酸基序的结构域。 Rhoa，被激活 通过激活磷脂酶D（PLD）和转录因子，PDZ-RHOGEFS具有重要的信号传导作用。 大麻素接收器CB1是一种绝对表达的GPCR 三个结合PDZ III类蛋白（包括PDZ-RHOGEF）的氨基酸）。我们的中心假设是CB1 受体激活，除了参与cAMP抑制和β-arrestin途径外，还启动了额外的 向下游至PDZ-RHOGEF的信号传导机制，导致RhoA激活并随后激活 pld。 PLD的激活产生了两个不同的第二信使磷脂酸，它激活MTOR 途径和胆碱，激活Sigma1受体。我们将使用多学科的方法，结合 最先进的分子和药物方法，以全面研究信号传导 通过激活CB1受体的C-MOTIF引起的途径和途径选择配体的鉴定。我们 将使用受体和PDZ-RHOGEF突变，测量第二使者水平（CAMP，胆碱和 磷脂酸）以及PLD激活的实时成像。我们提供了支持的扎实初步结果 该项目的可行性以及我们团队完成提议的工作的能力。该项目有两个目标： AIM 1。研究PDZ结合域在CB1诱导的信号传导中的作用；实验被设计为 表征由CB1-MOTIF激活的细胞内级联反应（CB1-PDZ结合域。AIM 2。研究 PDZ-RHOGEF/RHOA途径在培养的原代神经元和体内CB1受体信号传导中的作用。这 该项目的成功完成将增加当前的GPCR信号知识，并将作为 进一步开发配体的基础，选择性地靶向该途径。