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.

摘要 了解GPCR信号转导的潜在机制对于充分理解它们在细胞内的作用至关重要。 生理学和病理生理学。除了典型的第二信使（cAMP，cGMP和IP 3）和β- 抑制蛋白信号传导，小GTP酶蛋白，如Rho GTP酶主要参与GPCR介导的信号传导， 转导鸟嘌呤核苷酸交换因子（GEF）将Rho GTP酶从无活性（GDP结合） 状态转换为活动状态（GTP绑定）。Rho-GEF可被G β q、G β 12/13和G β s蛋白激活。但是，在这方面， 目前还没有证据表明G β i/0-WT可以直接激活RhoGEFs。PDZ结构域是结构蛋白 识别简单线性氨基酸基序的结构域，通常位于蛋白质C-末端（C-基序）。RhoA，激活 PDZ-RhoGEFs通过激活磷脂酶D（PLD）和转录因子，具有重要的信号传导作用。 大麻素受体CB 1是一种大量表达的GPCR，主要偶联到G β i/o，具有EAL C基序（最后 三个氨基酸）结合PDZ III类蛋白，包括PDZ-RhoGEF。我们的中心假设是CB 1 受体激活，除了参与cAMP抑制和β-arrestin途径，启动了额外的 PDZ-RhoGEF下游的信号传导机制导致RhoA的激活和随后的 PLD。PLD的激活产生两种不同的第二信使，磷脂酸，其激活mTOR 途径，胆碱，激活Sigma 1受体。我们将采用多学科方法， 国家的最先进的分子和药理学方法的全面调查的信号 通过激活CB 1受体的C基序和识别途径选择性配体引起的途径。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激活的活体成像。我们提供了坚实的初步结果支持 该项目的可行性和我们的团队完成工作的能力提出。该项目有两个目标： 目标1。研究PDZ结合结构域在CB 1诱导的信号传导中的作用;实验设计为 表征由CB 1-基序（CB 1-PDZ结合结构域）激活的细胞内级联。目标2.探讨 PDZ-RhoGEF/RhoA通路在培养的原代神经元和体内CB 1受体信号传导中的作用。的 该项目的成功完成将增加目前对GPCR信号传导的了解，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。