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

基本信息

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
项目类别：
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

项目摘要

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 信号转导的机制对于充分理解其作用至关重要除了典型的第二信使（cAMP、cGMP 和 IP3）和 β- 之外。抑制蛋白信号转导、小 GTP 酶蛋白（例如 Rho GTP 酶）很大程度上参与 GPCR 介导的信号鸟嘌呤核苷酸交换因子 (GEF) 将 Rho GTPases 从非活性（GDP 结合）转变为转导。 Rho-GEF 可以被 Gq、G12/13 和 Gs 蛋白激活。目前没有证据表明Gi/0-WT可以直接激活RhoGEFs结构蛋白。通常位于蛋白质 C 末端（RhoA，激活）的识别简单线性氨基酸基序的结构域。 PDZ-RhoGEFs 通过激活磷脂酶 D (PLD) 和转录因子发挥重要的信号传导作用。大麻素受体 CB1 是一种丰富表达的 GPCR，主要与 Gi/o 偶联，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 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 PDZ-RhoGEF 下游的信号传导机制导致 RhoA 激活并随后激活 PLD 的激活会产生两种不同的第二信使——磷脂酸，它会激活 mTOR。途径，以及激活 Sigma1 受体的胆碱，我们将采用多学科方法，结合起来。最先进的分子和药理学方法，用于全面研究信号传导通过激活 CB1 受体 C 基序和识别途径选择性配体而引发的途径。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，我们提供了可靠的初步结果支持。该项目的可行性以及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 诱导的信号传导实验中的作用；表征由 CB1-motifs（CB1-PDZ 结合域）激活的细胞内级联。目标 2. 研究 PDZ-RhoGEF/RhoA 通路在培养的原代神经元和体内 CB1 受体信号传导中的作用。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。