Cannabinoid Receptors and Associated Proteins-Renewal

大麻素受体和相关蛋白质更新

• 批准号: 10657170
• 负责人: ALLYN C HOWLETT
• 金额: $ 57.52万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10657170
• 关键词: 2-arachidonylglycerol; ADP ribosylation; Adolescent; Agonist; Amino Acid Sequence; Amino Acids; Attenuated; Auditory; Binding; Biological Assay; Brain; C-terminal; CNR1 gene; Calcium Channel; Carrier Proteins; Cell Fractionation; Cell membrane; Cell model; Cell physiology; Cells; Co-Immunoprecipitations; Complex; Crystallization; Cytosol; DNA Ligation; Disease; Epilepsy; Ethers; Family; Fluorescence Polarization; Functional disorder; Future; GTP-Binding Proteins; Gel; Health; Homologous Gene; Human; Hydrophobicity; In Vitro; Investigation; Knowledge; Length; Ligation; Lipids; Location; Marijuana; Mediating; Medical; Membrane; Mental Health; Modeling; Molecular; Monitor; Monomeric GTP-Binding Proteins; Mutation; Myristates; N-Myristoylation; N-terminal; Neurodegenerative Disorders; Neurons; Pain; Palmitates; Pathway interactions; Peptide Receptor; Peptides; Pertussis Toxin; Pharmaceutical Preparations; Phosphorylation; Process; Property; Protein Dephosphorylation; Protein Farnesylation; Proteins; Receptor Activation; Receptor Signaling; Recombinant Proteins; Recombinants; Regulation; Research Personnel; Resolution; Roentgen Rays; Role; SR141716; Scanning; Secure; Seizures; Seminal; Sensory; Signal Transduction; Site; Specificity; Structure; Surface; Synaptic Membranes; Testing; Tetrahydrocannabinol; Vision; X-Ray Crystallography; addiction; antagonist; beta barrel; brain cell; cannabinoid receptor; cannabinoid receptor interacting protein 1a; dimer; experimental study; fetal; genetic regulatory protein; human disease; human model; in vivo Model; insight; methanandamide; myristoylation; neuroblastoma cell; neurodevelopment; neuron development; neuroprotection; neurotransmitter release; novel; palmitoylation; pharmacologic; presynaptic; prevent; protein activation; protein aminoacid sequence; response; side effect; voltage

Project Summary The CB1 cannabinoid receptor (CB1R) regulates neuronal processes critical for retrograde signaling to reduce excessive neurotransmitter release, neuronal development in fetal and adolescent brain, and neuroprotection. Cannabinoid Receptor Interacting Protein 1a (CRIP1a) is associated with neurodevelopment, sensory function, seizures, and mental health (Oliver et al., 2020). CRIP1a is a 10-stranded -barrel protein in the family of lipidated-protein carriers (with UNC119 and PDE6δ) (Booth et al., 2021). Our studies demonstrate that CRIP1a binds myristoylated myrGi N-terminal 9-mer peptide and holo myrGi. Based upon our major advance in knowledge of the structure and function of CRIP1a, we hypothesize that CRIP1a functions to sequester or shuttle Gi proteins, and that the CRIP1a-Gi interaction can be regulated by the CB1R C-terminus during the agonist-stimulated G protein cycle, palmitoylation-depalmitoylation, Arl protein regulatory cycles, and phosphorylation-dephosphorylation to control loading and release of Gi cargo. We propose to investigate the CB1R associated proteins in the N18TG2 and SH-SY5Y human neuroblastoma cell models that endogenously express the CB1R, CRIP1a and G proteins, as well as using in vitro experiments using purified recombinant CRIP1a, myrGi, G and CB1R C-terminal, as well as peptides derived therefrom. The Aims are to determine: 1) selectivity for myrGi as CRIP1a cargo using fluorescence polarization to monitor the binding of peptides from lipidated Gz, Gs, Gq, and G12/13, various lipids (particularly myristate, palmitate), Gi peptide length and amino acid sequence, and to define the mechanism by pulldown assays and X-ray structure determination of these interactions using purified recombinant CRIP1a and peptides or full-length recombinant myrGi, G and CB1R C-terminal; 2) the interface of CRIP1a with the CB1R-G protein activation cycle (mechanism and cellular localization) by quantitating CRIP1a-Gi co-immunoprecipitation from neuronal cell homogenates treated with GTPS, full (2-AG ether, CP55940, WIN55212-2) and partial (methanandamide, Δ9-THC) agonists, competitive antagonist- inverse agonist (SR141716), and after Gi ADP-ribosylation with pertussis toxin. Cellular location of the CRIP1a- Gi interaction will be quantitated using proximity ligation assays and subcellular fractionation; 3) the mechanism(s) for loading and unloading myrGi into CRIP1a by quantitating the gel shifted CRIP1a- Giα by co-immunoprecipitation in intact neuronal cells or homogenates, and determining myrGi-CRIP1a interaction using recombinant proteins to assess: a) roles of palmitoylation and depalmitoylation, b) regulatory properties of Arl proteins, and c) regulation by phosphorylation of predicted sites on the surface of CRIP1a. The results of our investigation of CRIP1a and CB1R at the structural and functional level will inform future investigation of CRIP1a in models of human health and disease.

项目摘要 CB1大麻素受体(CB1R)调节对逆行信号转导至关重要的神经元过程。 过多的神经递质释放，胎儿和青少年大脑中的神经元发育，以及神经保护。 大麻素受体相互作用蛋白1a(CRIP1a)与神经发育、感觉功能、 癫痫发作和精神健康(Oliver等人，2020年)。CRIP1a是一种10链-Barrel蛋白，属于 脂化蛋白载体(使用UNC119和PDE6δ)(布斯等人，2021年)。我们的研究表明，CRIP1a 结合肉豆蔻酰化的MyrGiN-末端9肽和全息肉豆蔻基。基于我们在以下方面的主要进展 了解CRIP1a的结构和功能，我们假设CRIP1a的功能是隔离或 穿梭GI蛋白，以及CRIP1a-GI相互作用可以由CB1R C-末端在 激动剂刺激的G蛋白周期，棕榈酸化-脱氨酸化，ARL蛋白调节周期， 和磷酸化-去磷酸化，以控制GI货物的装载和释放。我们建议 研究人神经母细胞瘤N18TG2和SH-SY5Y细胞模型中CB1R相关蛋白的表达 内源性表达CB1R、CRIP1a和G蛋白，以及使用纯化的 重组CRIP1a、MyrGi、G和CB1RC-末端及其衍生的多肽。 目标是确定： 1)用荧光偏振法监测多肽与MyrGi结合的选择性 从脂化的Gz、Gs、Gq和G12/13，各种脂类(特别是肉豆蔻酸酯、棕榈酸酯)、胃肠肽长度和 氨基酸序列，并通过下拉分析和X射线结构测定确定其作用机制 这些相互作用使用纯化的重组CRIP1a与多肽或全长重组MyrGi、G和 CB1R C-末端； 2)CRIP1a与CB1R-G蛋白激活周期的接口(机制和细胞定位) 通过对神经细胞匀浆中CRIP1a-GI免疫共沉淀的定量研究，S，Full (2-AG醚，CP55940，WIN55212-2)和部分(甲烷胺，Δ9-THC)激动剂，竞争性拮抗剂- 反向激动剂(SR141716)，GIADP-核糖化后与百日咳毒素。CRIP1a的细胞定位- Gi相互作用将使用邻近连接分析和亚细胞分级来定量； 3)通过定量凝胶移位的-CRIP1a，探讨了MyrGi CRIP1a的装载和卸载机制(S)。 用免疫共沉淀法在完整神经细胞或匀浆中检测Giα，并测定MyrGi-CRIP1a 使用重组蛋白评估相互作用：a)棕榈酰化和去almito化作用，b)调节 ARL蛋白的性质，以及c)CRIP1a表面预测位点的磷酸化调节。 我们在结构和功能水平上对CRIP1a和CB1R的研究结果将为未来提供参考 CRIP1a在人类健康和疾病模型中的研究。