Cannabinoid Receptors and Associated Proteins-Renewal

大麻素受体和相关蛋白质更新

基本信息

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
项目类别：
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

项目摘要

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.

项目概要 CB1 大麻素受体 (CB1R) 调节对逆行信号传导至关重要的神经元过程，以减少神经递质过度释放、胎儿和青少年大脑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、癫痫发作和心理健康（Oliver et al., 2020）。脂化蛋白载体（带有 UNC119 和 PDE6δ）（Booth 等人，2021）。结合肉豆蔻酰化 myrGi N 端 9 聚体肽和 Holo myrGi 基于我们的重大进展。了解了 CRIP1a 的结构和功能后，我们发现 CRIP1a 的功能是隔离或穿梭 Gi 蛋白，并且 CRIP1a-Gi 相互作用可以在过程中受到 CB1R C 末端的调节激动剂刺激的 G 蛋白循环、棕榈酰化-去棕榈酰化、Arl 蛋白调节循环、我们建议通过磷酸化-去磷酸化来控制Gi货物的装载和释放。研究 N18TG2 和 SH-SY5Y 人神经母细胞瘤细胞模型中的 CB1R 相关蛋白内源表达 CB1R、CRIP1a 和 G 蛋白，以及使用纯化的体外实验重组 CRIP1a、myrGi、G 和 CB1R C 末端，以及由其衍生的肽。目标是确定： 1) 使用荧光偏振对 myrGi 作为 CRIP1a 货物的选择性来监测肽的结合衍生脂质 Gz、Gs、Gq 和 G12/13、各种脂质（特别是肉豆蔻酸酯、棕榈酸酯）、Gi 肽长度和氨基酸序列，并通过 Pulldown 分析和 X 射线结构测定来确定其机制使用纯化的重组 CRIP1a 和肽或全长重组 myrGi、G 和 CB1R C端； 2）CRIP1a与CB1R-G蛋白激活循环的界面（机制和细胞定位）通过对经 GTPS 处理的神经元细胞匀浆进行 CRIP1a-Gi 免疫共沉淀定量，完整（2-AG 醚、CP55940、WIN55212-2）和部分（甲烷酰胺、Δ9-THC）激动剂、竞争性拮抗剂- 反向激动剂 (SR141716)，以及用百日咳毒素进行 Gi ADP 核糖基化后的 CRIP1a- 的细胞定位。 Gi 相互作用将使用邻近连接和亚细胞分级分析进行定量； 3) 通过定量凝胶转移的 CRIP1a- 将 myrGi 装载和卸载到 CRIP1a 的机制通过在完整神经元细胞或匀浆中进行免疫共沉淀来检测 Giα，并测定 myrGi-CRIP1a 使用重组蛋白的相互作用来评估：a) 棕榈酰化和去棕榈酰化的作用，b) 调节 Arl 蛋白的特性，以及 c) 通过 CRIP1a 表面预测位点的磷酸化进行调节。我们对 CRIP1a 和 CB1R 在结构和功能层面的研究结果将为未来提供信息 CRIP1a 在人类健康和疾病模型中的研究。