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.

项目摘要 CB1大麻素受体（CB1R）调节逆行信号至关重要的神经元过程过多的神经递质释放，胎儿和青少年脑中的神经元发育以及神经保护作用。大麻素受体相互作用蛋白1a（CRIP1A）与神经发育，感觉函数，癫痫发作和心理健康（Oliver等，2020）。 CRIP1A是一种10链的桶蛋白脂化蛋白载体（带有UNC119和PDE6δ）（Booth等，2021）。我们的研究表明CRIP1A 结合Myristoyl的Myrgin末端9-Mer肽和HoloMyrgi。基于我们的重大进步了解CRIP1A的结构和功能，我们假设CRIP1A的功能是隔离或穿梭gi蛋白质，并且CRIP1A-GI可以由CB1R C端调节激动剂刺激的G蛋白周期，棕榈酰化 - 脱甲酰二酰化，ARL蛋白调节循环，和磷酸化 - 脱磷酸化，以控制GI货物的加载和释放。我们建议研究N18TG2和SH-SY5Y人类神经母细胞瘤细胞模型中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，以及从中衍生的Petides。目的是确定： 1）使用荧光极化来监测肽的结合，将Myrgi作为CRIP1A货物的选择性从脂化的GZ，GS，GQ和G12/13中，各种脂质（部分肉豆蔻酸酯，棕榈酸酯），Gi Peperpentend Rengus和氨基酸序列，并通过下拉分析和X射线结构确定机理这些相互作用使用纯化的重组CRIP1A和Pepperides或全长重组Myrgi，G和 CB1R C末端； 2）CRIP1A与CB1R-G蛋白激活周期的界面（机理和细胞定位）通过定量用GTP处理的神经元细胞匀浆的CRIP1A-GI共免疫沉淀，（2-ag Ether，CP55940，Win55212-2）和部分（甲基二酰胺，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。 crip1a-的蜂窝位置 GI相互作用将使用接近连接测定和亚细胞分级来定量； 3）通过量化凝胶移动的CRIP1A-，将Myrgi加载和卸载到CRIP1A中的机制 GIα通过完整神经元细胞或匀浆中的共免疫沉淀，并确定myrgi-Crip1a 使用重组蛋白的相互作用来评估：a）棕榈酰化和depalmitoylation的作用，b）调节 ARL蛋白质的性质，以及C）通过在CRIP1A表面的预测位点磷酸化来调节。我们调查在结构和功能层面上对CRIP1A和CB1R的调查结果将为未来提供信息对人类健康和疾病模型中CRIP1A的研究。