Cannabinoid Receptors and Associated Proteins-Renewal

项目摘要 CB 1大麻素受体（CB 1 R）调节对逆行信号传导至关重要的神经元过程， 神经递质的过度释放、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（CRIP 1a）与神经发育，感觉功能， 癫痫发作和精神健康（奥利弗等人，2020年）。CRIP 1a是CRIP 1a家族中的一种10链双桶蛋白， 脂质化蛋白载体（具有UNC 119和PDE 6 δ）（Booth等，2021年）。我们的研究表明CRIP 1a 结合肉豆蔻酰化myrGi β N-末端9-mer肽和全myrGi β。基于我们在 了解CRIP 1a的结构和功能，我们假设CRIP 1a的功能是螯合或 CRIP 1a-Gi蛋白的相互作用可以由CB 1 R C-末端调节， 激动剂刺激的G蛋白周期、棕榈酰化-脱棕榈酰化、Arl蛋白调节周期 和磷酸化-去磷酸化以控制Gi受体货物的装载和释放。我们建议 研究N18 TG 2和SH-SY 5 Y人神经母细胞瘤细胞模型中的CB 1 R相关蛋白， 内源性表达CB 1 R，CRIP 1a和G蛋白，以及使用纯化的 重组CRIP 1a、myrG 1 β、G1 β和CB 1 R C-末端，以及由其衍生的肽。 目的是确定： 1)使用荧光偏振监测肽的结合对myrGi作为CRIP 1a货物的选择性 从脂化的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，并通过下拉分析和X-射线结构测定来确定其机制。 使用纯化的重组CRIP 1a和肽或全长重组myrG 1 β、G2 β和 CB 1 R C端; 2)CRIP 1a与CB 1 R-G蛋白激活周期的界面（机制和细胞定位） 通过定量来自用GTP酶处理的神经元细胞匀浆的CRIP 1a-Gi共免疫沉淀，完全 (2-AG醚，CP 55940，WIN 55212 -2）和部分（甲雄酰胺，Δ9-THC）激动剂，竞争性拮抗剂- 反向激动剂（SR 141716），以及在用百日咳毒素进行Gi ADP-核糖基化之后。CRIP 1a的蜂窝定位- 将使用邻近连接测定和亚细胞分级分离来定量Gi-κ相互作用; 3)通过定量凝胶移位的CRIP 1a-1，将myrGi载体装载和卸载到CRIP 1a中的机制 通过完整神经元细胞或匀浆中的免疫共沉淀法测定Giα，并测定myrGi-CRIP 1a 使用重组蛋白评估相互作用：a）棕榈酰化和脱棕榈酰化的作用，B）调节 Arl蛋白的性质，和c）通过CRIP 1a表面上预测位点的磷酸化进行调节。 我们在结构和功能水平上对CRIP 1a和CB 1 R的研究结果将为未来的研究提供信息。 CRIP 1a在人类健康和疾病模型中的研究。

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.