Cannabinoid receptors and associated proteins

摘要 CB1受体(CB1R)是治疗成瘾、神经退行性疾病和 疼痛控制，但基于CB1R的药物化合物一直受到限制。的功能 CB1R调节发育中的神经元过程，神经传递中的逆行信号，以及细胞 神经保护机制对大脑功能至关重要。内源性大麻素配体2-花生四烯基甘油 和花生胺是突触活动的主要神经调节剂，但对CB1R如何 在特定的细胞类型中，信号可以由相关蛋白质调节，这一点才刚刚开始被认识到。这个 科学前提是CRIP1a对CB1R的调制可以从结构和结构上理解 功能水平，使得基于多肽或小分子干预的药物设计可以针对 CRIP1a-CB1R相互作用。我们最近发表的研究表明，CRIP1a可以降低 细胞表面CB1R的密度减弱激动剂依赖的内化，但不减弱结构性内化 通过与β-Drastins竞争结合C-末端位点来进行处理，并减少新- 延长WIN55212-2后合成CB1R到细胞表面，而不是CP55940。其他研究 证明CRIP1a在调节CB1R细胞信号转导中具有关键作用，它通过改变 从需要C-末端才能激活的Gi3和Go连接到不需要C-末端的Gi1和GI2。在未出版的 研究中，我们从X射线结晶学中确定了CRIP1a的高分辨结构，发现CRIP1a是 肉豆蔻酰化或异戊二烯基化蛋白质载体家族的成员。基于这一重大进展 在了解CRIP1a的结构和功能的基础上，我们假设CRIP1a的功能是 与CB1R-G-蛋白复合体相互作用的方式可受G-α和/或G-伽马调节 亚基特异性、磷酸化以及与其他已知的调节蛋白的相互作用 放行货物。我们建议在N18TG2神经母细胞瘤细胞中研究CB1R相关蛋白 内源性表达CB1R及其相关蛋白的模型及体外实验 纯化的重组蛋白及其衍生的多肽。该项目的目的是调查： CRIP1a与CB1R的相互作用；CRIP1与G-R的结构和功能相互作用 蛋白质；以及通过货物释放蛋白和磷酸化调节CRIP1a的功能。这个 拟议的调查结果应证明对外地具有变革性，因为它提供了以下证据 CB1R和相关的CRIP1a相互作用，引导细胞信号通路。从这种理解来看，小说 多肽和小分子可被开发为治疗神经系统疾病的药物，其中 CB1R和CRIP1a在神经元中共存。

Summary The CB1 receptor (CB1R) a therapeutic target for treatment of addictions, neurodegenerative disorders and pain management, but medicinal compounds based upon the CB1R have been limited. The functions of the CB1R to regulate neuronal processes in development, retrograde signaling in neurotransmission, and cellular mechanisms of neuroprotection are critical to brain function. Endocannabinoid ligands 2-arachidonoylglycerol and anandamide are the primary neuromodulators of synaptic activity, but the full understanding of how CB1R signaling can be regulated by associated proteins in specific cell types is just beginning to be appreciated. The Scientific Premise is that CRIP1a modulation of the CB1R can be understood at the structural and functional level such that drug design based on peptide or small molecule interventions can target the CRIP1a-CB1R interaction. Our recently published studies have demonstrated that CRIP1a reduces the density of cell surface CB1R, attenuates the agonist-dependent but not the constitutive internalization processes by competing with β-arrestins for binding to C-terminal sites, and curtails the trafficking of newly- synthesized CB1R to the cell surface after prolonged WIN55212-2 but not CP55940. Other studies demonstrated that CRIP1a has a critical role in regulating CB1R cellular signaling by altering the preference for coupling from Gi3 & Go, which require the C-terminus for activation, to Gi1 & Gi2, which do not. In unpublished studies, we have determined the high resolution structure from X-ray crystallography, and found that CRIP1a is a member of the family of carriers for myristoylated or isoprenylated proteins. Based upon this major advance in knowledge of the structure and function of CRIP1a, we hypothesize that the function of CRIP1a is to interact with the CB1R–G-protein complex in ways that can be regulated by G-alpha and/or G-gamma subunit specificity, phosphorylation, and interaction with other regulatory proteins that are known to release cargo. We propose to investigate the CB1R associated proteins in the N18TG2 neuroblastoma cell model which endogenously expresses the CB1R and its associated proteins, as well as in in vitro experiments of purified recombinant proteins and peptides derived therefrom. The aims of this project are to investigate: the interaction of CRIP1a with the CB1R; the structural and functional interaction of CRIP1 with G- proteins; and the regulation of CRIP1a function by cargo-releasing proteins and phosphorylation. The results of the proposed investigation should prove to be transformative for the field by providing evidence that CB1R and associated CRIP1a interact to direct cellular signaling pathways. From this understanding, novel peptides and small molecules could be developed as therapeutic agents for neurological diseases in which both CB1R and CRIP1a co-exist in neurons.