Cannabinoid Receptor Interacting Protein 1a (CRIP1a) in cell signaling and cargo transport

项目总结 这项新的F31提议是为了支持Erin Hughes的职业发展，他将在 大麻素受体生化神经药理学与豪利特和洛瑟博士合著。近年来，我们看到了 含有精神活性成分Δ9-四氢大麻酚的大麻在全球范围内的使用增加 CB1大麻素受体(CB1R)部分激动剂。CB1R是一种发现的G蛋白偶联受体 在中枢神经系统中，并调节神经前体的发育、神经承诺和迁移， 和神经递质的释放。Gpr的功能依赖于改变构象和 通过G蛋白转导反应。这一过程需要Gα-GDP亚单位与Gβγ亚单位的关联 形成与gpr结合的异三聚体G蛋白复合体。CB1R的刺激启动了这种交换 GTP与G-α结合，激活G-蛋白异三聚体，释放G-α和G-βγ亚基。这个 释放的G蛋白亚基通过各种相互作用传播信号级联。大麻素受体 相互作用蛋白1a(CRIP1a)是一种在人脑不同区域的神经元中发现的蛋白质，它抑制 CB1R细胞信号转导通过调节G蛋白的激活。科学前提是CRIP1a调制 可以理解CB1R的作用，为我们提供了一个深入了解CB1R在神经细胞中作用的视角 发信号。CRIP1a可以调节与Cb1R结合的Gα亚基的类型，但其转换机制 是未知的。先前的免疫沉淀研究表明CB1R C-末端区域与 因此，对CRIP1a介导的G-α交换的一种可能的解释可能涉及形成一个 Cb1R、亚型特异性Gα蛋白和CRIP1a之间的三元复合体。洛瑟-豪利特合作 用X-射线结晶学测定了CRIP1a的高分辨结构，发现CRIP1a是一个 肉豆蔻酰化或异戊二烯基化蛋白质载体家族的成员。根据我们所知道的 CRIP1a的结构和功能，我推测CRIP1a与N-末端肉豆蔻酰化的货物结合 蛋白质，如GαI蛋白。我建议研究N18TG2神经母细胞瘤中CRIP1a的相互作用 内源性表达CB1R及相关蛋白的细胞模型。这个项目的目的是 测定(I)激动剂刺激和异三聚体过程中CRIP1a-Gα相互作用的调节 G蛋白解离和这种相互作用的细胞定位；以及(Ii)棕榈酰化的作用 肉豆蔻酰化对G蛋白与CRIP1a相互作用的影响。拟议研究的结果将证明 通过表征一类新的载货蛋白质并提供证据，为该领域带来变革 这将促进我们对CRIP1a相互作用如何、何时、何地调控CB1R通路的理解。

PROJECT SUMMARY This new F31 proposal is to support the career development of Erin Hughes who will receive training in cannabinoid receptor biochemical neuropharmacology with Drs. Howlett and Lowther. Recent years have seen a global increase in the usage of marijuana containing the psychoactive constituent Δ9-tetrahydrocannabinol, a partial agonist of the CB1 cannabinoid receptor (CB1R). The CB1R is a G-protein coupled receptor (GPCR) found in the central nervous system and modulates neuroprogenitor development, neural commitment and migration, and neurotransmitter release. GPCR function is dependent upon the ability to change conformations and to transduce a response via G-proteins. This process requires association of a Gα-GDP subunit with Gβγ subunits to form a heterotrimeric G-protein complex that binds to the GPCR. Stimulation of CB1R initiates the exchange of GDP for GTP bound to Gα, activating the G-protein heterotrimer, and releasing the Gα and Gβγ subunits. The released G-protein subunits propagate the signaling cascade through various interactions. Cannabinoid receptor interacting protein 1a (CRIP1a) is a protein found in human neurons at various brain regions, which suppresses CB1R cellular signaling via modulating G-protein activation. The Scientific Premise is that CRIP1a modulation of CB1R can be understood providing us with an in-depth view of the role of CB1R in neuronal cell signaling. CRIP1a can modulate the type of Gα subunit bound to CB1R, however the mechanism of switching is unknown. Previous immunoprecipitation studies demonstrated that the CB1R C-terminal region interacts with CRIP1a, and therefore one possible explanation for CRIP1a-mediated Gα switching may involve formation of a ternary complex between CB1R, subtype-specific Gα proteins, and CRIP1a. The Lowther-Howlett collaboration has determined the high-resolution structure of CRIP1a by X-ray crystallography and found that CRIP1a is a member of the family of carriers for myristoylated or isoprenylated proteins. Based on the knowledge of the structure and function of CRIP1a, I hypothesize that CRIP1a binds N-terminally myristoylated cargo proteins, such as Gαi proteins. I propose to investigate CRIP1a interactions in the N18TG2 neuroblastoma cell model which endogenously expresses the CB1R and associated proteins. The aims of this project are to determine (i) the regulation of the CRIP1a- Gα interaction during agonist stimulation and heterotrimeric G protein dissociation and the cellular localization of such interactions; and (ii) the role of palmitoylation and myristoylation on G-protein interaction with CRIP1a. The results of the proposed studies will prove to be transformative for the field by characterizing a new class of cargo carrying proteins and providing evidence that will advance our understanding of how, when and where CRIP1a interactions modulate CB1R pathways.