用近距离标记分析脂质GPCR分子相互作用的分析
Xian Zhao1,2, Hideru Obinata3
1Department of Biochemistry, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.
Methods in molecular biology (Clifton, N.J.)
|July 8, 2024
概括
这项研究引入了使用TurboID或miniTurbo的近距离标记方法,以识别与G蛋白结合受体 (GPCR) 相互作用的蛋白质. 这种技术克服了研究疏水性GPCR及其信号合作伙伴的挑战.
科学领域:
- 生物化学 生物化学
- 细胞生物学 细胞生物学
- 蛋白质组学是指蛋白质组学.
背景情况:
- G蛋白结合受体 (GPCR) 是关键的跨膜蛋白,参与细胞信号通路.
- 研究GPCR分子相互作用至关重要,但由于它们的疏水性和动态相互作用,具有挑战性.
- 传统的共同免疫沉方法经常与GPCR相互作用蛋白的亲和性净化作斗争.
研究的目的:
- 用近距离标记来描述与GPCRs相互作用的蛋白质映射的协议.
- 调整TurboID和miniTurbo系统以识别活细胞中的GPCR相关蛋白质.
- 为了能够分析脂质作用的GPCRs的分子相互作用.
主要方法:
- 利用TurboID和miniTurbo,工程化生物素结合酶,用于接近目标GPCRs附近的蛋白质的靠近依赖生物化.
- 开发了一种标记,生物化蛋白质的净化和样本准备用于蛋白质组分析的协议.
- 作为模型脂质GPCR,使用了斯芬哥辛-1-酸盐受体1 (S1PR1).
主要成果:
- 成功地证明了TurboID/miniTurbo的应用,用于标记与GPCR非常接近的蛋白质.
- 能够有效净化与模型GPCR,S1PR1.1.相互作用的生物化蛋白质.
- 建立了活细胞中GPCR相互作用伙伴的蛋白质组分析管道.
结论:
- 使用TurboID/miniTurbo的近距离标记提供了一种强大的方法来克服研究GPCR分子相互作用的挑战.
- 这种方法有助于全面地绘制与脂质GPCRs相关的蛋白质.
- 描述的协议促进了对GPCR信号网络的理解.
更多相关视频
16:16Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors
Published on: September 13, 2013
15.2K
14:02Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells
Published on: April 9, 2018
8.5K
相关概念视频
G Protein-coupled Receptors
11.7K
G Protein-Coupled Receptors or GPCRs are membrane-bound receptors that transiently associate with heterotrimeric G proteins and induce an appropriate response to sensory stimuli such as light, odors, hormones, cytokines, or neurotransmitters.
GPCRs are also called heptahelical, 7TM, or serpentine receptors, and consist of seven (H1-H7) transmembrane alpha-helices that span the bilayer to form a cylindrical core. The transmembrane helices are connected by three extracellular loops and three...
GPCRs are also called heptahelical, 7TM, or serpentine receptors, and consist of seven (H1-H7) transmembrane alpha-helices that span the bilayer to form a cylindrical core. The transmembrane helices are connected by three extracellular loops and three...
11.7K
G-protein Coupled Receptors
117.9K
G-protein coupled receptors are ligand binding receptors that indirectly affect changes in the cell. The actual receptor is a single polypeptide that transverses the cell membrane seven times creating intracellular and extracellular loops. The extracellular loops create a ligand specific pocket which binds to neurotransmitters or hormones. The intracellular loops holds onto the G-protein.
117.9K
Transducer Mechanism: G Protein–Coupled Receptors
1.9K
G Protein–Coupled Receptors (GPCRs) are membrane-bound receptors that transiently associate with heterotrimeric G proteins and induce an appropriate response to various stimuli. GPCRs regulate critical physiological pathways and are excellent drug targets for treating diseases such as diabetes, cancer, obesity, depression, or Alzheimer's. Nearly 35% of approved drugs implement their therapeutic effects by selectively interacting with specific GPCRs.
GPCRs are also called heptahelical,...
GPCRs are also called heptahelical,...
1.9K
GPCR Desensitization
5.9K
G protein-coupled receptor (GPCR) signaling plays a crucial role in cell functioning. GPCR desensitization is an equally essential process. It allows cells to respond to changing environments and regain sensitivity to new stimuli while preventing unnecessary stimulation when no longer needed. Prolonged exposure to stimuli leads to GPCR desensitization. It involves blocking the receptors from binding and activating additional G proteins. This inhibits activation of downstream effectors, thereby...
5.9K
Protein-protein Interfaces
12.5K
Many proteins form complexes to carry out their functions, making protein-protein interactions (PPIs) essential for an organism's survival. Most PPIs are stabilized by numerous weak noncovalent chemical forces. The physical shape of the interfaces determines the way two proteins interact. Many globular proteins have closely-matching shapes on their surfaces, which form a large number of weak bonds. Additionally, many PPIs occur between two helices or between a surface cleft and a...
12.5K
