监测GPCR-β-阻丁1/2实时生命系统中的相互作用,加速药物发现
Summary
GPCR-β-阻尼相互作用是GPCR药物发现中的一个新兴领域。精确、精确和易于设置的方法对于监控生活系统中的这种相互作用是必要的。我们展示了一种结构补体测定,以监测实时活细胞中的GPCR-β-阻尼蛋白相互作用,并可扩展到任何GPCR。
Abstract
G-蛋白耦合受体(GPCRs)和β-失能素之间的相互作用是具有非常重要生理影响的重要过程。目前,在GPCR药物发现领域,特别是在GPCR偏置症研究期间,新药物的表征与β-逮捕蛋白和其他细胞蛋白的相互作用具有极其宝贵的价值。在这里,我们展示了一种新型结构补体测定的应用,以准确监测实时生命系统中的受体-β-逮捕素相互作用。该方法简单、准确,易于扩展到任何感兴趣的GPCR,而且它的优点是克服了由于每个向量系统中存在低表达启动子而导致的非特定交互。这种结构补加测定提供了关键特性,能够准确、准确地监测受体-β-失素相互作用,使其适用于研究任何GPCR系统的偏置性痛苦以及GPCR c-终点的磷酸化由不同的GPCR-激酶(GRKs)和后翻译修改的arrestins,稳定或稳定受体-β-逮捕复合物的代码。
Introduction
GPCRs代表了市场上近35%的当前药物的目标1、2,而对其药理学的清晰了解对于开发新型治疗药物3至关重要。GPCR药物发现的关键方面之一,特别是在偏置激动剂的发育过程中,是新配体对受体-β-逮捕素相互作用4和β-逮捕蛋白与其他细胞蛋白相互作用的表征,例如作为克拉特林5。
据记载,β-arrestin依赖信号在神经紊乱(如双相情感障碍、严重抑郁症和精神分裂症6)中起关键作用,在一些药物(如吗啡7)中也有严重的副作用。
目前用于监测这些相互作用的方法通常不代表研究中蛋白质的实际内源水平,在某些情况下,它们显示信号弱、光漂白,并且根据GPCR,建立8可能具有技术挑战性。这种新颖的结构补体测定使用低表达促进因子载体来模拟内源性生理水平,与目前的方法9相比,具有较高的灵敏度。使用这种方法,可以容易地描述Galanin受体-β-逮捕1/2和β-逮捕2-克拉林相互作用10。这种方法可以广泛用于任何特别感兴趣的GPCR,其中β-逮捕蛋白发挥关键的生理功能或其信号在某些疾病中相关。
Protocol
1. 引性设计策略 设计引源,将感兴趣的基因引入 pBiT1.1-C [TK/LgBiT]、pBiT2.1-C [TK/SmBiT]、pBiT1.1-N [TK/LgBiT] 和 pBiT2.1-N [TK/SmBiT] 矢量图。 选择这三个位点中至少一个作为定向克隆所需的两种唯一限制酶之一,因为存在一个帧内停止通子,该块子将多克隆位点分开,如图11所示。 如表1所示,将核苷酸序列纳入引物中,以编码表2</strong…
Representative Results
使用此处介绍的程序,监测了典型GPCR和两种β-逮捕蛋白异体之间的相互作用。胰高血糖素类似肽受体(GLP-1r)结构是使用含有NheI和EcoRI酶限制性位点的引基体,并克隆到载体pBiT1.1-C[TK/LgBiT]和pBiT2.1-C[TK/SmBiT],而在β-阻尼的情况下,另外两个载体是在β-逮捕2和NheI和XhoI的情况下使用pBiT1-N[TK/LgBiT]和pBiT2.1-N[TK/SmBiT]在β-逮捕1的情况下使用酶抑制位BgIII和EcoRI。HEK293细胞在N-或C端接端使用GLP-1r-LgBiT/SmBiT和50 ng的β-阻…
Discussion
使用此处介绍的方法,使用此 GPCR-β-逮捕蛋白结构补充测定,可在实时生命系统中监控任何 GPCR 和 β-阻尼1/2之间的相互作用。在这方面,我们能够观察到GLP-1r(一种典型的B类GPCR)的两种β-逮捕素异种的招募,我们还观察到受体-β-逮捕复合物在达到最大值后几分钟内分离发光信号。
为了在结构补性测定系统中具有最佳灵敏度,在受体和每个β-阻尼素异构体之间对它进行了四种不同的空…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作得到了韩国国家研究基金会(NRF)(NRF-2015M3A9E7029172)的资助,该研究由科学、ICT和未来规划部资助。
Materials
| Antibiotics penicillin streptomycin | Welgene | LS202-02 | Penicillin/Streptomycin |
| Bacterial Incubator | JEIO Tech | IB-05G | Incubator (Air-Jacket), Basic |
| Cell culture medium | Welgene | LM 001-05 | DMEM Cell culture medium |
| Cell culture transfection medium | Gibco | 31985-070 | Optimem 1X cell culture medium |
| CO2 Incubator | NUAIRE | NU5720 | Direct Heat CO2 Incubator |
| Digital water bath | Lab Tech | LWB-122D | Digital water bath lab tech |
| DNA Polymerase proof reading | ELPIS Biotech | EBT-1011 | PfU DNA polymerase |
| DNA purification kit | Cosmogenetech | CMP0112 | miniprepLaboPass Purificartion Kit Plasmid Mini |
| DNA Taq Polymerase | Enzynomics | P750 | nTaq DNA polymerase |
| Enzyme restriction BglII | New England Biolabs | R0144L | BglII |
| Enzyme restriction buffer | New England Biolabs | B72045 | CutSmart 10X Buffer |
| Enzyme restriction EcoRI | New England Biolabs | R3101L | EcoRI-HF |
| Enzyme restriction NheI | New England Biolabs | R01315 | NheI |
| Enzyme restriction XhoI | New England Biolabs | R0146L | XhoI |
| Fetal Bovine Serum | Gibco Canada | 12483020 | Fetal Bovine Serum |
| Gel/PCR DNA MiniKit | Real Biotech Corporation | KH23108 | HiYield Gel/PCR DNA MiniKit |
| Ligase | ELPIS Biotech | EBT-1025 | T4 DNA Ligase |
| Light microscope | Olympus | CKX53SF | CKX53 Microscope Olympus |
| lipid transfection reagent | Invitrogen | 11668-019 | Lipofectamine 2000 |
| Luminometer | Biotek/Fisher Scientific | 12504386 | Synergy 2 Multi-Mode Microplate Readers |
| NanoBiT System | Promega | N2014 | NanoBiT PPI MCS Starter System |
| Nanoluciferase substrate | Promega | N2012 | Nano-Glo Live Cell assay system |
| PCR Thermal cycler | Eppendorf | 6336000015 | Master cycler Nexus SX1 |
| Poly-L-lysine | Sigma Aldrich | P4707-50ML | Poly-L-lysine solution |
| Trypsin EDTA | Gibco | 25200-056 | Trysin EDTA 10X |
| White Cell culture 96 well plates | Corning | 3917 | Assay Plate 96 well plate |
Referencias
- Sriram, K., Insel, P. A. GPCRs as targets for approved drugs: How many targets and how many drugs?. Molecular Pharmacology. 93 (4), 251-258 (2018).
- Hauser, A. S., Attwood, M. M., Rask-Andersen, M., Schiöth, H. B., Gloriam, D. E. Trends in GPCR drug discovery: new agents, targets and indications. Nature Reviews Drug Discovery. 16 (12), 829-842 (2017).
- Langmead, C. J., Summers, R. J. Molecular pharmacology of GPCRs. British Journal of Pharmacology. 175 (21), 1754005-1754008 (2018).
- Lohse, M. J., Hoffmann, C. Arrestin Interactions with G Protein-Coupled Receptors. Handbook of Experimental Pharmacology. 219, 15-56 (2014).
- Kang, D. S., et al. Structure of an arrestin2-clathrin complex reveals a novel clathrin binding domain that modulates receptor trafficking. Journal of Biological Chemistry. 284, 29860-29872 (2009).
- Park, S. M., et al. Effects of β-Arrestin-Biased Dopamine D2 Receptor Ligands on Schizophrenia-Like Behavior in Hypoglutamatergic Mice. Neuropsychopharmacology. 41 (3), 704-715 (2016).
- Zhu, L., Cui, Z., Zhu, Q., Zha, X., Xu, Y. Novel Opioid Receptor Agonists with Reduced Morphine-like Side Effects. Mini-Reviews in Medicinal Chemistry. 18 (19), 1603-1610 (2018).
- Smith, J. S., Lefkowitz, R. J., Rajagopal, S. Biased signalling: from simple switches to allosteric microprocessors. Nature Reviews Drug Discovery. 17 (4), 243-260 (2018).
- Dixon, A. S. NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chemical Biology. 11 (2), 400-408 (2016).
- Reyes-Alcaraz, A., Lee, Y. N., Yun, S., Hwang, J. I., Seong, J. Y. Conformational signatures in β-arrestin2 reveal natural biased agonism at a G-protein-coupled receptor. Communications Biology. 3, 1-128 (2018).
- Promega. . Nanobit Protein Protein Interaction System Protocol. , (2019).
- Life Biomedical. . HiYield Gel/PCR Fragments Extraction Kit. , (2019).
- New England BioLabs. . Ligation Calculator. , (2019).
- . . Cosmo Genetech. , (2019).
- Baggio, L. L., Drucker, D. J. Biology of incretins: GLP-1 and GIP. Gastroenterology. 132, 2131-2157 (2007).
- ProMega. . NanoGLO Endurazine and Vivazine Live Cell Substrates Technical Manual. , (2019).
- Ali, R., Ramadurai, S., Barry, F., Nasheuer, H. P. Optimizing fluorescent protein expression for quantitative fluorescence microscopy and spectroscopy using herpes simplex thymidine kinase promoter sequences. FEBS Open Bio. 8 (6), 1043-1060 (2018).
Citar este artículo
Reyes-Alcaraz, A., Lee, Y., Yun, S., Hwang, J., Seong, J. Y. Monitoring GPCR-β-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery. J. Vis. Exp. (148), e59994, doi:10.3791/59994 (2019).