配体激活的G蛋白偶联受体内在化的检测通过共聚焦显微镜
Summary
这个协议描述了在哺乳动物细胞中的G蛋白偶联受体（GPCR）的内化共聚焦显微镜检测。它包括基本细胞培养，转染，和共聚焦显微镜的程序和提供了一个高效和容易解释的方法来检测融合表达GPCR的亚细胞定位和内化。
Abstract
Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.
Introduction
细胞具有贩卖货物机械运送细胞外材料-如配体，微生物，营养素，和跨膜蛋白，成信息，能源和其他目的1，2中的单元格。它是细胞内环境稳定，组织的功能和整体细胞存活的关键。荧光融合蛋白的基于细胞的表达是一个功能强大的方法来研究的定位和跨膜受体，如G蛋白偶联受体（GPCR），内化在信号通路3。
从水母Aequorea victoria的标记有绿色荧光蛋白（GFP）的融合蛋白的表达，与直接荧光检测技术相结合，在已获得广泛的接受蛋白靶向研究4，5，6。基于GFP-detecti上具有允许活细胞，同时避免固定的工件7的实时成像的优点。一系列通用的克隆载体的已被构造成便于在各种细胞中8蛋白质融合到GFP的表达，9。的质粒pEGFP-N1载体是编码增强型绿色荧光蛋白（EGFP），它已被从野生型GFP优化用于在哺乳动物细胞更亮的荧光和更高的表达一种广泛使用的并商业化的质粒载体。观察EGFP的荧光信号在哺乳动物细胞中表达时，荧光显微术，应在488nm处激发和在507 nm处的发射，最好用共聚焦显微镜下进行。
监测的亚细胞定位和受体配体-介导的内是一种常用的技术来研究的GPCR 10的信号转导和功能</s向上>，11。在大多数情况下，内化导致的GPCR脱敏（尽管激动剂的存在下的刺激反应的衰减）12，13。内化受体可以结合到溶酶体和降解，或它们可以被再循环回到细胞表面，这取决于受体和在实验中使用的细胞系的性质。
促性腺激素释放激素受体（GnRHRs）属于GPCR家族，并具有典型的GPCR蛋白结构，具有细胞外氨基末端，一个细胞内-COOH终端，和七个跨膜（TM）结构域14。重组质粒Sj的 GnRH受体-EGFP的转染（从曼氏粳稻的促性腺激素释放激素受体基因）和EGFP标记的GnRH受体Sj的 （在HEK293细胞中表达）的共焦显微镜检测报道上10sly和GFP荧光确立为跨膜蛋白定位试验15个记者。现在，SJ GnRH受体的内在化，通过S.粳稻促性腺激素释放激素（GnRH的Sj的 ）激活，表现在通过共聚焦显微镜时间和剂量依赖性。
Protocol
Representative Results
Discussion
这里介绍的协议提供了一种有效和容易解释的方法来检测在HEK293细胞中的亚细胞定位和表达GPCR融合蛋白的内在化。该技术可以容易地适用于许多不同基因和细胞类型，诸如用于从家蚕在HEK293和家蚕细胞16 corazonin受体的细胞定位和内化。
这个功能强大的试验的成功应用依赖于几个关键因素。最重要的是细胞的身体健康，这是转染效率和数据质量至关重?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors of this paper would like to thank Prof. Jiayan Xie for technical assistance and equipment usage. This work was financially supported by the National Natural Science Foundation of China (41406137).
Materials
| HEK293 cell line | |||
| DMEM/High glucose | Hyclone | SH30022.01 | High glucose 4.0 mM L-glutamine |
| Foetal Bovine Serum (FBS) | PuFei | 1101-100 | |
| Phosphate Buffered Saline (PBS) | Genom | GNM10010 | |
| Penicillin-Streptomycin | Genom | GNM15140 | |
| 0.25% Trypsin & 0.02% EDTA | Genom | GNM25200 | |
| Opti-MEM (1X) | GIBCO, by Life Technologies | 31985-062 | reduced serum media |
| Lipofectamine 2000 Transfection Reagent | Invitrogen | 11668027 | Reagent 1 |
| X-tremeGENE HP DNA Transfection Reagent | Roche | 6366236001 | Reagent 2 |
| DAPI Staining Solution | Beyotime | C1006 | |
| DiI | Beyotime | C1036 | |
| Antifade Mounting Medium | Beyotime | P0126 | |
| Paraformaldehyde | Sinopharm Chemical Reagent Co.(SCRC) | 80096618 | |
| 100 mm cell culture | CORNING | 430167 | |
| 6-well plate | ExCell Bio | CS016-0092 | |
| 12-well plate | ExCell Bio | CS016-0093 | |
| Cover Glass | NEST | 801007 | |
| Microscope Slides | Citoglas | 10127105P-G | |
| Thermo Scientific Forma CO2 Incubator | Thermo | 3111 | |
| TCS SP5II laser scanning confocal microscope | Leica | 5100001311 |
References
- Mellman, I. Endocytosis and molecular sorting. Annu. Rev. Cell Dev. Biol. 12, 575-625 (1996).
- Hausott, B., Klimaschewski, L. Membrane turnover and receptor trafficking in regenerating axons. Eur J Neuro. 43, 309-317 (2016).
- Kallal, L., Benovic, J. L. Using green fluorescent proteins to study G-protein-coupled receptor localization and trafficking. Trends Pharmacol Sci. 21, 175-180 (2000).
- Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., Cormier, M. J. Primary structure of the Aequorea victoria green-fluorescent protein. Gene. 111, 229-233 (1992).
- Spector, D. L., Goldman, R. D. Constructing and Expressing GFP Fusion Proteins. CSH protocols. 2006, (2006).
- Drew, D. E., von Heijne, G., Nordlund, P., de Gier, J. W. Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli. FEBS Lett. 507, 220-224 (2001).
- von Arnim, A. G., Deng, X. W., Stacey, M. G. Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants. Gene. 221, 35-43 (1998).
- Webb, C. D., Decatur, A., Teleman, A., Losick, R. Use of green fluorescent protein for visualization of cell-specific gene expression and subcellular protein localization during sporulation in Bacillus subtilis. J Bacteriol. 177, 5906-5911 (1995).
- Kain, S. R., et al. Green fluorescent protein as a reporter of gene expression and protein localization. BioTechniques. 19, 650-655 (1995).
- Fukunaga, S., Setoguchi, S., Hirasawa, A., Tsujimoto, G. Monitoring ligand-mediated internalization of G protein-coupled receptor as a novel pharmacological approach. Life Sci. 80, 17-23 (2006).
- Yang, J., et al. Agonist-Activated Bombyx Corazonin Receptor Is Internalized via an Arrestin-Dependent and Clathrin-Independent Pathway. Biochemistry. 55, 3874-3887 (2016).
- Kohout, T. A., Lefkowitz, R. J. Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization. Mol Pharmacol. 63, 9-18 (2003).
- Ferguson, S. S., et al. Role of beta-arrestin in mediating agonist-promoted G protein-coupled receptor internalization. Science. 271, 363-366 (1996).
- Stojilkovic, S. S., Reinhart, J., Catt, K. J. Gonadotropin-releasing hormone receptors: structure and signal transduction pathways. Endocr Rev. 15, 462-499 (1994).
- Yan, Y. J., et al. Identification and Expression Profile of the Gonadotropin-Releasing Hormone Receptor in Common Chinese Cuttlefish, Sepiella japonica. J Exp Zool A Ecol Genet Physiol. , (2016).
- Yang, J., et al. Specific activation of the G protein-coupled receptor BNGR-A21 by the neuropeptide corazonin from the silkworm, Bombyx mori, dually couples to the G(q) and G(s) signaling cascades. J Biol Chem. 288, 11662-11675 (2013).
- Lu, Z. M., et al. Cloning, Characterization, and Expression Profile of Estrogen Receptor in Common Chinese Cuttlefish, Sepiella japonica. J Exp Zool A Ecol Genet Physiol. 325, 181-193 (2016).
