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Adenosine A2A Receptor Ligand Binding Experiments by Using Real-time Single-cell FRET
采用实时单细胞荧光共振能量转移(FREET)法进行腺苷A2A受体配体结合实验   

编审
Cheng Zhang Cheng Zhang
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Abstract

We designed a fluorescence resonance energy transfer (FRET)-based approach to study the ligand binding constants of the adenosine A2A receptor (A2AR). Our assay is based in the interaction of a fluorescent A2AR agonist ligand (MRS5424) with an A2AR tagged with the cyan fluorescent protein (CFP) at the N-terminus (i.e. A2ARCFP) and expressed in living cells. Thus, upon fast superfusion of the A2ARCFP expressing cells with MRS5424, the ligand-receptor interaction is determined by single-cell FRET in a real-time mode. Accordingly, our approach allowed immediate ‘real-time’ readout of the ligand-receptor interaction, thus allowing kinetic binding experiments, a feature impossible to achieve using conventional radioisotope-labelled ligands. In addition, since our assay permitted the visual confirmation of receptor localization it also allowed localized saturation binding experiments.

Keywords: FRET(烦恼), Fluorescent ligands(荧光配体), GPCR(G蛋白偶联受体), Real-time binding(实时结合), Kinetics(动力学)

Materials and Reagents

  1. Cell line (i.e. HEK-293 cells)
  2. Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich)
  3. Sodium pyruvate
  4. L-glutamine
  5. Antibiotics: streptomycin and penicillin
  6. Fetal bovine serum
  7. TransFectinTM Lipid Reagent (Bio-Rad Laboratories)
  8. Hank’s balanced salt solution (HBSS) (see Recipes)
  9. Cell culture medium (see Recipes)

Equipment

  1. 18 mm diameter glass coverslips
  2. Attofluor holder
  3. Inverted Axio Observer microscope (ZEISS) equipped with a 63x oil immersion objective
  4. Polychrome V (TILL Photonics)
  5. Avalanche photodiodes (TILL Photonics)
  6. Focal drug application system (ALA Scientific Instruments, OCTAFLOWTM)
  7. Digidata 1440A analog/digital converter (Molecular Devices)

Software

  1. pCLAMP (Molecular Devices)
  2. GraphPad Prism (GraphPad Software)

Procedure

  1. Two days before the experiment, the cells (i.e. HEK-293 cells) were seeded onto 18 mm diameter glass coverslips and transiently transfected with an A2AR construct tagged with the CFP at its N-terminal tail (A2ARCFP) (Figure 1).


    Figure 1. Cell surface localisation of the A2ARCFP construct. HEK-293 cells were transiently transfected with A2ARCFP, fixed and analyzed by confocal microscopy. The A2ARCFP was mainly targeted to the cell surface and scarcely accumulated at the intracellular level (Fernández-Dueñas et al., 2013). Scale bar: 10 µm

  2. The day of the experiment the transiently transfected cells were mounted in an Attofluor holder and placed on an inverted Axio Observer microscope equipped with a 63x oil immersion objective and a dual-emission photometry system.
  3. Then, cells were continuously superfused with a FRET-compatible A2AR fluorescent ligand (i.e. MRS5424) (Fernández-Dueñas et al., 2012) dissolved in HBSS and applied with the aid of a focal drug application system.
  4. A Polychrome V was used as the light source in our dual-emission photometry system. Upon excitation with the corresponding donor excitation wavelength (i.e. A2ARCFP) the fluorescent signals of the donor and acceptor fluorophores were detected by avalanche photodiodes and digitized using a Digidata 1440A analog/digital converter.
  5. pCLAMP and GraphPad Prism softwares were used for data collection and analysis.
  6. Accordingly, a FRET signal was measured upon donor (i.e. A2ARCFP) excitation at 430 ± 10 nm [beam splitter dichroic long-pass (DCLP) 460 nm] and an illumination time set to 10 ms at 10 Hz. Then, the emission light intensities were determined at 535 ± 15 nm (F535; MRS5424 emission) and 480 ± 20 nm (F480; A2ARCFP emission) with a beam splitter DCLP of 505 nm. No corrections for spillover between channels or direct MRS5424 excitation were made.
  7. The increase in FRET ratio (F535/F480) was fitted to the equation: r(t) = A x (1 - e-t/τ), where τ is the time constant (in seconds) and A is the magnitude of the FRET signal (Figure 2). When necessary for calculating τ, agonist-independent changes in FRET due to photobleaching were subtracted (Fernández-Dueñas et al., 2012, Fernández-Dueñas et al., 2013).


    Figure 2. Example of FRET ratio fitting. Time-resolved changes in A2ARCFP and MRS5424 fluorescence emission signals in single cells transfected with A2ARCFP (see Figure 1). The ratio (blue trace) of the emission intensities of the MRS5424 (F535) and CFP (F480) in response to MRS5424 application was recorded from single HEK293 cells expressing the A2ARCFP (see Figure 1). Shown are the changes induced by rapid superfusion with 2 μM MRS5424. The increase of the ratio F535/F480 was fitted by a simple monoexponential curve (r(t) = A x (1 - e-t/τ)) using the GraphPad Prism software which gave a time constant (τ) in this experiment of 14 ± 1 sec. This assay is well suited for competitive ligand binding experiments using non-fluorescent compounds (Fernández-Dueñas et al., 2012, Fernández-Dueñas et al., 2013).

Recipes

  1. HBSS
    137 mM NaCl
    5.4 mM KCl
    0.3 mM Na2HPO4
    0.4 mM KH2PO4
    4.2 mM NaHCO3
    1.3 mM CaCl2
    0.5 mM MgCl2
    0.6 mM MgSO4
    5.6 mM glucose
    pH 7.4
  2. Cell culture medium
    Dulbecco’s modified Eagle’s medium (DMEM) supplemented with:
    1 mM sodium pyruvate
    2 mM L-glutamine
    100 U/ml streptomycin
    100 mg/ml penicillin
    5% (v/v) fetal bovine serum

Acknowledgments

This work was supported by grants SAF2011-24779, Consolider-Ingenio CSD2008-00005 and PCIN-2013-019-C03-03 from Ministerio de Economía y Competitividad and ICREA Academia-2010 from the Catalan Institution for Research and Advanced Studies (to FC), by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Intramural Research Program (to KAJ). FC belong to the “Neuropharmacology and Pain” accredited research group (Generalitat de Catalunya, 2014 SGR 1251). We thank E. Castaño and B. Torrejón from the Scientific and Technical Services (SCT) group at the Bellvitge Campus of the University of Barcelona for their technical assistance.

References

  1. Fernandez-Duenas, V., Gomez-Soler, M., Jacobson, K. A., Kumar, S. T., Fuxe, K., Borroto-Escuela, D. O. and Ciruela, F. (2012). Molecular determinants of A2AR-D2R allosterism: role of the intracellular loop 3 of the D2R. J Neurochem 123(3): 373-384.
  2. Fernández-Dueñas, V., Gómez-Soler, M., Morato, X., Núñez, F., Das, A., Kumar, T. S., Jaumà, S., Jacobson, K. A. and Ciruela, F. (2013). Dopamine D2 receptor-mediated modulation of adenosine A2A receptor agonist binding within the A2AR/D2R oligomer framework. Neurochem Inter 63(1): 42-46.

简介

我们设计了基于荧光共振能量转移(FRET)的方法来研究腺苷A 2A受体(A 2A R)的配体结合常数。 我们的测定基于荧光A 2A 2A R激动剂配体(MRS5424)与在青色荧光蛋白(CFP)标记的A 2A 2A R的相互作用, (即 A R CFP )并在活细胞中表达。 因此,在用MRS5424快速超表达A 2A,R sup CFP表达细胞时,通过单细胞FRET以实时模式确定配体 - 受体相互作用。 因此,我们的方法允许配体 - 受体相互作用的立即"实时"读出,因此允许动力学结合实验,使用常规放射性同位素标记的配体不可能实现的特征。 此外,因为我们的测定允许受体定位的视觉确认,其还允许局部饱和结合实验。

关键字:烦恼, 荧光配体, G蛋白偶联受体, 实时结合, 动力学

材料和试剂

  1. 细胞系(即 HEK-293细胞)
  2. Dulbecco改良的Eagle培养基(DMEM)(Sigma-Aldrich)
  3. 丙酮酸钠
  4. L-谷氨酰胺
  5. 抗生素:链霉素和青霉素
  6. 胎牛血清
  7. TransFectin TM 脂质试剂(Bio-Rad Laboratories)
  8. 汉克平衡盐溶液(HBSS)(参见配方)
  9. 细胞培养基(参见配方)

设备

  1. 18毫米直径的玻璃盖片
  2. Attofluor支架
  3. 配备63x油浸物镜的倒置Axio观察显微镜(ZEISS)
  4. 彩色V(TILL Photonics)
  5. 雪崩光电二极管(TILL Photonics)
  6. 局部药物应用系统(ALA Scientific Instruments,OCTAFLOW TM
  7. Digidata 1440A模拟/数字转换器(Molecular Devices)

软件

  1. pCLAMP(Molecular Devices)
  2. GraphPad Prism(GraphPad软件)

程序

  1. 在实验前两天,将细胞(即HEK-293细胞)接种在18mm直径的玻璃盖玻片上,并用标记有CFP的A 2A 2A R构建体瞬时转染 在其N-末端尾(A 2A 2A R CFP )(图1)。


    图1. A 2A R CFP 构建。将HEK-293细胞瞬时转染A 2A sub R CFP ,固定并通过共聚焦显微镜分析。 A/2A/R/sup> CFP 主要靶向细胞表面,并且在细胞内水平几乎不积累(Fernández-Dueñas等人,2013) 。比例尺:10μm

  2. 实验当天,将瞬时转染的细胞安装在Attofluor保持器中,并置于装备有63x油浸物镜和双发射光度测定系统的倒置Axio Observer显微镜上。
  3. 然后,用FRET相容性A 2A 2A R荧光配体(即EMS MRS5424)连续超表达细胞(Fernández-Dueñaset al。,2012) )溶解在HBSS中并且在局部药物施用系统的辅助下施用。
  4. 在我们的双发射测光系统中使用多色V作为光源。在用相应的施主激发波长(即A 2A 2A R CFP )激发时,供体和受体荧光团的荧光信号通过雪崩光电二极管并使用Digidata 1440A模拟/数字转换器进行数字化。
  5. pCLAMP和GraphPad Prism软件用于数据收集和分析。
  6. 因此,在430±10nm处的供体(即 A R CFP )激发下测量FRET信号[分束器二向色长通(DCLP)460nm],并且照明时间在10Hz设置为10ms。然后,在535±15nm(F <535; MRS5424发射)和480±20nm(F <480> A <2A>)的条件下测定发射光强度,使用505nm的分束器DCLP。没有对通道之间的溢出或直接MRS5424激发进行校正。
  7. 将FRET比率(F 535/F 480)的增加拟合到等式:r(t)= A×(1-e -t /τ ),其中τ是时间常数(以秒为单位),A是FRET信号的幅度(图2)。当需要计算τ时,减去由于光漂白引起的FRET中激动剂无关的变化(Fernández-Dueñas等人,2012,Fernández-Dueñas等人,2013) 。


    图2.FRET比率拟合的实施例。在A细胞转染的单个细胞中,A R CFP 和MRS5424荧光发射信号的时间分辨的变化(参见图1)。记录响应于MRS5424应用的MRS5424(F 535)和CFP(F ))的发射强度与单个HEK293细胞的比率(蓝色曲线) (参见图1)。显示了用2μMMRS5424通过快速表面灌流诱导的变化。比率F 535/F 480的增加通过简单的单指数曲线拟合(r(t)= A×(1-e -t /τ )),使用GraphPad Prism软件,其在本实验中给出14±1秒的时间常数(τ)。该测定法非常适合于使用非荧光化合物的竞争性配体结合实验(Fernández-Dueñaset al。,2012,Fernández-Dueñaset al。,2013)。< br />

食谱

  1. HBSS
    137 mM NaCl 5.4 mM KCl
    0.3mM Na 2 HPO 4
    0.4mM KH 2 PO 4 sub/
    4.2mM NaHCO 3 3/v/v 1.3mM CaCl 2 0.5mM MgCl 2 sub 0.6mM MgSO 4 5.6mM葡萄糖 pH 7.4
  2. 细胞培养基
    Dulbecco改良的Eagle培养基(DMEM),补充有:
    1mM丙酮酸钠 2mM L-谷氨酰胺 100U/ml链霉素 100mg/ml青霉素
    5%(v/v)胎牛血清

致谢

这项工作得到了来自加拿大研究和高等研究机构(FC)的经济部长奖学金SAF2011-24779,Consolider-Ingenio CSD2008-00005和PCIN-2013-019-C03-03以及加泰罗尼亚研究与高等研究学院ICREA学术成员2010 ,由国家糖尿病和消化和肾脏疾病研究所(NIDDK)校内研究计划(KAJ)。 FC属于"神经药理学和疼痛"认证研究组(Generalitat de Catalunya,2014 SGR 1251)。我们感谢巴塞罗那大学Bellvitge校区科学技术服务(SCT)小组的E.Castaño和B.Torrejón的技术援助。

参考文献

  1. Fernandez-Duenas,V.,Gomez-Soler,M.,Jacobson,K.A.,Kumar,S.T.,Fuxe,K.,Borroto-Escuela,D.O.和Ciruela,F。(2012)。 A 2A RD 2 的分子决定因素 R变构性:D 2 r R的细胞内环3的作用。神经化学123(3):373-384。
  2. Fernández-Dueñas,V.,Gómez-Soler,M.,Morato,X.,Núñez,F.,Das,A.,Kumar,T. S.,Jaumà,S.,Jacobson,K.A。和Ciruela, 多巴胺D 2受体介导的腺苷A 2A的调节 受体激动剂结合在A 2A R/D 2 R寡聚体框架内。神经化学杂志63(1) :42-46。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Fernández-Dueñas, V., Jacobson, K. A. and Ciruela, F. (2014). Adenosine A2A Receptor Ligand Binding Experiments by Using Real-time Single-cell FRET. Bio-protocol 4(6): e1070. DOI: 10.21769/BioProtoc.1070.
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