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GPCRs Interaction Measurement by Fluorescence Resonance Energy Transfer (FRET)
通过荧光共振能量转移(FRET)测定G卵白偶联受体家族(GPCR)的相互作用   

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Abstract

This is a protocol to determine the physical interaction of a G-protein coupled receptor (GPCR) with itself (homodimerization) or with other GPCR (heterodimerazation) using fluorescence resonance energy transfer (FRET). FRET is a distance-dependent interaction between the electronic excited states of two dye molecules (in this case, CFP and YFP) in which excitation is transferred from a donor molecule (CFP) to an acceptor (YFP) molecule without emission of a photon that can be used to determine interaction among YFP- and CFP-tagged GPCRs. Nowadays, FRET microscopy technique can be used to determine interaction between any proteins that retain biological function when expressed as a fusion to the fluorescent protein.

Keywords: Fluorescent microscopy(荧光显微镜), Hormonal receptors(激素受体), Protein interaction(蛋白质相互作用), Somatostatin(生长抑素), High-resolution(高分辨率)

Materials and Reagents

  1. A cell line lacking the expression of the GPCR of interest
  2. Expression plasmid containing E-CFP
  3. Expression plasmid containing E-YFP
  4. Expression plasmid containing E-YFP and E-GFP coupled in frame (CFP-YFP or Positive control)
  5. Expression plasmid containing the first GPCR of interest tagged with E-CFP (GPCR1-CFP)
  6. Expression plasmid containing the second GPCR of interest tagged with E-YFP (GPCR2-YFP)
  7. Lipofectamine 2000
  8. 4% paraformaldehyde
  9. PBS
  10. Fluoromount
  11. Poly-l-lysine

Equipment

  1. Round coverslips coated with poly-l-lysine
  2. Nikon Eclipse TE2000 E scope equipped with a 400 DCLP dichroic filter (Chroma)
  3. ORCA II BT digital camera

Software

  1. MetaMorph software (Imaging Corporation)
  2. Image J software

Procedure

  1. Plate cells at 100,000 cells/ml onto round coverslips previously coated with poly-l-lysine.
  2. Transfect the cells with 1 μg of each plasmid using Lipofectamine 2000, 48 h after plating. The following transfections are needed:
    1. GPCR1-CFP + GPCR2-YFP
    2. CFP-YFP (Positive control)
    3. CFP (Donor)
    4. YFP (Acceptor)
    5. CFP + YFP (Negative control)
  3. 24 h after transfection, eliminate the culture medium and fix the cells for 5 min in 4% paraformaldehyde.
  4. Rinsed twice in PBS.
  5. Mount onto a slide using Fluoromount to reduce photobleaching.
  6. Acquire images of cells transfected with each pair of receptors of interest and controls with an inverted Nikon Eclipse TE2000 E scope equipped with a 400 DCLP dichroic filter and recorded with an ORCA II BT digital camera, both controlled with MetaMorph software (Imaging Corporation). Specifically, acquire three sequential images at 1 sec of exposure with the suitable filters sets for the Donor (E-CFP; excitation at 440 and emission at 510 nm), Acceptor (E-YFP/F46L; excitation at 495 and emission at 540 nm) and raw FRET (excitation at 440 and emission at 540 nm) and under a 60x oil immersion objective.
  7. Quantify the signal intensity of each cell using the Image J software in order to calculate the net FRET by using the three filters method with the methodology developed previously (Farhan et al., 2004). Specifically, Raw FRET images are corrected with the fully specified bleedthrough method, using the following equation: FRET = Raw FRET - [Acceptor - (DA * Donor)] * [AF] - [Donor - (AD* Acceptor)] * [DF], where DA is the proportion in which the donor signal contributes to the acceptor, AD is the proportion in which the acceptor signal contributes to the donor, AF is the proportion in which the acceptor contributes to the raw FRET signal, and DF is the proportion in which the donor contributes to the raw FRET signal. These coefficients were calculated from cells expressing Donor (E-CFP) or Acceptor (E-YFP) alone. Specifically, DA is the proportion of Acceptor signal to Donor signal in cells expressing Donor (E-CFP) alone; AD is the proportion of Donor signal to Acceptor signal in cells expressing Acceptor (E-YFP) alone; DF is the proportion of raw FRET to Donor signal in cells expressing Donor (E-CFP) alone; AF is the proportion of raw FRET to Acceptor signal in cells expressing Acceptor (E-YFP) alone.
  8. FRET efficiency is calculated in relation to the positive control consisting in a vector with E-CFP and E-YFP coupled in frame (CFP-YFP), which provided the upper FRET efficiency limit.
  9. For image analysis and coefficient calculation, background is always subtracted in each picture. A 1:1 E-YFP/E-CFP ratio and equal E-YFP and E-CFP intensities between all samples need to be used for FRET measurements.

Acknowledgments

This protocol is adapted from Duran-Prado et al. (2007) and Duran-Prado et al. (2012).

References

  1. Duran-Prado, M., Bucharles, C., Gonzalez, B. J., Vazquez-Martinez, R., Martinez-Fuentes, A. J., Garcia-Navarro, S., Rhodes, S. J., Vaudry, H., Malagon, M. M. and Castano, J. P. (2007). Porcine somatostatin receptor 2 displays typical pharmacological sst2 features but unique dynamics of homodimerization and internalization. Endocrinology 148(1): 411-421.
  2. Duran-Prado, M., Gahete, M. D., Hergueta-Redondo, M., Martinez-Fuentes, A. J., Cordoba-Chacon, J., Palacios, J., Gracia-Navarro, F., Moreno-Bueno, G., Malagon, M. M., Luque, R. M. and Castano, J. P. (2012). The new truncated somatostatin receptor variant sst5TMD4 is associated to poor prognosis in breast cancer and increases malignancy in MCF-7 cells. Oncogene 31(16): 2049-2061.
  3. Farhan, H., Korkhov, V. M., Paulitschke, V., Dorostkar, M. M., Scholze, P., Kudlacek, O., Freissmuth, M. and Sitte, H. H. (2004). Two discontinuous segments in the carboxyl terminus are required for membrane targeting of the rat gamma-aminobutyric acid transporter-1 (GAT1). J Biol Chem 279(27): 28553-28563.

简介

这是使用荧光共振能量转移(FRET)确定G蛋白偶联受体(GPCR)与其自身(同源二聚化)或与其它GPCR(异源二聚体化)的物理相互作用的方案。 FRET是两种染料分子(在这种情况下,CFP和YFP)的电子激发态之间的距离依赖性相互作用,其中激发从供体分子(CFP)转移到受体(YFP)分子而不发射光子 可以用于确定YFP-和CFP-标记的GPCR之间的相互作用。 现在,FRET显微技术可以用于确定当表达为与荧光蛋白融合时保留生物学功能的任何蛋白质之间的相互作用。

关键字:荧光显微镜, 激素受体, 蛋白质相互作用, 生长抑素, 高分辨率

材料和试剂

  1. 缺乏感兴趣的GPCR表达的细胞系
  2. 含有E-CFP的表达质粒
  3. 含有E-YFP的表达质粒
  4. 含有E-YFP和E-GFP偶联的表达质粒(CFP-YFP或阳性对照)
  5. 包含用E-CFP(GPCR1-CFP)标记的感兴趣的第一GPCR的表达质粒
  6. 包含用E-YFP(GPCR2-YFP)标记的感兴趣的第二GPCR的表达质粒
  7. Lipofectamine 2000
  8. 4%多聚甲醛
  9. PBS
  10. Fluoromount
  11. 聚-l-赖氨酸

设备

  1. 涂有聚-l-赖氨酸的圆形盖玻片
  2. Nikon Eclipse TE2000 E示波器配有400 DCLP二向色滤光片(Chroma)
  3. ORCA II BT数码相机

软件

  1. MetaMorph软件(Imaging Corporation)
  2. Image J软件

程序

  1. 将细胞以100,000细胞/ml接种到先前涂覆有聚-l-赖氨酸的圆形盖玻片上
  2. 在接种后48小时,使用Lipofectamine 2000,用1μg每种质粒转染细胞。 需要以下转染:
    1. GPCR1-CFP + GPCR2-YFP
    2. CFP-YFP(阳性对照)
    3. CFP(捐助者)
    4. YFP(Acceptor)
    5. CFP + YFP(阴性对照)
  3. 转染后24小时,消除培养基,并在4%多聚甲醛中固定细胞5分钟
  4. 在PBS中冲洗两次。
  5. 使用Fluoromount安装到载玻片上以减少光漂白
  6. 获得用每对感兴趣的受体和对照,用配备有400DCLP二向色滤光片的倒置Nikon Eclipse TE2000 E显微镜转染的细胞的图像,并用ORCA II BT数字照相机记录,二者均用MetaMorph软件(Imaging Corporation)控制。具体地,使用合适的用于供体的过滤器组(E-CFP;在440处激发和在510nm处发射),受体(E-YFP/F46L;在495激发和540nm处发射)获得在1秒曝光的三个连续图像)和原始FRET(在440激发和在540nm发射)和在60x油浸物镜下
  7. 使用Image J软件对每个细胞的信号强度进行定量,以便通过使用具有先前开发的方法的三个滤波器方法来计算净FRET(Farhan等人,2004)。具体地,使用以下公式用完全指定的渗透法校正原始FRET图像:FRET =原始FRET- [受体 - (DA *供体)] * [AF] - [供体 - (AD *受体)] * [DF ],其中DA是供体信号对受体有贡献的比例,AD是受体信号对供体有贡献的比例,AF是受体对原始FRET信号有贡献的比例,DF是供体对原始FRET信号有贡献的比例。这些系数由单独表达供体(E-CFP)或受体(E-YFP)的细胞计算。具体来说,DA是单独表达供体(E-CFP)的细胞中受体信号与供体信号的比例; AD是单独表达受体(E-YFP)的细胞中供体信号与受体信号的比例; DF是单独表达供体(E-CFP)的细胞中原始FRET与供体信号的比例; AF是单独表达受体(E-YFP)的细胞中原始FRET与受体信号的比例
  8. FRET效率相对于阳性对照计算,所述阳性对照在具有E-CFP和E-YFP联合框架(CFP-YFP)的载体中,其提供了较高的FRET效率极限。
  9. 对于图像分析和系数计算,在每个图像中总是减去背景。 所有样品之间需要使用1:1的E-YFP/E-CFP比和相等的E-YFP和E-CFP强度进行FRET测量。

致谢

该协议改编自Duran-Prado等人(2007)和Duran-Prado等人(2012)。

参考文献

  1. Duran-Prado,M.,Bucharles,C.,Gonzalez,B.J.,Vazquez-Martinez, R.,Martinez-Fuentes,A.J.,Garcia-Navarro,S.,Rhodes,S.J.,Vaudry, H.,Malagon,M.M.and Castano,J.P。(2007)。 猪  生长抑素受体2显示典型的药理学sst2特征 但是同源二聚化和内化的独特动力。内分泌学 148(1):411-421。
  2. Duran-Prado,M.,Gahete,MD,Hergueta-Redondo,M.,Martinez-Fuentes,AJ,Cordoba-Chacon,J.,Palacios,J.,Gracia-Navarro,F.,Moreno-Bueno, Malagon,MM,Luque,RMand Castano,JP(2012)。 新的截短的生长抑素受体变体sst5TMD4与乳腺癌的预后不良有关,并增加了MCF- 7 cells。 Oncogene 31(16):2049-2061。
  3. Farhan,H.,Korkhov,V.M.,Paulitschke,V.,Dorostkar,M.M.,Scholze,P.,Kudlacek,O.,Freissmuth,M.and Sitte,H.H。(2004)。 羧基末端的两个不连续片段是大鼠γ-氨基丁酸转运蛋白的膜靶向所必需的, 1(GAT1)。 J Biol Chem 279(27):28553-28563。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gahete, M. D., Luque, R. M. and Castaño, J. P. (2012). GPCRs Interaction Measurement by Fluorescence Resonance Energy Transfer (FRET). Bio-protocol 2(22): e293. DOI: 10.21769/BioProtoc.293.
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