丁香实验_LOGO
登录
提问
我要登录
|免费注册
点赞
收藏
wx-share
分享

Monitoring Protein‐Protein Interactions in Living Cells by Bioluminescence Resonance Energy Transfer (BRET)

互联网

1226
  • Abstract
  • Table of Contents
  • Materials
  • Figures
  • Literature Cited

Abstract

 

Bioluminescence resonance energy transfer (BRET) allows monitoring of protein?protein interactions in real time in living cells. One candidate interacting protein is fused to a luminescent energy donor, such as Renilla luciferase, and the other to a fluorescent energy acceptor, such the green fluorescent protein (GFP), and the two are then coexpressed in the same cells. If the two proteins interact, their close proximity allows nonradiative energy transfer (BRET) between the luciferase and the GFP. BRET does not occur if the two proteins are separated by more than 100 Å, making the technique ideal for monitoring protein?protein interactions in biological systems. This unit describes the use of BRET to study constitutive and agonist?promoted interactions among signaling molecules, as illustrated by the homodimerization of the CXCR4 receptor and the recruitment of ??arrestin2 to agonist?activated G?protein?coupled receptors. This noninvasive and homogeneous assay provides a robust and sensitive proteomic platform with applications for basic science research and drug discovery.

Keywords: protein?protein interaction; GFP; energy transfer; luminescence

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Detection of Constitutive Protein‐Protein Interactions by BRET
  • Alternate Protocol 1: Measurement of Dynamic Protein‐Protein Interactions by BRET: Example Of β‐Arrestin2 Recruitment to Agonist‐Activated GPCRs as a Functional Readout of Receptor Activation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Detection of Constitutive Protein‐Protein Interactions by BRET

  Materials
  • CXCR4 template DNA (entire coding sequence)
  • Control template DNA: coding sequence of γ‐aminobutyric acid type B (GBR2) receptor
  • Coding sequence of EYFP
  • Mammalian expression plasmids:
    • Plasmids encoding a codon‐humanized R. reniformis luciferase (hRluc): phRluc‐N (allows cloning of candidate proteins upstream of hRluc) and phRluc‐C (allows cloning of candidate proteins downstream of hRluc); available in all translation frames from Perkin‐Elmer; humanized Rluc plasmids are also available from Promega
    • Plasmids encoding EYFP: pEYFP‐N (to clone upstream of EYFP) and pEYFP‐C (to clone downstream of EYFP), available from BD Biosciences
  • HEK293T cells (ATCC #CRL 11554)
  • HEK293T cell culture medium (see recipe )
  • Transfection reagent (also see appendix 11 ), e.g., Fugene 6 (Roche Applied Science)
  • Phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2A ) containing 0.5 mM MgCl 2
  • PBS, pH 7.4 ( appendix 2A ) containing 5 mM EDTA
  • PBS, pH 7.4 ( appendix 2A ) containing 0.5 mM MgCl 2
  • PBS, pH 7.4 ( appendix 2A ) containing 0.5 mM MgCl 2 and 0.1% (w/v) glucose
  • 50 µM coelenterazine‐h (Nanolight) in PBS, pH 7.4 ( appendix 2A ); prepare fresh from 1 mM stock (see recipe )
  • Test compound (e.g., receptor agonist or antagonist)
  • 6‐well tissue culture plates
  • White opaque‐bottom polystyrene 96‐well plates (for luminescence and BRET measurement; Costar or Perkin‐Elmer)
  • White clear‐bottom polystyrene 96‐well plates (for fluorescence measurements; Costar or Perkin‐Elmer)
  • Plate reader for luminescence, fluorescence, and BRET detection (see discussion of instrumentation in Critical Parameters and Troubleshooting)
  • Software for data analysis; Microsoft Excel or Graph Pad Prism (unit 7.5 )
  • Additional reagents and equipment for PCR (CPMB UNIT ), cloning (CPMB Chapter 3 ), mammalian cell culture ( appendix 3B ), and cell counting ( appendix 3B ) or protein assay (CPMB UNIT )

Alternate Protocol 1: Measurement of Dynamic Protein‐Protein Interactions by BRET: Example Of β‐Arrestin2 Recruitment to Agonist‐Activated GPCRs as a Functional Readout of Receptor Activation

  • V2R, β2AR, and β‐arrestin2 coding sequences
  • 8‐arginine‐vasopressin (8‐AVP)
  • Isoproterenol
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   Figure 5.23.1 Principle of BRET. (A ) To study the interaction between two proteins (P1 and P2), one is fused to Rluc and the other to a fluorescent protein (FP; EYFP for BRET1 or GFP2 for BRET2 ). If the two proteins do not interact (>100 Å apart; left panel), the energy generated by the Rluc after oxidizing its substrate coelenterazine (coelenterazine‐ h for BRET1 , λmax ∼480 nm; or DeepBlueC for BRET2 , λmax ∼400 nm) is not transferred to the FP. If the two proteins interact (<100 Å apart; right panel), the energy is transferred from the Rluc to the FP, which in turn emits light at a longer wavelength (λmax ∼530 nm for EYFP in BRET1 ; λmax ∼510 nm for GFP2 in BRET2 ). (B ) BRET1 and BRET2 spectra in response to addition of the substrates coelenterazine‐ h (left panel) and DeepBlueC (right panel), respectively. In BRET1 , significant overlap exists between the emission of Rluc and the emission of EYFP (left panel: solid line and gray line, respectively) that results in a higher background signal. In BRET2 , the emission spectra of Rluc and GFP2 (right panel: solid line and gray line, respectively) are better separated than in BRET1 , resulting in a lower background signal. The overlap between the Rluc emission spectrum (solid line) and the excitation spectrum of the FP (dashed line) is a prerequisite for BRET to occur.
    View Image
  •   Figure 5.23.2 Detection of CXCR4 homodimerization by BRET1 . HEK293T cells were cotransfected with the indicated plasmids. Transfection with CXCR4‐hRluc alone, pCXCR4‐hRluc/pGBR2‐EYFP, or pCXCR4‐EYFP/pGBR2‐hRluc served as negative controls, while transfection with pEYFP‐hRluc fusion plasmid was used as a positive control for BRET1 . After the addition of coelenterazine‐ h , the BRET1 signal was measured with the Mithras LB940. The BRET1 ratio was calculated as the emission in the EYFP channel/Rluc channel, as described in the . Cells expressing the negative controls yielded a background BRET ratio of ∼0.73, while the cells expressing the positive control or coexpressing pCXCR4‐hRluc and pCXCR4‐EYFP yielded higher BRET ratios, indicative of energy transfer and protein‐protein interaction. The BRETnet ratio was calculated by subtracting the BRET ratio obtained from cells coexpressing the EYFP and hRluc fusion proteins from the BRET ratio obtained from cells expressing the hRluc fusion protein alone (see inset).
    View Image
  •   Figure 5.23.3 Monitoring agonist‐promoted β‐arrestin2‐Rluc recruitment to β2AR‐EYFP and V2R‐EYFP by BRET1 . (A ) Real time BRET1 kinetics: HEK293T cells were cotransfected with pβ‐arrestin2‐hRluc and pβ2AR‐EYFP or pV2R‐EYFP. The cells were dispensed into white 96‐well opaque plates at a density of 50,000 cells/well, and coelenterazine‐ h and saturating concentrations of the receptor‐specific agonists (0.1 µM AVP for V2R‐EYFP and 1 µM isoproterenol for β2AR‐EYFP) were added. BRET measurements were recorded on the FUSION plate reader over a period of 30 min. at room temperature. BRET values in the absence of the agonist were not different from the background BRET readings. (B ) Agonist‐promoted BRET dose response. HEK293T cells transiently expressing β‐arrestin2‐Rluc and β2AR‐EYFP or V2R‐EYFP were dispensed into white opaque 96‐well plates and incubated with increasing concentrations of receptor‐specific agonists for 15 min. The agonist EC50 (∼1 nM for AVP and V2R‐EYFP; ∼20 nM for isoproterenol and β2AR‐EYFP) was derived from the nonlinear regression curve fitting of the obtained data points using the software PRISM 4.0 (GraphPad).
    View Image
  •   Figure 5.23.4 BRET efficiency is dependent on the distance between the energy donor and acceptor. BRET is maximal when the distance between the two candidate proteins is short (zone 1), and decreases with the increase in distance as a function of Förster's equation: efficiency of energy transfer = [1+( R / R o )6 ]−1 (zones 2 and 3). R represents the distance between the energy donor and acceptor, while R 0 is the distance between the energy donor and acceptor resulting in half‐maximal BRET; this relationship greatly influences the ability of BRET to detect changes in the distance between the energy donor and acceptor. In zone 1, distance changes between the energy donor and acceptor would go undetected in BRET because the energy transfer signal is already maximal, while in zone 3, distance changes may be undetectable because of the very low energy transfer in this region.
    View Image
  •   Figure 5.23.5 BRET titration curve. The BRET signal increases with the increase in the ratio of energy acceptor to energy donor and reaches a plateau when all the energy donor proteins are saturated with the energy acceptor. In the case of a specific protein‐protein interaction, the BRET ratio increases hyperbolically and rapidly saturates (solid line; specific BRET), while in the case of nonspecific interaction resulting from random collisions, the “bystander BRET” signal increases almost linearly and may eventually saturate at very high expression levels of the energy acceptor protein. A BRET50 can be calculated from a BRET saturation curve, giving a relative affinity index between the test proteins. A decrease in the affinity between the test proteins should result in a right shift in the curve and in an increase in the BRET50 (dashed line), which may or may not be accompanied by a change in the maximum BRET signal. Changes in the conformation of the two proteins that do not alter the affinity of their association may result in changes in the maximal BRET without affecting the BRET50 (dotted line).
    View Image

Videos

Literature Cited

Literature Cited
   Angers, S, Salahpour, A., Joly, E., Hilairet, S., Chelsky, D., Dennis, M., and Bouvier, M. 2000. Detection of beta 2‐adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl. Acad. Sci. U.S.A. 97:3684‐3689.
   Arai, R., Nakagawa, H., Tsumoto, K., Mahoney, W., Kumagai, I., Ueda, H., and Nagamune, T. 2001. Demonstration of a homogeneous noncompetitive immunoassay based on bioluminescence resonance energy transfer. Anal. Biochem. 289:77‐81.
   Bertrand, L., Parent, S., Caron, M., Legault, M., Joly, E., Angers, S., Bouvier, M., Brown, M., Houle, B., and Menard, L. 2002. The BRET2/arrestin assay in stable recombinant cells: A platform to screen for compounds that interact with G protein‐coupled receptors (GPCRS). J. Recept. Signal Transduct. Res. 22:533‐541.
   Boute, N., Boubekeur, S., Lacasa, D., and Issad, T. 2003. Dynamics of the interaction between the insulin receptor and protein tyrosine‐phosphatase 1B in living cells. EMBO Rep. 4:313‐319.
   Boute, N., Pernet, K., and Issad, T. 2001. Monitoring the activation state of the insulin receptor using bioluminescence resonance energy transfer. Mol. Pharmacol. 60:640‐645.
   Charest, P.G., Terrillon, S., and Bouvier, M. 2005. Monitoring agonist‐promoted conformational changes of beta‐arrestin in living cells by intramolecular BRET. EMBO Rep. 6:334‐340.
   Eidne, K.A., Kroeger, K.M., and Hanyaloglu, A.C. 2002. Applications of novel resonance energy transfer techniques to study dynamic hormone receptor interactions in living cells. Trends Endocrinol. Metab. 13:415‐421.
   Gales, C., Rebois, R.V., Hogue, M., Trieu, P., Breit, A., Hebert, T.E., and Bouvier, M. 2005. Real‐time monitoring of receptor and G‐protein interactions in living cells. Nat. Methods 2:177‐184.
   Germain‐Desprez, D., Bazinet, M., Bouvier, M., and Aubry, M. 2003. Oligomerization of transcriptional intermediary factor 1 regulators and interaction with ZNF74 nuclear matrix protein revealed by bioluminescence resonance energy transfer in living cells. J. Biol. Chem. 278:22367‐22373.
   Griesbeck, O., Baird, G.S., Campbell, R.E., Zacharias, D.A., and Tsien, R.Y. 2001. Reducing the environmental sensitivity of yellow fluorescent protein: Mechanism and applications. J. Biol. Chem. 276:29188‐29194.
   Hamdan, F.F., Audet, M., Garneau, P., Pelletier, J., and Bouvier, M. 2005. High‐throughput screening of G protein‐coupled receptors antagonists using a BRET1‐based β‐arrestin2 recruitment assay. J Biomol. Screen. 10:463‐475.
   Hu, C.D., Chinenov, Y., and Kerppola, T.K. 2002. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol. Cell 9:789‐798.
   Kaihara, A., Kawai, Y., Sato, M., Ozawa, T., and Umezawa, Y. 2003. Locating a protein‐protein interaction in living cells via split Renilla luciferase complementation. Anal. Chem. 75:4176‐4181.
   Mercier, J.F., Salahpour, A., Angers, S., Breit, A., and Bouvier, M. 2002. Quantitative assessment of beta 1‐ and beta 2‐adrenergic receptor homo‐ and heterodimerization by bioluminescence resonance energy transfer. J. Biol. Chem. 277:44925‐44931.
   Michelini, E., Mirasoli, M., Karp, M., Virta, M., and Roda, A. 2004. Development of a bioluminescence resonance energy‐transfer assay for estrogen‐like compound in vivo monitoring. Anal. Chem. 76:7069‐7076.
   Nagai, T., Ibata, K., Park, E.S., Kubota, M., Mikoshiba, K., and Miyawaki, A. 2002. A variant of yellow fluorescent protein with fast and efficient maturation for cell‐biological applications. Nat. Biotechnol. 20:87‐90.
   Oakley, R.H., Laporte, S.A., Holt, J.A., Caron, M.G., and Barak, L.S. 2000. Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein‐coupled receptors delineate two major classes of receptors. J. Biol. Chem. 275:17201‐17210.
   Otsuji, T., Okuda‐Ashitaka, E., Kojima, S., Akiyama, H., Ito, S., and Ohmiya, Y. 2004. Monitoring for dynamic biological processing by intramolecular bioluminescence resonance energy transfer system using secreted luciferase. Anal. Biochem. 329:230‐237.
   Percherancier, Y., Berchiche, Y.A., Slight, I., Volkmer‐Engert, R., Tamamura, H., Fujii, N., Bouvier, M., and Heveker, N. 2005. Bioluminescence resonance energy transfer reveals ligand‐induced conformational changes in CXCR4 homo‐ and heterodimers. J. Biol. Chem. 280:9895‐9903.
   Perroy, J., Pontier, S., Charest, P.G., Aubry, M., and Bouvier, M. 2004. Real‐time monitoring of ubiquitination in living cells by BRET. Nat. Methods 1:203‐208.
   Pfleger, K.D. and Eidne, K.A. 2004. Monitoring the formation of dynamic G‐protein coupled receptor/protein complexes in living cells. Biochem. J. 385:625‐637.
   Subramanian, C., Kim, B.H., Lyssenko, N.N., Xu, X., Johnson, C.H., and von Arnim, A.G. 2004. The Arabidopsis repressor of light signaling, COP1, is regulated by nuclear exclusion: Mutational analysis by bioluminescence resonance energy transfer. Proc. Natl. Acad. Sci. U.S.A. 101:6798‐6802.
   Terrillon, S., Durroux, T., Mouillac, B., Breit, A., Ayoub, M.A., Taulan, M., Jockers, R., Barberis, C., and Bouvier, M. 2003. Oxytocin and vasopressin V1a and V2 receptors form constitutive homo‐ and heterodimers during biosynthesis. Mol. Endocrinol. 17:677‐691.
   Verhaegent, M. and Christopoulos, T.K. 2002. Recombinant Gaussia luciferase: Overexpression, purification, and analytical application of a bioluminescent reporter for DNA hybridization. Anal. Chem. 74:4378‐4385.
   Vrecl, M., Jorgensen, R., Pogacnik, A., and Heding, A. 2004. Development of a BRET2 screening assay using beta‐arrestin 2 mutants. J. Biomol. Screen. 9:322‐333.
   Wilson, T. and Hastings, J.W. 1998. Bioluminescence. Annu. Rev. Cell. Dev. Biol. 14:197‐230.
   Xu, Y., Piston, D.W., and Johnson, C.H. 1999. A bioluminescence resonance energy transfer (BRET) system: Application to interacting circadian clock proteins. Proc. Natl. Acad. Sci. U.S.A. 96:151‐156.
   Zacharias, D.A., Violin, J.D., Newton, A.C., and Tsien, R.Y. 2002. Partitioning of lipid‐modified monomeric GFPs into membrane microdomains of live cells. Science 296:913‐916.
   Zapata‐Hommer, O. and Griesbeck, O. 2003. Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP. BMC Biotechnol. 3:5.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library
 
提问
扫一扫
丁香实验小程序二维码
实验小助手
丁香实验公众号二维码
扫码领资料
反馈
TOP
打开小程序