Monitoring Protein‐Protein Interactions in Living Cells by Bioluminescence Resonance Energy Transfer (BRET)
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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
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PDF or HTML at Wiley Online LibraryTable 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
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Basic Protocol 1: Detection of Constitutive Protein‐Protein Interactions by BRET
Materials
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
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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
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