Trends in Endocrinology & Metabolism
ReviewApplications of novel resonance energy transfer techniques to study dynamic hormone receptor interactions in living cells
Section snippets
Fluorescence resonance energy transfer (FRET)
FRET is the current method used to monitor protein interactions, both spatially and temporally, by means of imaging techniques. This enables dynamic protein interactions to be assessed at a resolution that is much higher than that from any microscope. However, FRET can also be measured quantitatively by the use of fluorometric plate readers. An ever-increasing range of intrinsically fluorescent proteins, which can be genetically fused to almost any protein of interest 2., 3., 4., together with
Intensity-based FRET
Intensity-based methods of detecting FRET can either involve measurement of acceptor emission alone, or a ratiometric measurement of fluorescence intensity of acceptor over donor [6]. The acceptor:donor fluorescence ratio in a cell coexpressing both fusion proteins is compared with a cell expressing only the donor fusion protein. If an interaction occurs between the tagged proteins, the resultant energy transfer increases the FRET ratio (Fig. 1). However, this method has various drawbacks and
Fluorescence decay kinetics-based FRET
In kinetic-based approaches of FRET, the excited-state kinetics of the donor or acceptor fluorescence can be measured by two approaches: (1) photobleaching FRET (pbFRET) and (2) fluorescence lifetime imaging microscopy (FLIM). These approaches are independent of donor and/or acceptor concentration, and are appropriate for the detection and cellular location of interactions between individual proteins 2., 7., 8..
Bioluminescence resonance energy transfer
The natural phenomenon of bioluminescence in marine organisms, such as the sea pansy Renilla reniformis and the jellyfish Aequora victoria, has always been of fascination to humans. The engineering of naturally occurring proteins to produce bioluminescent light in mammalian cells, and its adaptation to the direct study of protein interactions, is truly innovative. Although relatively few studies have employed BRET to date, it has great universal potential, because it incorporates the attractive
Applications of RET to study receptor interactions
Over recent years, an increasing number of studies has successfully employed resonance energy transfer (ret) to study receptor interactions, both in heterologous expression systems and in vivo (Table 1).
FRET or BRET? Advantages and disadvantages
There is no perfect RET method, because each comes with its own advantages and drawbacks. For example, BRET avoids the need for excitation (which is a prerequisite for FRET), thus circumventing problems associated with measuring light-sensitive proteins, such as those encoded by the circadian clock genes from cyanobacteria [12]. Problems relate to autofluorescence, photobleaching and cell damage, and can result in loss of signal. The lower background fluorescence associated with BRET makes it
Future advances and applications in RET technology
Discovery of new bioluminescent and fluorescent molecules will undoubtedly drive the development of RET technologies, which will, in turn, expand current applications. New smaller fluorophores provide obvious advantages over the use of GFP, and the discovery of genetically encoded fluorophores will be an important improvement. An alternative is the incorporation of the FLASH-EDT2 fluorescent label inside living cells. This cell-permeable label covalently labels recombinant proteins containing
Conclusion
Although the evidence so far clearly indicates that RET procedures provide a powerful tool for studying receptor signaling in endocrine pathways, the potential of these techniques is far from being fully realized. Theoretically, any protein–protein interaction could be measured by either FRET or BRET which thus could embrace countless applications, in both applied and basic research. However, an important caveat is the possibility of recording non-specific interactions as a result of
Glossary
- Aequorin:
- Photoprotein found in luminescent jellyfish, Aequorea victoria, and other marine organisms. The aequorin complex comprises a 22 000-kDa apoaequorin protein, molecular oxygen and the luciferin, coelenterazine. When three Ca2+ ions bind to this complex, coelenterazine is oxidized to coelenteramide, with a concomitant release of carbon dioxide and blue light.
- Bioluminescence:
- similar to chemiluminescence (where the production of light occurs when the excitation energy has come from a
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