Review
Applications of novel resonance energy transfer techniques to study dynamic hormone receptor interactions in living cells

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Abstract

Many aspects of hormone receptor function that are crucial for controlling signal transduction of endocrine pathways can be monitored more accurately with the use of non-invasive, live cell resonance energy transfer (RET) techniques. Fluorescent RET (FRET), and its variation, bioluminescent RET (BRET), can be used to assess the real-time responses to specific hormonal stimuli, whilst preserving the cellular protein network, compartmentalization and spatial arrangement. Both FRET and BRET can be readily adapted to the study of membrane proteins. Here, we focus on their applications to the analysis of interactions involving the superfamily of hormone G-protein-coupled receptors. RET is also emerging as a significant tool for the determination of protein function in general. Such techniques will undoubtedly be of value in determining the functional identities of the vast array of proteins that are encoded by the human genome.

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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