Trends in Biotechnology
ReviewLighting up multiprotein complexes: lessons from GPCR oligomerization
Section snippets
Background
Biological processes proceed through a sequence of specific protein–protein interactions (PPIs) along intracellular signaling cascades. Characterization of these interactions is thus essential to the understanding of cellular mechanisms. Using genetic approaches (e.g. yeast two-hybrid screens) 1, 2, it is possible to reveal new PPIs, which are subsequently confirmed and validated by additional biochemical approaches, such as immobilized PPI assays (e.g. co-immunoprecipitation and pull-down
Fluorescence RET (FRET)
Classical RET techniques, including fluorescence RET (FRET) and bioluminescence RET (BRET), use the nonradiative transfer of energy (Box 1) between donor and acceptor fluorescent molecules as a measure of their proximity. For example, cyan (CFP) and yellow (YFP) variants of the green fluorescent protein (GFP) can be used for FRET in live cells. When CFP is excited and FRET occurs, CFP emission decreases and YFP emission increases (Figure 1a). The presence of specific receptor homo- or
PPIs at the surface of a living cell
The difficulties encountered with classical RET techniques for analysis of GPCR homo- and heterocomplex assembly at the cell surface can be circumvented using the SNAP-tag technology (Table 1) [33]. The SNAP-tag method is based on irreversible and specific reaction of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine (BG) derivatives, which can be selectively labeled with a variety of chemical fluorophores (Figure 1c). The first remarkable characteristic of
Protein-fragment complementation assays toward the study of protein oligomerization
Protein-fragment complementation assays (PCAs) facilitate direct detection of PPIs and the study of their dynamic events in living cells 39, 40. In brief, PCAs consist of the structural and functional reconstitution of an active protein, typically an enzyme or FP, from two inactive halves that are genetically fused to the interacting proteins of interest. When FP or LP fragments are used for complementation, these assays are known as bimolecular fluorescence or luminescence complementation
Detection of higher-order protein complexes
The RET techniques described above are well-suited for detection of interactions between two proteins that form homo- or heterodimer complexes; however, a given protein can also be part of multiprotein complexes involving numerous interactions with different receptor partners. The formation of highly organized protein structures can be extremely specific, especially if they dictate the final functional output of a specific receptor oligomer. Thus, the existence of high-order oligomer complexes
Concluding remarks
Fluorescence- and luminescence-based assays are useful tools for the visualization and characterization of noncovalent PPIs in many cell types and organisms. In recent years, new optical techniques based on RET and protein-fragment complementation have enabled researchers to detect specific PPIs in diverse biological fields. Thus, as the use of these approaches increases dramatically, it is timely to revise their significant strengths and weaknesses to encourage reliable biotechnological
Acknowledgements
This work was supported by grants SAF2008-01462 and Consolider-Ingenio CSD2008-00005 from Ministerio de Ciencia e Innovación (to F.C.) and a start-up package from the Department of Pharmacology and Chemical Biology, University of Pittsburgh and by the National Institutes of Health grants DK087688 (J.-P.V.). F.C. and V.F.D. belong to the Neuropharmacology and Pain accredited research group (Generalitat de Catalunya, 2009 SGR 232). We thank Benjamín Torrejón and Esther Castaño from the Scientific
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