Demonstration of a Homogeneous Noncompetitive Immunoassay Based on Bioluminescence Resonance Energy Transfer

Arai, Ryoichi; Nakagawa, Hideyuki; Tsumoto, Kouhei; Mahoney, Walt; Kumagai, Izumi; Ueda, Hiroshi; Nagamune, Teruyuki

doi:10.1006/abio.2000.4924

Cited by 73 publications

(32 citation statements)

References 16 publications

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“…The lack of incident illumination and the large spectral shift in the BRET 2 system between the bioluminescent emission (390 nm) and the fluorescence emission of the GFP 2 acceptor (515 nm) (45) were expected to lead to higher sensitivity and less need for corrections compared with FRET. An earlier version of BRET has been reported to be 10-fold more sensitive than FRET (46).…”

Section: Resultsmentioning

confidence: 99%

Oligomerization of the Yeast α-Factor Receptor

Gehret

Bajaj

Naider

et al. 2006

Journal of Biological Chemistry

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Oligomerization of G protein-coupled receptors is commonly observed, but the functional significance of oligomerization for this diverse family of receptors remains poorly understood. We used bioluminescence resonance energy transfer (BRET) to examine oligomerization of Ste2p, a G protein-coupled receptor that serves as the receptor for the ␣-mating pheromone in the yeast Saccharomyces cerevisiae, under conditions where the functional effects of oligomerization could be examined. Consistent with previous results from fluorescence resonance energy transfer (Overton, M. C., and Blumer, K. J. (2000) Curr. Biol. 10, 341-344), we detected efficient energy transfer between Renilla luciferase and a modified green fluorescent protein individually fused to truncated ␣-factor receptors lacking the cytoplasmic C-terminal tail. In addition, the low background of the BRET system allowed detection of significant, but less efficient, energy transfer between full-length receptors. The reduced efficiency of energy transfer between full-length receptors does not appear to result from different levels of receptor expression. Instead, attachment of fluorescent reporter proteins to the full-length receptors appears to significantly increase the distance between reporters. Mutations that were previously reported to block dimerization of truncated ␣-factor receptors reduce but do not completely eliminate BRET transfer between receptors. Dominant negative effects of mutant alleles of ␣-factor receptors appear to be mediated by receptor oligomerization since these effects are abrogated by introduction of additional mutations that reduce oligomerization. We find that heterodimers of normal and dominant negative receptors are defective in their ability to signal. Thus, signal transduction by oligomeric receptors appears to be a cooperative process requiring an interaction between functional monomers. G protein-coupled receptors (GPCRs)3 comprise a large family of cellular receptors responsible for transducing signals from a wide variety of extracellular stimuli including peptides, neurotransmitters, hormones, and light. All GPCRs are transmembrane proteins consisting of an extracellular N-terminal domain, seven transmembrane ␣-helical segments, and a cytoplasmic C-terminal tail. A large body of evidence indicates that GPCRs form homo-and/or hetero-oligomeric complexes in cells (2-5). Although the implications of oligomerization for receptor function remain poorly understood, in some cases oligomerization is capable of affecting biogenesis and membrane targeting of receptors (6, 7). In addition, cooperation between different monomers appears to be responsible for mediating or modulating the signaling function of some GPCRs (8, 9).The possibility of artifactual aggregation (or dis-aggregation) of GPCRs during solubilization and extraction from the membranes makes it desirable to monitor the oligomeric state of receptors that are maintained in their native cellular membranes. Because detection of nonradiative energy transfer between fluoresc...

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Section: Resultsmentioning

confidence: 99%

Oligomerization of the Yeast α-Factor Receptor

Gehret

Bajaj

Naider

et al. 2006

Journal of Biological Chemistry

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“…Moreover, the analysis of FRET signal is complicated by the direct excitation of the acceptor molecule, resulting in a higher background signal. As this background can be eliminated in BRET, this method is theoretically more sensitive than FRET, which was demonstrated by a noncompetitive homogenous bioluminescent immunoassay that was ten times more sensitive than a comparable FRET assay (Arai et al 2001). The disadvantages include the fact that since BRET is always measured between fusion proteins, it can only be performed in recombinant systems.…”

Section: Bretmentioning

confidence: 99%

Dimerization and oligomerization of G-protein-coupled receptors: debated structures with established and emerging functions

Szidonya

Cserző

Hunyady

2008

Journal of Endocrinology

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Dimerization or oligomerization of G-protein-coupled receptors (GPCRs) is a novel concept, which may lead to the reevaluation of the actions of pharmacological ligands, hormones, neurotransmitters, and other mediators acting on GPCRs. Although a large number of data obtained using different biophysical, biochemical and structural methods, and functional approaches argue for dimerization or oligomerization of these receptors, several publications criticized the applied methods and challenged the concept. The aim of this paper is to review the data that support the concept of receptor oligomerization, and the most important arguments against it. We conclude that it will require major methodical improvements to obtain decisive proof, whether GPCRs exist in their native membrane environments as homo- or heterodimeric or oligomeric complexes, in which receptor monomers have stable direct interactions. However, overwhelming amounts of data suggest that many GPCRs exhibit functional properties that require direct or indirect interactions between clustered receptors. Although it is difficult to conclude, about the exact nature of these interactions, dimerization or oligomerization of GPCRs is a useful paradigm for pharmacologists to study properties of receptors, which require functionally important clustering of receptors, such as trafficking of newly synthesized receptors to the cell surface, allosteric modulation of ligand binding, signaling specificity, co-internalization, or cross-inhibition of GPCRs.

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“…After Ueda's work, this method was extended to hapten molecules detection and new formats such as homogeneous assays were also developed. These homogeneous assay systems are either based on enzymatic complementation or resonance energy transfer (fluorescence (Ueda et al 1999;Arai et al 2000;Wei et al 2006) or bioluminescence (Arai et al 2001)). …”

Section: Open Sandwich Immunoassay (Osia)mentioning

confidence: 99%

Recent Progress in Noncompetitive Hapten Immunoassays: A Review

Fan¹

2012

Trends in Immunolabelled and Related Techniques

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Demonstration of a Homogeneous Noncompetitive Immunoassay Based on Bioluminescence Resonance Energy Transfer

Cited by 73 publications

References 16 publications

Oligomerization of the Yeast α-Factor Receptor

Oligomerization of the Yeast α-Factor Receptor

Dimerization and oligomerization of G-protein-coupled receptors: debated structures with established and emerging functions

Recent Progress in Noncompetitive Hapten Immunoassays: A Review

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