**7. Conclusions**

94 Bioluminescence – Recent Advances in Oceanic Measurements and Laboratory Applications

Heterologous - no interaction

 Dimer - homologous Dimer - heterologous Trimer - homologous

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[*C*]

Fig. 3. BRET competition assay. In homologous assay the same receptor is used as a competitor, whereas in heterologous assay different receptor is used. For the latter case a hetero-dimer with lower association constant than that of the homo-dimer is presented.

**6. Other BRET-based approaches to identify 7TMR hetero-dimerization** 

To overcome certain limitations of the classical BRET assays described above, some other BRET-based approaches have been developed to study 7TMR oligomerization/ heterodimerization. Sequential-BRET-FRET (SRET) enables identification of oligomers formed by three different proteins. In SRET, the oxidation of the RLuc substrate by an RLuc-fusion protein triggers the excitation of the acceptor GFP2 by BRET2 and subsequent energy transfer to the acceptor YFP by FRET. Combination of bimolecular fluorescence complementation (BiFC) and BRET techniques is based on the ability to produce a fluorescent complex from non-fluorescent constituents if a protein-protein interaction occurs. Two receptors are fused at their C-termini with either N-terminal or C-terminal fragments of YFP, respectively, and receptor hetero-dimerization causes YFP reconstitution. Then, if there is hetero-trimerization, BRET can be obtained when the cells also co-express the third receptor fused to Rluc (reviewed by (Ferré & Franco, 2010)). GPCR-Heteromer Identification Technology (GPCR-HIT) utilizes BRET and ligand-dependent recruitment of a 7TMR-specific interaction partners (such as a β-arrestin, PKC or G-protein) to enable 7TMR heteromer discovery and characterization (Mustafa & Pfleger, 2011; See et al., 2011). In this set up, only one receptor subtype is fused to Rluc and the second receptor subtype is untagged. A third protein capable of interacting specifically with one or both receptors in a

[*D*]=0.1 [*A*]=1.0

0,0

0,2

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BRET-based techniques are extremely powerful, provided that they are conducted with the appropriate controls and correctly interpreted. Quantitative BRET assays allow us to support the ability of receptor for homo-dimer and hetero-dimer. Homologous saturation assay provide us with the oligomerisation state of receptors. Data interpretation is more difficult for hetero-oligomers and the mixtures of monomer, dimer and higher oligomer populations. For the quantitative approach we also need to know the relative concentrations of all receptors used in the experiment, which can be obtained from radioligand binding, Western blot or ELISA assays.
