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Received for publication March 12, 2004.
Revised May 12, 2004.
Accepted for publication May 13, 2004.
Heterodimerization has been documented for several members of the G protein-coupled receptor (GPCR) super-family, including the closely related MT1 and MT2 melatonin receptors. However, the relative abundance of hetero- versus homodimers and the specific properties, which can be attributed to each form, are difficult to determine. Using a bioluminescence resonance energy transfer (BRET) donor saturation assay, we show that half maximal MT1/MT2 heterodimer formation is reached for expression levels as low as 
4000 receptors/cell. The relative propensity of MT1 homodimer and MT1/MT2 heterodimer formation are similar, whereas that for the MT2 homodimer formation is 3-4 fold lower. These data indicate that both the relative expression level of each receptor isoform and the affinities between monomers may determine the actual proportion of homo- and heterodimers. The specific interaction of ligands with the MT1/MT2 heterodimer was studied using a BRET-based assay as a read out for the conformational changes of the heterodimer. A MT1/MT2 heterodimer-specific profile as well as ligands selective for the MT1/MT2 heterodimer compared to the MT2 homodimer could be identified. Classical radioligand binding and BRET studies suggest that heterodimers contain two functional ligand binding sites that maintain their respective selectivity for MT1 and MT2 ligands. Occupation of either binding site is sufficient to induce a conformational change within the heterodimer. Taken together, we show that the probability of GPCR heterodimer formation may be equal or even higher than that of the corresponding homodimers and that specific properties of heterodimers can be revealed using a BRET-based ligand/receptor interaction assay.
Key words:
Melatonin, Fluorescence techniques, Receptor binding studies
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