Abstract

Heterodimerization has been documented for several members of the G protein-coupled receptor (GPCR) superfamily, 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 per cell. The relative propensity of MT₁ homodimer and MT₁/MT₂ heterodimer formation are similar, whereas that for the MT₂ homodimer formation is 3- to 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 readout for the conformational changes of the heterodimer. An MT₁/MT₂ heterodimer-specific profile and ligands selective for the MT₁/MT₂ heterodimer compared with the MT₂ homodimer could be identified. Classic 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, these results show that the probability of GPCR heterodimer formation may be equal to 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.