Endothelin Receptor Dimers Evaluated by FRET, Ligand Binding, and Calcium Mobilization

Endothelin-1 (ET-1) mediates physiological responses via endothelin A (ETA) and B (ETB) receptors, which may form homo- and heterodimers with unknown function. Here, we investigated ET-receptor dimerization using fluorescence resonance energy transfer (FRET) between receptors tagged with CFP (donor) and receptors tagged with tetracysteine-FlAsH (fluorescein arsenical hairpin) (acceptor) expressed in HEK293 cells. FRET efficiencies were 15%, 22%, and 27% for ETA/ETA, ETB/ETB and ETA/ETB, respectively, and dimerization was further supported by coimmunoprecipitation. For all dimer pairs, the natural but nonselective ligand ET-1 rapidly (≤30 s) reduced FRET by >50%, but did not detectably reduce coimmunoprecipitation. ET-1 stimulated a transient increase in intracellular Ca2+ ([Ca2+]i) lasting 1–2 min for both homodimer pairs, and these ET-1 actions on FRET and [Ca2+]i elevation were blocked by the appropriate subtype-selective antagonist. In contrast, ETA/ETB heterodimers mediated a sustained [Ca2+]i increase lasting >10 min, and required a combination of ETA and ETB antagonists to block the observed FRET and [Ca2+]i responses. The sensitive CFP/FlAsH FRET assay used here provides new insights into endothelin-receptor dimer function, and represents a unique approach to characterize G-protein-coupled receptor oligomers, including their pharmacology.