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

Department of Physiology, University of Wisconsin, Madison, Wisconsin

Correspondence: Address reprint requests to Jeffery W. Walker, Dept. of Physiology, 1300 University Ave., Madison, WI 53706. Tel.: 608-262-6941; Fax: 608-265-5512; E-mail: jwalker{at}physiology.wisc.edu.

Endothelin-1 (ET-1) mediates physiological responses via endothelin A (ET_A) and B (ET_B) 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 ET_A/ET_A, ET_B/ET_B, and ET_A/ET_B, 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, ET_A/ET_B heterodimers mediated a sustained [Ca²⁺]_i increase lasting >10 min, and required a combination of ET_A and ET_B 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.