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

¹ University of Wisconsin-Madison

^* To whom correspondence should be addressed. E-mail: jwalker{at}physiology.wisc.edu.

Submitted on August 8, 2007
Revised on September 19, 2007
Accepted on 7 March 2008

	Abstract

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 co-immunoprecipitation. For all dimer pairs, the natural but non-selective ligand, ET-1, rapidly (30s) reduced FRET by >50%, but did not detectably reduce co-immunoprecipitation. ET-1 stimulated a transient increase in intracellular Ca²⁺ ([Ca²⁺]_i) lasting 1-2 minutes 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.

Key Words: FlAsH, GPCR, co-immunoprecipitation, heterodimers