Endothelin-1 (ET-1) is a potent vasoactive peptide that acts on endothelin A (ETEndothelins (ET-1, ET-2, and ET-3) 1 are important regulators in the vascular system. They act via two receptors: the endothelin A (ET A ) and endothelin B (ET B ) receptors (1, 2). Although human ET A and ET B receptors share 59% amino acid sequence identity (exceeding 75% at the cytoplasmic face), both receptor subtypes couple to different G proteins and differ in their ligand-induced internalization and intracellular trafficking. Whereas the ET A receptor stimulates G proteins of the G q/11 and G 12/13 families, the ET B receptor activates mainly G proteins of the G i and G q/11 families (3, 4). Whether ET B receptors also stimulate proteins of the G 12/13 family is still controversial and may depend on expression levels or cell types investigated (5, 6). Upon ligand binding, both receptor subtypes are rapidly desensitized by phosphorylation through the G protein-coupled receptor kinase type 2 (7). Following internalization via caveolae and/or clathrin-coated pits, the ET A receptor is recycled back to the cell surface (8, 9). In contrast, the ET B receptor is exclusively internalized via a clathrin-dependent pathway and transported to late endosomes and lysosomes (9, 10).The ET A receptor is mainly expressed in vascular smooth muscle cells. Its activation elicits a long-lasting contraction via an increase in cytosolic Ca 2ϩ concentrations and activation of Rho proteins (11,12). The ET B receptor is predominantly expressed in endothelial cells and stimulates the release of NO and prostacyclin, thereby causing relaxation of vascular smooth muscle cells (13). In addition, ET A and ET B receptors are co-expressed in numerous cells, e.g. astrocytes, cardiomyocytes, epithelial cells of the choroid plexus and the anterior pituitary, and certain vascular smooth muscle cells (14 -16). In disease states, such as atherosclerosis and hypercholesterolemia, vascular smooth muscle cells co-express ET A and ET B receptors (17). Because atypical ligand binding was observed for cells co-expressing ET A and ET B receptors, e.g. astrocytes, epithelial cells of the anterior pituitary, or vascular smooth muscle cells, it was suggested that the two receptor subtypes form heterodimers (15,16,18). For example, in epithelial cells of the anterior pituitary, ET B receptor-selective ligands such as sarafotoxin 6c, ET-3, and IRL1620 were competitors of 125 I-ET-1 binding only in the presence of the ET A receptor-selective antagonist BQ123 (16). In astrocytes, ET A and ET B receptors cooperatively control ET-1 clearance, because only the combi-

Endothelin-1 is a potent vasoactive peptide that exerts its action via endothelin-A (ETA) and endothelin-B (ETB) receptors. Both types of receptor belong to the rhodopsin-like (class A) G protein-coupled receptors. Since there is an increasing body of evidence that G protein-coupled receptors function as homo- and heterodimers, we sought to prove whether endothelin receptor subtypes also exist as homodimers. To this end we generated plasmids encoding fusion proteins of the endothelin-A and endothelin-B receptor with cyan (ETA.CFP, ETB.CFP) or yellow fluorescent protein (ETA.YFP, ETB.YFP), suitable for fluorescence resonance energy transfer experiments. HEK293 cells transiently co-expressing either ETA.CFP and ETA.YFP or ETB.CFP and ETB.YFP showed significant fluorescence resonance energy transfer. The calculated efficiencies of fluorescence resonance energy transfer were between 12 and 18% for both endothelin-A and endothelin-B receptor homodimers. When cells co-expressing ETA.CFP and ETA.YFP or ETB.CFP and ETB.YFP were treated with endothelin-1 for 30 minutes at 37degreesC, no change in fluorescence resonance energy transfer efficiencies was found. Similarly, the endothelin-A receptor-selective antagonist, BQ123, and the endothelin-B receptor-selective antagonist, BQ788, did not change the fluorescence resonance energy transfer efficiencies of ETA.CFP/ ETA.YFP and ETB.CFP/ETB.YFP homodimers, respectively. The data demonstrate that endothelin-A and endothelin-B receptors exist as constitutive homodimers, which are not altered in the presence of agonists or antagonists.

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