Endothelin-1 (ET-1) is a potent vasoactive peptide that acts on endothelin A

(ETA) and endothelin B (ETB) receptors. Although both receptor subtypes are

co-expressed in numerous cells, little is known about their ability to form

heterodimers. In this study it was shown that both receptors were coimmunoprecipitated

with an ETB-specific antibody using extracts from HEK293

cells stably co-expressing a fusion protein consisting of a myc-tagged ETA

receptor and CFP (ETAmyc.CFP) and a fusion protein consisting of an ETB

receptor and YFP (ETB.YFP). Co-immunoprecipitation was also observed with

extracts from HEK293 cells transiently co-expressing FLAG-tagged ETB and

myc-tagged ETA receptors, thereby excluding that heterodimerisation is

mediated by the CFP/YFP moieties. Heterodimerisation was further confirmed

in fluorescence resonance energy transfer (FRET) analysis of HEK293 cells

transiently co-expressing ETAmyc.CFP and ETB.YFP receptors. Additionally

homodimerisation of ETA and ETB receptors could be demonstrated in FRET

analysis of HEK293 cells transiently co-expressing ETAmyc.CFP and

ETAmyc.YFP or ETB.CFP and ETB.YFP receptors. FRET efficiencies were

between 12 and 18% in untreated and antagonist- or ET-1-treated cells,

indicating constitutive homo- and heterodimerisation. Prolonged stimulation

(30 min) with the ETB receptor-selective agonist BQ3020 decreased FRET

efficiency by 50%. This decrease was not observed when internalisation was

inhibited by co-expression of dominant-negative K44A.dynamin I or incubation

with 450 mM sucrose. Enzyme-linked immunosorbent assay and laser scanning

microscopy of cell clones stably co-expressing ETAmyc.CFP/ETBflag.YFP

receptors revealed a slower sequestration of the ETB.flag.YFP receptors upon

stimulation with ET-1 than with BQ3020. No difference in ET-1 or BQ3020-

mediated sequestration was observed with cell clones expressing ETBflag.YFP

alone. The data suggest that ETA and ETB receptors form constitutive

heterodimers, which show a slower sequestration upon stimulation with ET-1

than with BQ3020. Heterodimer dissociation along the endocytic pathway only

occurs upon ETB receptor- selective stimulation.