Ligand-dependent differences in the internalization of endothelin A and endothelin B receptor heterodimers

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. Here we show that both receptors were co-immunoprecipitated 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 (ET(A)myc.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 heterodimerization is mediated by the CFP/YFP moieties. Heterodimerization was further confirmed in fluorescence resonance energy transfer ( FRET) analysis of HEK293 cells transiently co-expressing ET(A)myc.CFP and ETB.YFP receptors. FRET efficiencies were between 12 and 18% in untreated and antagonist- or ET-1-treated cells, indicating constitutive heterodimerization. Prolonged stimulation (30 min) with the ETB receptor-selective agonist BQ3020 decreased FRET efficiency by 50%. This decrease was not observed when internalization 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 ET(A)myc.CFP/ET(B)flag.YFP receptors revealed a slower sequestration of the ET(B)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 ET(B)flag.YFP receptors 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-selective stimulation.