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 (ET(A)) and endothelin B (ET(B)) 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 ET(B)-specific antibody using extracts from HEK293 cells stably co-expressing a fusion protein consisting of a myc-tagged ET(A) receptor and CFP (ET(A)myc.CFP) and a fusion protein consisting of an ET(B) receptor and YFP (ET(B).YFP). Co-immunoprecipitation was also observed with extracts from HEK293 cells transiently co-expressing FLAG-tagged ET(B) and myc-tagged ET(A) 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 ET(B).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 ET(B) 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 ET(A) and ET(B) 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 ET(B)-selective stimulation.
Gregan
Bernd
B
Institut für Pharmakologie, Charité Universitätsmedizin Berlin, Campus Benjamin Franklin, Thielallee 67-73, 14195 Berlin, Germany.
Jürgensen
Jana
J
Papsdorf
Gisela
G
Furkert
Jens
J
Schaefer
Michael
M
Beyermann
Michael
M
Rosenthal
Walter
W
Oksche
Alexander
A
D001667:Binding, Competitive; D002460:Cell Line; D019281:Dimerization; D016232:Endothelins / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D011233:Precipitin Tests; D011869:Radioligand Assay; D044022:Receptor, Endothelin A / Q000819:agonists / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D044023:Receptor, Endothelin B / Q000819:agonists / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
D016232:Endothelins; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D011993:Recombinant Fusion Proteins