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

Item Type:Article
Title:Ligand-dependent differences in the internalization of endothelin A and endothelin B receptor heterodimers
Creators Name:Gregan, B. and Juergensen, J. and Papsdorf, G. and Furkert, J. and Schaefer, M. and Beyermann, M. and Rosenthal, W. and Oksche, A.
Abstract: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.
Keywords:Competitive Binding, Cell Line, Dimerization, Endothelins, Fluorescence Resonance Energy Transfer, Immunoblotting, Kinetics, Precipitin Tests, Radioligand Assay, Endothelin A Receptor, Endothelin B Receptor, Recombinant Fusion Proteins, Subcellular Fractions, Transfection
Source:Journal of Biological Chemistry
Publisher:American Society for Biochemistry and Molecular Biology
Page Range:27679-27687
Date:25 June 2004
Official Publication:https://doi.org/10.1074/jbc.M403601200
PubMed:View item in PubMed

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