Open AccessEvidence for Downregulation of the Endothelin-B-Receptor By the Use of Fluorescent Endothelin-1 and a Fusion Protein Consisting of the Endothelin-B-Receptor and the Green Fluorescent Protein
Author(s) -
Alexander Oksche,
Gregor Boese,
Angelika Horstmeyer,
G Papsdorf,
Jens Furkert,
Michael Beyermann,
Michael Bienert,
Walter Rosenthal
Publication year - 2000
Publication title -
journal of cardiovascular pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.762
H-Index - 100
eISSN - 1533-4023
pISSN - 0160-2446
DOI - 10.1097/00005344-200036051-00016
Subject(s) - internalization , endothelin receptor , receptor , green fluorescent protein , fusion protein , endosome , endothelin 1 , microbiology and biotechnology , biology , downregulation and upregulation , g protein coupled receptor , ligand (biochemistry) , fluorescence microscope , lipid bilayer fusion , fluorescence , membrane , chemistry , biochemistry , recombinant dna , gene , physics , quantum mechanics
We generated fusion proteins consisting of the endothelin-B (ET(B))-receptor and the enhanced green fluorescent protein (EGFP) to visualize receptor internalization. In Madin Darby canine kidney (MDCK) clones expressing ET(B)/EGFP fusion proteins, single class high affinity binding sites for [125I]endothelin-1 (ET-1) were found (for two different clones apparent K(D) values were 31 +/- 15 pM and 30 +/- 7 pM). Pretreatment of membranes with GTPgammaS prior to saturation analysis did not alter these values. We also labelled ET-1 with cyanine-dyes (Cy3/ET-1, Cy5/ET-1). In displacement analyses with membranes of MDCK ET(B)/EGFP clones using [125I]ET-1, we found reduced affinity for Cy3/ET-1 and Cy5/ET-1 (about 5- to 10-fold, respectively), but normal efficacy when compared to unlabelled ET-1. Both fluorescent ligands and the ET(B)/EGFP fusion protein were suitable for analysis of receptor trafficking in living cells and cells fixed at different timepoints. Laser scanning microscopy of MDCK ET(B)/EGFP clones incubated with Cy3/ET-1 or Cy5/ET-1 revealed rapid internalization of ligand/receptor complexes, which clustered in large, perinuclear structures (most probably late endosomes). Our data argue against recycling of the ET(B) receptor and favour its targeting to the lysosomal pathway.