Evidence 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

Item Type:	Article
Title:	Evidence 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
Creators Name:	Oksche, A. and Boese, G. and Horstmeyer, A. and Papsdorf, G. and Furkert, J. and Beyermann, M. and Bienert, M. and Rosenthal, W.
Abstract:	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.
Keywords:	Cell Line, Down-Regulation, Endothelin-1, Green Fluorescent Proteins, Luminescent Proteins, Fluorescence Microscopy, Endothelin Receptors, Recombinant Fusion Proteins, Animals, Dogs
Source:	Journal of Cardiovascular Pharmacology
ISSN:	0160-2446
Publisher:	Lippincott Williams & Wilkins
Volume:	36
Number:	5 Suppl 1
Page Range:	S44-S47
Date:	November 2000
PubMed:	View item in PubMed

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