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

We generated fusion proteins consisting of the endothelin-B (ETB)-receptor and the enhanced green fluorescent protein (EGFP) to visualize receptor int ernalization. In Madin Darby canine kidney (MDCK) clones expressing ETB/EGF P fusion proteins, single class high affinity binding sites for [I-125]endo thelin-1 (ET-1) were found (for two different clones apparent K-D values we re 31 +/- 15 pM and 30 +/- 7 pM). Pretreatment of membranes with GTP gammaS 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 ETB/EGFP clones using [I-125]ET-1, we found reduced affin ity for Cy3/ET-1 and Cy5/ET-1 (about 5-to 10-fold, respectively), but norma l efficacy when compared to unlabelled ET-1. Both fluorescent ligands and t he ETB/EGFP fusion protein were suitable for analysis of receptor trafficki ng in living cells and cells fixed at different timepoints. Laser scanning microscopy of MDCK ETB/EGFP clones incubated with Cy3/ET-1 or Cy5/ET-1 reve aled rapid internalization of ligand/receptor complexes, which clustered in large, perinuclear structures (most probably late endosomes). Our data arg ue against recycling of the ETB receptor and favour its targeting to the ly sosomal pathway.