We generated fusion proteins consisting of the endothelin-B (ETB)-receptor and the enhanced green fluorescent protein (EGFP) to visualize receptor internalization. In Madin Darby canine kidney (MDCK) clones expressing ETB/EGFP fusion proteins, single class high affinity binding sites for [125I]endothelin-1 (ET-1) were found (for two different clones apparent KD values were 31 ± 15 pM and 30 ± 7 pM). Pretreatment of membranes with GTPγS 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 [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 ETB/EGFP fusion protein were suitable for analysis of receptor trafficking 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 revealed rapid internalization of ligand/receptor complexes, which clustered in large, perinuclear structures (most probably late endosomes). Our data argue against recycling of the ETB receptor and favour its targeting to the lysosomal pathway.
*Research Institute for Molecular Pharmacology, Berlin and †Institute of Pharmacology, Freie University Berlin, Berlin, Germany
Address correspondence and reprint requests to Alexander Oksche, Forschungsinstitut für Molekulare Pharmakologie, Alfred-Kowalke-Strasse 4, D-10315 Berlin, Germany. E-mail: [email protected]
© 2000 Lippincott Williams & Wilkins, Inc.