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Late endosomal/lysosomal targeting and lack of recycling of the ligand-occupied endothelin B receptor

Item Type:Article
Title:Late endosomal/lysosomal targeting and lack of recycling of the ligand-occupied endothelin B receptor
Creators Name:Oksche, A. and Boese, G. and Horstmeyer, A. and Furkert, J. and Beyermann, M. and Bienert, M. and Rosenthal, W.
Abstract:A fusion protein consisting of the endothelin B (ET(B)) receptor and the enhanced green fluorescent protein (EGFP) in conjunction with Cyanin3- or fluorescein-conjugated endothelin 1 (Cy3-ET1, Fluo-ET1) was used to investigate the ligand-mediated internalization of the ET(B) receptor. The ET(B) receptor and the ET(B)/EGFP fusion protein displayed very similar pharmacological properties when expressed in Chinese hamster ovary cells. The integrity of the fusion protein was verified by low temperature PAGE analysis of the (125)I-ET1-bound ET(B) receptor and the (125)I-ET1-bound ET(B)/EGFP fusion protein. Fluorescence microscopy of Chinese hamster ovary cells expressing the ET(B)/EGFP fusion protein demonstrated strong signals at the plasma membrane. On addition of Cy3-ET1, internalization of ligand and receptor occurred within 5 min via a sucrose-sensitive (i.e., clathrin-mediated) pathway. On further incubation, ET(B)/EGFP and Cy3-ET1 fluorescences were found in the perinuclear region, colocalized with fluorescent low density lipoproteins, a marker of the late endosomal/lysosomal pathway, but not with fluorescent transferrin, a marker of the recycling pathway. No dissociation of Cy3-ET1 from the receptor was seen within 4 h. Using (125)I-ET1 or Cy3-ET1, binding sites were again demonstrable at the cell surface within 2 h. The reappearance of binding sites was abolished by prior treatment of the cells with cycloheximide, an inhibitor of protein synthesis. The data demonstrate that the ligand-occupied ET(B) receptor is internalized; however, it does not recycle like most of the G protein-coupled receptors but is sorted to the late endosomal/lysosomal pathway in a manner similar to that of the family of protease-activated receptors.
Keywords:Amino Acid Sequence, CHO Cells, Down-Regulation, Endosomes, Endothelin-1, Ligands, Lysosomes, Fluorescence Microscopy, Molecular Sequence Data, Phagocytosis, Endothelin B Receptor, Endothelin Receptors, Animals, Cricetinae
Source:Molecular Pharmacology
Publisher:American Society for Pharmacology and Experimental Therapeutics
Page Range:1104-1113
Date:June 2000
Official Publication:http://molpharm.aspetjournals.org/cgi/content/abstract/57/6/1104
PubMed:View item in PubMed

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