PT  - JOURNAL ARTICLE
AU  - Oksche, Alexander
AU  - Boese, Gregor
AU  - Horstmeyer, Angelika
AU  - Furkert, Jens
AU  - Beyermann, Michael
AU  - Bienert, Michael
AU  - Rosenthal, Walter
TI  - Late Endosomal/Lysosomal Targeting and Lack of Recycling of the Ligand-Occupied Endothelin B Receptor
DP  - 2000 Jun 01
TA  - Molecular Pharmacology
PG  - 1104--1113
VI  - 57
IP  - 6
4099  - http://molpharm.aspetjournals.org/content/57/6/1104.short
4100  - http://molpharm.aspetjournals.org/content/57/6/1104.full
SO  - Mol Pharmacol2000 Jun 01; 57
AB  - A fusion protein consisting of the endothelin B (ETB) 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 ETB receptor. The ETBreceptor and the ETB/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 125I-ET1-bound ETB receptor and the 125I-ET1-bound ETB/EGFP fusion protein. Fluorescence microscopy of Chinese hamster ovary cells expressing the ETB/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, ETB/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 125I-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 ETB 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. The American Society for Pharmacology and Experimental Therapeutics