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

A fusion protein consisting of the endothelin B (ETB) receptor and the enha nced green fluorescent protein (EGFP) in conjunction with Cyanin3- or fluor escein-conjugated endothelin 1 (Cy3-ET1, Fluo-ET1) was used to investigate the ligand-mediated internalization of the ETB receptor. The ETB receptor a nd the ETB/EGFP fusion protein displayed very similar pharmacological prope rties when expressed in Chinese hamster ovary cells. The integrity of the f usion protein was verified by low temperature PAGE analysis of the I-125-ET 1-bound ETB receptor and the I-125-ET1-bound ETB/EGFP fusion protein. Fluor escence microscopy of Chinese hamster ovary cells expressing the ETB/EGFP f usion protein demonstrated strong signals at the plasma membrane. On additi on of Cy3-ET1, internalization of ligand and receptor occurred within 5 min via a sucrose-sensitive (i.e., clathrin-mediated) pathway. On further incu bation, ETB/EGFP and Cy3-ET1 fluorescences were found in the perinuclear re gion, colocalized with fluorescent low density lipoproteins, a marker of th e late endosomal/lysosomal pathway, but not with fluorescent transferrin, a marker of the recycling pathway. No dissociation of Cy3-ET1 from the recep tor was seen within 4 h. Using I-125-ET1 or Cy3-ET1, binding sites were aga in 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-occup ied ETB receptor is internalized; however, it does not recycle like most of the G protein-coupled receptors but is sorted to the late endosomal/lysoso mal pathway in a manner similar to that of the family of protease-activated receptors.