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 enhanced green fluorescent protein (EGFP) in conjunc-tion with Cyanin3- or fluorescein-conjugated endothelin 1 (Cy3-ET1, Fluo-ET1) was used to investigate the ligand-mediated internalization of the ETB receptor. The ETB receptor and the ETB/EGFP fusion protein displayed very similar pharmacologi-cal properties when expressed in Chinese hamster ovary cells. The integrity of the fusion protein was verified by low temper-ature PAGE analysis of the 125I-ET1-bound ETB receptor and the 125I-ET1-bound ETB/EGFP fusion protein. Fluorescence mi-croscopy 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.