Endothelins (ETs) are 21 amino acid peptides which bind to ETA- and ETB-receptors to evoke diverse physiological responses. This report studies the internalization of ETA-receptor in Chinese hamster ovary (CHO) cells which were stably transfected with ETA-receptor cDNA. ET-1 binding induced ETA internalization in a time-dependent manner with 40% of ETA-receptors internalized at 37°C after 30 min. To localize internalized ETA-receptor, cells were immunostained using a polyclonal antibody against the extracellular loop between IV and V transmembrane segments of the ETA-receptor. To examine the fate of internalized ET-1, cells were treated with 10 nM biotinylated ET-1 coupled with Texas Redlabeled streptavidin. In the absence of ET-1, a majority of ETA was localized on the surface of cells. After ET-1 treatment for 60 min, internalized ETA-receptors were localized in a perinuclear structure. ET-1 remained bound to ETA-receptor after internalization for up to 60 min and then dissociated from the receptor. After dissociation, ET-1 possibly became degraded and ETA recycled back to the cell surface. Protein kinase inhibitors such as KT5926 and staurosporine partially inhibited ETA-receptor internalization. The results of this study may facilitate the understanding of pathways involved in ET-1-induced receptor internalization.
Address correspondence and reprint requests to Jinshyun WuWong, Abbott Laboratories, 5440 Patrick Henry Drive, Santa Clara, CA 95054, U.S.A.
© 2000 Lippincott Williams & Wilkins, Inc.