Internalization of type-A endothelin receptor.

Endothelins (ETs) are 21 amino acid peptides which bind to ET(A)- and ET(B)-receptors to evoke diverse physiological responses. This report studies the internalization of ET(A)-receptor in Chinese hamster ovary (CHO) cells which were stably transfected with ET(A)-receptor cDNA. ET-1 binding induced ET(A) internalization in a time-dependent manner with 40% of ET(A)-receptors internalized at 37 degrees C after 30 min. To localize internalized ET(A)-receptor, cells were immunostained using a polyclonal antibody against the extracellular loop between IV and V transmembrane segments of the ET(A)-receptor. To examine the fate of internalized ET-1, cells were treated with 10 nM biotinylated ET-1 coupled with Texas Red-labeled streptavidin. In the absence of ET-1, a majority of ET(A) was localized on the surface of cells. After ET-1 treatment for 60 min, internalized ET(A)-receptors were localized in a perinuclear structure. ET-1 remained bound to ET(A)-receptor after internalization for up to 60 min and then dissociated from the receptor. After dissociation, ET-1 possibly became degraded and ET(A) recycled back to the cell surface. Protein kinase inhibitors such as KT5926 and staurosporine partially inhibited ET(A)-receptor internalization. The results of this study may facilitate the understanding of pathways involved in ET-1-induced receptor internalization.