The Endothelin Subtype A Receptor Undergoes Agonist- and Antagonist-Mediated Internalization in the Absence of Signaling¹

The potent vasoconstrictor and mitogen to smooth muscle cells, endothelin-1 (ET-1), acts via two distinct G protein-coupled receptors, subtype A (ETAR) and subtype B, that are coupled primarily to the Gq-phospholipase C signaling pathway. It is known that ET-1 binding to ETAR promotes internalization, with subsequent degradation of at least a portion of the bound ligand. To investigate whether signaling is required for endocytosis, we developed stably transfected lines of human embryonic kidney 293 cells expressing wild-type ETAR and a receptor chimera (ETARC) in which the C-terminal cytoplasmic tail to ETAR was replaced with that of the lutropin receptor, another G protein-coupled receptor, but one which signals through the Gs-adenylyl cyclase pathway. ETARC binds ET-1 like ETAR, but is deficient in signaling. Using a combined concanavalin A/sucrose gradient centrifugation technique to separate plasma membranes from other cellular membranes, we found that [125I]ET-1 is rapidly internalized into ETAR-expressing cells at 37 C (t1/2 for internalization = 5 min; endocytic rate constant = 0.1 min(-1); ETARC-expressing cells also internalize [125I]ET-1, albeit at a somewhat slower rate than wild-type receptor (t1/2 for internalization = 15 min; endocytic rate constant = 0.03 min(-1). Using immunofluorescence confocal microscopy and an antibody developed to the N-terminal region of ETAR, qualitatively similar results were obtained. In addition, it was found using confocal microscopy that the ETAR-selective antagonist, BQ123, also promoted rapid internalization in cells expressing ETAR. These results establish that inositol 1,4,5-trisphosphate signaling is not required for ligand-mediated internalization of ETAR and suggest that a receptor conformational change is necessary. Moreover, the finding that BQ123 promotes ETAR internalization is novel and has potentially important implications in its clinical use.