THE ENDOTHELIN SUBTYPE-A RECEPTOR UNDERGOES AGONIST-MEDIATED AND ANTAGONIST-MEDIATED INTERNALIZATION IN THE ABSENCE OF SIGNALING

The potent vasoconstrictor and mitogen to smooth muscle cells, endothe lin-1(ET-1), acts via two distinct G protein-coupled receptors, subtyp e A (ETAR) and subtype B, that are coupled primarily to the G(q)-phosp holipase C signaling pathway. It is known that ET-1 binding to ETAR pr omotes internalization, with subsequent degradation of at least a port ion of the bound ligand. To investigate whether signaling is required for endocytosis, we developed stably transfected lines of human embryo nic 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 G(s)-adenylyl cyclase pathway. ETA RC binds ET-1 like ETAR, but is deficient in signaling. Using a combin ed concanavalin A/sucrose gradient centrifugation technique to separat e plasma membranes from other cellular membranes, we found that [I-125 ] ET-1 is rapidly internalized into ETAR-expressing cells at 37 C (t(1 /2) for internalization = 5 min; endocytic rate constant = 0.1 min(-1) ); ETARC-expressing cells also internalize [I-125]ET-1, albeit at a so mewhat slower rate than wild-type receptor (t(1/2) for internalization = 15 min; endocytic rate constant = 0.03 min(-1)). Using immunofluore scence confocal microscopy and an antibody developed to the N-terminal region of ETAR, qualitatively similar results were obtained. In addit ion, it was found using confocal microscopy that the ETAR-selective an tagonist, BQ123, also promoted rapid internalization in cells expressi ng ETAR. These results establish that inositol 1,4,5-trisphosphate sig naling 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.