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

Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229

Address all correspondence and requests for reprints to: Dr. David Puett, Department of Biochemistry and Molecular Biology, Life Sciences Building, University of Georgia, Athens, Georgia 30602-7229. E-mail: puett{at}bchiris.bmb.uga.edu

The potent vasoconstrictor and mitogen to smooth muscle cells, endothelin-1 (ET-1), acts via two distinct G protein-coupled receptors, subtype A (ET_AR) and subtype B, that are coupled primarily to the G_q-phospholipase C signaling pathway. It is known that ET-1 binding to ET_AR 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 ET_AR and a receptor chimera (ET_ARC) in which the C-terminal cytoplasmic tail to ET_AR was replaced with that of the lutropin receptor, another G protein-coupled receptor, but one which signals through the G_s-adenylyl cyclase pathway. ET_ARC binds ET-1 like ET_AR, 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 ET_AR-expressing cells at 37 C (t_1/2 for internalization = 5 min; endocytic rate constant = 0.1 min^-1); ET_ARC-expressing cells also internalize [¹²⁵I]ET-1, albeit at a somewhat slower rate than wild-type receptor (t_1/2 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 ET_AR, qualitatively similar results were obtained. In addition, it was found using confocal microscopy that the ET_AR-selective antagonist, BQ123, also promoted rapid internalization in cells expressing ET_AR. These results establish that inositol 1,4,5-trisphosphate signaling is not required for ligand-mediated internalization of ET_AR and suggest that a receptor conformational change is necessary. Moreover, the finding that BQ123 promotes ET_AR internalization is novel and has potentially important implications in its clinical use.

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