entaaification of Atrial Natriuretic Factor Receptor of Neuroblastoma N4TG1 Cells: Binding Characteristics and Photoaffinity Labeling |
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Authors: | Kailash N. Pandey Kunio S. Misono Ryoichi Takayanagi Spyros N. Pavlou Tadashi Inagami |
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Affiliation: | Department of Biochemistry, Vanderbilt University, School of Medicine, Nashville, Tennessee, U.S.A.;Department of Medicine, Vanderbilt University, School of Medicine, Nashville, Tennessee, U.S.A. |
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Abstract: | We have found specific receptors for atrial natriuretic factor (ANF) in cultured neuroblastoma cells (N4TG1) of peripheral ganglionic origin. Scatchard analysis of the displacement binding revealed noninteracting, single-class binding sites with a KD of 1 X 10(-10) M and a density (Bmax) of 110,000-150,000 sites/cell. The cell-bound 125I-ANF was displaced by unlabeled ANF in a dose-dependent manner. Hormones unrelated to ANF such as angiotensins, adrenocorticotropic hormone, or arginine vasopressin were ineffective in displacing the cell-bound radioactivity. Using azidobenzoyl-125I-ANF as a photoaffinity ligand, an ANF receptor with an apparent Mr of 138,000 was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The addition of unlabeled ANF (1 microM) to the incubation medium completely abolished the labeling of this protein band, but atriopeptin I (1 microM) or angiotensins I, II, and III (each 1 microM) were not effective in inhibiting the affinity labeling. The treatment of the neuroblastoma cells with ANF stimulated intracellular cyclic GMP levels in a dose-dependent manner with an EC50 of 5 nM. ANF (1 X 10(-7) M) stimulated cyclic GMP accumulation in less than 5 min by 30-fold as compared to the controls. |
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Keywords: | Atrial natriuretic factor receptor Photoaffinity labeling Cyclic GMP Peripheral neuroblastoma cell |
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