entaaification of Atrial Natriuretic Factor Receptor of Neuroblastoma N4TG1 Cells: Binding Characteristics and Photoaffinity Labeling

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.