COVALENT CROSS-LINKING ANALYSIS OF ANGIOTENSIN RECEPTORS ON DIFFERENTIATED NG108-15 CELLS

Neuroblastoma × glioma hybrid cells (NG108-15) were used as a model system to characterize neuronal-glial type angiotensin (ANG) receptors by covalent crosslinking analysis. After differentiation with 1.5% DMSO and 0.5% fetal bovine serum for four to five days, saturation analysis revealed a single high affinity site with a Kd = 1.35 ± 0.42 nM and a Bmax = 468 ± 106 fmol/mg protein. Using the homobifunctional crosslinking reagent bis(sulfosuccinimidyl) suberate (BS3), a site with an estimated Mr of 78 kDa was specifically labeled with 125I-ANG II as determined by SDS-polyacrylamide gel electrophoresis. Both ANG II and ANG III (10-6 M) inhibited specific labeling. The Ki for ANG III binding was similar by both pharmacologic (Ki = 3.33 ± 0.98 nM) and gel densitometric (Ki = 2.65 ± 0.32 nM) analyses. We conclude that the 78 kDa protein represents a high affinity ANG binding site with similar affinities for both ANG II and ANG III. © 1990.