Covalent crosslinking 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 K_d = 1.35 ± 0.42 nM and a B_max = 468 ± 106 fmol/mg protein. Using the homobifunctional crosslinking reagent bis(sulfosuccinimidyl) suberate (BS³), a site with an estimated M_r 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 K_i for ANG III binding was similar by both pharmacologic (K_i = 3.33 ± 0.98 nM) and gel densitometric (K_i = 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.