IDENTIFICATION AND CHARACTERIZATION OF ANGIOTENSIN-II RECEPTOR SUBTYPES IN CULTURED BOVINE AND HUMAN ADRENAL FASCICULATA CELLS AND PC12W CELLS

Angiotensin-II (Ang II) receptor subtypes (AT1 and AT2) were analyzed in bovine adrenal cells (BAC) by binding and cross-linking experiments using [I-125]Ang II nd [I-125]CGP42112, a specific ligand of AT2 receptors. [I-125] Ang II binding was reduced by 80% and 20% in the presence of maximal concentrations of the AT1 antagonist losartan (DuP 753) and CGP42112, respectively, whereas [I-125]CGP42112 binding was inhibited by Ang 11 or CGP42112, but not by losartan. In the presence of the reducing agent dithio-1,4-erythritol, the binding of [I-125] CGP42112 was increased 2-fold; this was due to an increase in the binding affinity (K(d), 8 +/- 4 x 10(-10) vs. 4.8 +/- 1.2 x 10(-10) m). Cross-linking of [I-125]Ang II to BAC in the presence of disuccinimidyl suberate, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed a band of 70,000 +/- 8,000 mol wt (Mr) under both reducing and nonreducing conditions. This band disappeared when the incubation was performed in the presence of 10(-6) m Ang II or 5 x 10(-8) M CGP42112, but not in the presence of 10(-5) m losartan. Dithio-1,4-erythritol (10 mm) markedly enhanced the band. After cross-linking with 1,5-difluoro-2,4-dinitrobenzene and solubilization of the cells in the presence of protease inhibitors, two radioactive bands were observed with Mr of 70,000 and 50,000. The first disappeared after the addition of Ang II or CGP42112, whereas the second disappeared in the presence of Ang II or losartan, but not in the Presence of CGP42112. Cross-linking of [I-125]Ang II to either human adrenal fasciculata-reticularis cells, which contain only AT1 sites, or COS-7 cells transfected with human AT1 cDNA revealed a major band of 50,000 Mr that was blunted by Ang 11 or losartan, but not by CGP42112. Moreover, cross-linking of [I-125]Ang II to PC12W cells, which contain only the AT2 receptor subtype, revealed a single radioactive band of 70,000 Mr that was blunted by CGP42112 but not by losartan. Thus, in both BAC and PC12W cells, the AT2 receptor has a Mr of 70,000, whereas the AT1 receptor in BAC, human adrenal cells, and cells transfected with human AT1 receptor cDNA has a Mr of 50,000. Therefore, the heterogeneity of the size of the Ang II receptor previously reported after photoaffinity or cross-linking was probably due not only to a variation in the degree of glycosylation between tissues and species, but also to the presence of two different receptor subtypes.