Biochemical Characterization of Two Distinct Angiotensin AT 2 Receptor Populations in Murine Neuroblastoma N1E‐115 Cells

The murine neuroblastoma N1E‐115 cell line possesses a high density of angiotensin II (Angll) receptors that can be solubilized with the zwitterionic detergent 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate. These solubilized binding sites exhibited high affinity for CGP‐42112A and not Losartan, indicating that they were of the AT 2 subtype. However, displacement of 125 I‐Angll with the AT 2 nonpeptide antagonist PD‐123319 resulted in a biphasic curve, suggesting heterogeneity of the AT 2 receptor population in N1E‐115 cells. In support of this view, separation of two receptor populations was accomplished with heparin‐Sepharose chromatography. More specifically, three distinct protein peaks eluted from the heparin‐Sepharose column, two of which bound 125 I‐Angll with high affinity and saturability. One of these binding peaks (peak I) eluted rapidly and represented ∼80% of the total binding activity, whereas the remaining binding activity was contained within a second peak (peak III) that required the addition of 1.5 M NaCI for its complete elution. Pharmacological analysis revealed that both peaks of binding activity were exclusively AT 2 receptors insofar as they exhibited high affinity for CGP‐42112A and little or no affinity for the AT 1 ‐selective antagonist Losartan. However, whereas the nonpeptidic AT 2 ‐selective antagonist PD‐123319 completely displaced the binding of 126 I‐Angll from peak I in a monophasic fashion (IC 50 = 9.1 ± 4.1 n M ; mean ± SEM; n = 3), PD‐123319 was much less effective in displacing 125 I‐Angll from peak III (IC 50 = 196 β 27 n M ; mean β SEM; n = 3). Treatment of individual peaks with the reducing agent dithiothreitol caused a large increase in 125 I‐Angll specific binding in peak III, whereas a decrease in binding was observed in peak I. Moreover, GTPγS significantly reduced high‐affinity agonist binding in peak I but not peak III, further suggesting heterogeneity in the AT 2 receptor family. Finally, immunoblotting studies with polyclonal antisera raised against peak I specifically detected two proteins of 110 and 66 kDa, as is true in crude solubilized membranes, whereas no immunospecific proteins were detected in peak III. These same antisera immunoprecipitated 125 I‐Angll binding activity in peak I but were ineffective in peak III. Collectively, these results suggest that heparin‐Sepharose chromatography can efficiently separate two pharmacologically, biochemically and immunologically distinct populations of AT 2 receptors.