Angiotensin II stabilizes a multimeric type 2 (AT2) receptor complex in murine neuroblastoma N1E-115 cells

Previous work has demonstrated that crosslinking of [125I]AngII to CHAPS solubilized angiotensin Type 2 receptors (AT2) in N1E-115 neuroblastoma cells identifies two radiolabeled proteins of 110 and 66 kDa. Similarly, affinity purification of AT2 receptors using AngII yields two proteins of 110 and 66 kDa. In the present study, anti-AT2 receptor antisera were used to examine the relationship between these two proteins. Agonist treatment (AngII) of intact cells increased the 110 kDa band while decreasing the 66 kDa protein. In intact or solubilized membranes, the ratio of 110 kDa/66 kDa proteins was significantly higher in the presence of an agonist and substantially lower with the antagonist Sar1,Ile8-AngII, suggesting that AngII stabilizes a large 110 kDa multimeric complex that may include the 66 kDa protein. To directly examine this hypothesis, anti-AT2 antisera were further purified against either the 110 or 66 kDa proteins. Both purified antibodies displayed crossreactivity with the two proteins. Moreover, when harshly reduced and denatured, the 110 kDa protein released a prominent immunoreactive 66 kDa protein, as well as other smaller proteins. Collectively, these results suggest that the 110 kDa protein consists, in part, of the 66 kDa protein and, as such, that an AT2 receptor subtype may exist as a multimeric complex that is stabilized by agonist occupancy.