ANGIOTENSIN-II AT2 RECEPTORS ARE FUNCTIONALLY COUPLED TO PROTEIN-TYROSINE DEPHOSPHORYLATION IN N1E-115 NEUROBLASTOMA-CELLS

Murine N1E-115 neuroblastoma cells are shown to express a single class of angiotensin II (Ang II) receptors that display all the pharmacolog ical properties defining the Ang II receptor subtype 2 (AT2): high aff inity for I-125-labelled AT2-selective agonist CGP 42112 (K-d 91 +/- 1 9 pM); expected rank order of potency (CGP 42112= (Sar(1),Ile(8))Ang I I greater than or equal to Ang II>PD 123319>>DUP 753) for several Ang II analogues; increased binding in the presence of the reducing reagen t dithiothreitol (DTT); and insensitivity to analogues of GTP. Molecul ar cloning of cDNA encoding AT2 receptors from N1E-115 cells reveals n ucleotide sequence identity with the AT2 subtype expressed in fetal ti ssue. Murine AT2 receptors transiently expressed in COS cells display the same pharmacological profile as endogenous Ang II receptors of N1E -115 cells. Taken together, these data reveal the exclusive presence o f the AT2 receptor subtype in N1E-115 cells. Incubation of N1E-115 cel ls with Ang II leads to a marked decrease in the level of tyrosine pho sphorylation of several proteins with apparent molecular masses of 80, 97, 120, 150 and 180 kDa respectively. Tyrosine dephosphorylation of the same set of proteins is observed after treatment with the AT2-spec ific agonist CGP 42112. The response to both effecters is rapid and tr ansient, showing a maximum between 5 and 10 min, and returning to basa l levels after 20-30 min. In both cases, tyrosine dephosphorylation ca n be prevented by co-incubation with an excess of the antagonist Saril e. These data thus establish that AT2 receptor activation leads to pro tein tyrosine dephosphorylation in N1E-115 cells, and support a possib le role for AT2 receptors in the negative regulation of cell prolifera tion.