Cloning and expression of angiotensin II type 2 (AT(2)) receptors from murine neuroblastoma N1E-115 cells: Evidence for AT(2) receptor heterogeneity

Homology-based PCR was used to isolate an angiotensin II type 2 (AT(2)) receptor cDNA from murine neuroblastoma N1E-115 cells. Despite subtle differences in the nucleotide sequence (the N1E-115 clone coded for Phe(133) as TTC and Gln(326) as GAG; base substitutions are in bold-italics), the AT(2) receptor protein was identical to other reported murine AT(2) clones. When transfected into COS-1 cells, the expressed AT(2) receptor displayed high affinity for AngII and for AT(2)-selective compounds, GTP gamma S-insensitive agonist binding and enhanced agonist binding by dithiothreitol. Previously, we have demonstrated that N1E-115 cells possess two distinct subpopulations of AT, receptors, defined as peak I and peak LU receptors, that can be separated by heparin-sepharose chromatography. The two subpopulations differ pharmacologically, biochemically and immunologically. The binding properties of the cloned AT(2) receptor closely resembled that of peak III receptors. Moreover, antisera raised against peak I AT(2) receptor failed to immunoreact to either peak III receptors or cloned AT(2) receptors expressed in COS-1 cells. Collectively, these data suggest that the cloned AT(2) receptor is identical to peak III receptors from N1E-115 cells and that a novel AT(2) receptor (peak I) remains to be cloned.