Expression and characterization of mouse angiotensin II type 1a receptor tagging hemagglutinin epitope in cultured cells

The octapeptide angiotensin II mediates the physiological actions of the re nin-angiotensin system through activation of several angiotensin II recepto r (AT) subtypes, in particular AT1 (AT1a and AT1b in the case of rodents). Although we and others have generated mutant mice in which the AT1a gene wa s disrupted, the function of mouse AT1 remains to be fully elucidated, due to the lack of effective tools involving antibodies against AT1 for detecti ng biological responses in cellular conditions. To avoid these problems, we constructed the hemagglutinin (HA)-tagged mouse AT1a, and stably introduce d this recombinant receptor into human embryonic kidney 293-T cells. Radiol igand binding of [I-125] angiotensin II to AT1a was specific, saturable, an d reversible. Scatchard analysis demonstrated that the transfected receptor had a dissociation constant of 1.7 nM with a density of 1.2 x 10(5) sites/ cells. Angiotensin II stimulated a rapid increase in cytosolic free calcium , and angiotensin II-induced phosphorylation of extracellular signal-regula ted kinases (Erk) was found in a dose-dependent manner. After solubilizatio n, Western blot analysis showed specific interactions between an anti-HA an tibody and HA-tagged mouse AT1a. Furthermore, a significant proportion of H A-tagged mouse AT1a was specifically immunoprecipitated with this antibody. In the immunocytochemical and electronmicroscopic studies, treatment of th is cell line with angiotensin II resulted in decrease in signals of the sur face receptors. Based on these results, the cell line established here prov ides an excellent tool for studying angiotensin II actions mediated through mouse AT1a, at sub-nanomolar concentrations.