Characterization of a rat type 2 angiotensin II receptor stably expressed in 293 cells

A cDNA clone for the rat type 2 (AT2) angiotensin II receptor was stably transfected into human embryonic kidney 293 cells. Binding characteristics of CGP42112A (Kd = 0.18 nM, Bmax = 10.8 pmol/mg protein) and ligand specificity were indistinguishable from those obtained with the whole rat fetus and with transiently transfected COS-7 cells. Non-hydrolyzable guanine nucleotide analogs did not affect the ligand binding curve; interestingly, the guanine nucleotide analog's effect was observed in the presence of sulfhydryl reducing agent, suggesting that a certain redox condition may affect G protein coupling to this receptor. Using the established cell line, several second messenger systems were assessed. None of cAMP levels, cGMP levels, arachidonic acid release, or phosphotyrosine phosphatase activity was affected by angiotensin II stimulation of this receptor. Furthermore, the AT2 receptor did not undergo agonist-stimulated internalization. These results using the cloned receptor suggest that the transfected AT2 receptor fails to effectively couple to the major G protein-mediated signaling mechanisms and ligand-activated internalization in transfected 293 cells.