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(Circulation Research. 2000;87:753.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Functional Reconstitution of the Angiotensin II Type 2 Receptor and G_i Activation

Jakob Lerche Hansen, Guy Servant, Thomas J. Baranski, Toshiro Fujita, Taroh Iiri, Søren P. Sheikh

From the Laboratory for Molecular Cardiology and the Department of Medicine B, University of Copenhagen (J.L.H., S.H., S.P.S.), Denmark; the Department of Cellular and Molecular Pharmacology and The Cardiovascular Research Institute, University of California (G.S.), San Francisco, Calif; the Departments of Internal Medicine and Molecular Biology and Pharmacology, Division of Endocrinology, Diabetes and Metabolism, Washington University School of Medicine (T.J.B.), St. Louis, Mo; and the Department of Endocrinology and Nephrology and University of Tokyo School of Medicine (T.I.), Tokyo, Japan.

Correspondence to Søren P. Sheikh, Laboratory of Molecular Cardiology, Rigshospitalet 9312, University of Copenhagen, Juliane Mariesvej 20, DK-2100 Copenhagen, Denmark. E-mail sheikh{at}molheart.dk\\ © 2000 American Heart Association, Inc.

Abstract—On the basis of the patterns of conserved amino acid sequence, the angiotensin II type 2 (AT₂) receptor belongs to the family of serpentine receptors, which relay signals from extracellular stimuli to heterotrimeric G proteins. However, the AT₂ receptor signal transduction mechanisms are poorly understood. We have measured AT₂-triggered activation of purified heterotrimeric proteins in urea-extracted membranes from cultured COS-7 cells expressing the recombinant receptor. This procedure removes contaminating GTP-binding proteins without inactivating the serpentine receptor. Binding studies using [¹²⁵I] angiotensin (Ang) II revealed a single binding site with a K_d=0.45 and a capacity of 627 fmol/mg protein in the extracted membranes. The AT₂ receptor caused a rapid activation of _i and _o but not of _q and _s, as measured by radioactive guanosine 5'-3-O-(thio)triphosphate (GTPS) binding. Activation required the presence of activated receptors, ß, and subunits. As a first step aimed at developing an in vitro assay to examine AT₂ receptor pharmacology, we tested a battery of Ang II–related ligands for their ability to promote AT₁ or AT₂ receptor–catalyzed G_i activation. Two proteolytic fragments of Ang II, Ang III and Ang1–7, also promoted activation of _i through the AT₂ receptor. Furthermore, we found that [Sar¹,Ala⁸]Ang II is an antagonist for both AT₁ and AT₂ receptors and that CPG42112 behaves as a partial agonist for the AT₂ receptor. In combination with previous observations, these results show that the AT₂ receptor is fully capable of activating G_i and provides a new tool for exploring AT₂ receptor pharmacology and interactions with G-protein trimers.

Key Words: AT₂ • angiotensin II type 2 receptor • G_i activation

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