© 2000 American Heart Association, Inc.
Molecular Medicine |
Functional Reconstitution of the Angiotensin II Type 2 Receptor and Gi Activation
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
Kd=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 (GTP
S) 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 Gi 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
Gi and provides a new tool for exploring
AT2 receptor pharmacology and interactions with
G-protein trimers.
Key Words: AT2 • angiotensin II type 2 receptor • Gi activation
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