Vol. 298, Issue 2, 665-673, August 2001

Identification of Angiotensin II Type 2 (AT₂) Receptor Domains Mediating High-Affinity CGP 42112A Binding and Receptor Activation

John Hines, Jennifer N. Heerding, Steven J. Fluharty and Daniel K. Yee

Departments of Pharmacology (J.H.), Animal Biology (S.J.F., J.N.H., D.K.Y.), and Institute of Neurological Sciences (S.J.F.), University of Pennsylvania, Philadelphia, Pennsylvania

Chimeric angiotensin II (AngII) receptors constructed of portions of the AT₂ receptor substituted into the AT₁ receptor revealed the AT₂ third extracellular loop and seventh transmembrane-spanning domain as major determinants for the ability to bind and activate in response to the AT₂ receptor-selective agonist CGP 42112A. Radioligand binding experiments showed that chimeric AngII receptors possessing the AT₂ third extracellular loop and seventh transmembrane-spanning domain bound CGP 42112A with high affinity approaching that of the wild-type AT₂ receptor. The presence of the AT₂ third extracellular loop appeared sufficient for high-affinity CGP 42112A binding, which was further enhanced by the additional presence of the AT₂ seventh transmembrane-spanning domain. Experiments with PD 123319, losartan, and [Sar¹,Ile⁸]-AngII showed that increases in binding affinity associated with these domains were specific for CGP 42112A. Use of phosphoinositide hydrolysis as a functional index to measure activation of these chimeric AngII receptors further demonstrated that the AT₂ seventh transmembrane-spanning domain was especially critical for CGP 42112A to act as an agonist. The absence of the AT₂ seventh transmembrane-spanning domain prohibited CGP 42112A-induced activation of these receptors, even in the presence of high concentrations of CGP 42112A sufficient to saturate the binding sites. This study is the first to identify binding determinants of the AT₂ receptor that are selective for CGP 42112A, and indicates that these determinants are at least partially distinct from those for the AT₂-selective antagonist PD 123319. These differences may be a factor in the pharmacodynamic difference between these two ligands.