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

• Published in: The Journal of pharmacology and experimental therapeutics Vol. 298; no. 2; p. 665
• Main Authors: Hines, J, Heerding, J N, Fluharty, S J, Yee, D K
• Format: Journal Article
• Language: English
• Published: United States American Society for Pharmacology and Experimental Therapeutics 01.08.2001
• Subjects: Animals; Anti-Arrhythmia Agents - pharmacology; COS Cells; DNA, Complementary - biosynthesis; DNA, Complementary - genetics; Imidazoles - pharmacology; Inosine Triphosphate - metabolism; Losartan - pharmacology; Mutagenesis - genetics; Oligopeptides - metabolism; Pyridines - pharmacology; Radioligand Assay; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Receptors, Angiotensin - agonists; Receptors, Angiotensin - genetics; Receptors, Angiotensin - metabolism; Recombinant Fusion Proteins - metabolism; Thermodynamics; Transfection
• ISSN: 0022-3565

Chimeric angiotensin II (AngII) receptors constructed of portions of the AT 2 receptor substituted into the AT 1 receptor revealed the AT 2 third extracellular loop and seventh transmembrane-spanning domain as major determinants for the ability to bind and activate in response to the AT 2 receptor-selective agonist CGP 42112A. Radioligand binding experiments showed that chimeric AngII receptors possessing the AT 2 third extracellular loop and seventh transmembrane-spanning domain bound CGP 42112A with high affinity approaching that of the wild-type AT 2 receptor. The presence of the AT 2 third extracellular loop appeared sufficient for high-affinity CGP 42112A binding, which was further enhanced by the additional presence of the AT 2 seventh transmembrane-spanning domain. Experiments with PD 123319, losartan, and [Sar 1 ,Ile 8 ]-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 AT 2 seventh transmembrane-spanning domain was especially critical for CGP 42112A to act as an agonist. The absence of the AT 2 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 AT 2 receptor that are selective for CGP 42112A, and indicates that these determinants are at least partially distinct from those for the AT 2 -selective antagonist PD 123319. These differences may be a factor in the pharmacodynamic difference between these two ligands.