Identification of angiotensin II type 2 (AT2) receptor domains mediating high-affinity CGP 42112A binding and receptor activation.
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.
Hines
J
J
Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6046, USA.
Heerding
J N
JN
Fluharty
S J
SJ
Yee
D K
DK
D000818:Animals; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D019556:COS Cells; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D007093:Imidazoles / Q000494:pharmacology; D007293:Inosine Triphosphate / Q000378:metabolism; D019808:Losartan / Q000494:pharmacology; D016296:Mutagenesis / Q000235:genetics; D009842:Oligopeptides / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D011869:Radioligand Assay; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000819:agonists / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013816:Thermodynamics; D014162:Transfection
D000889:Anti-Arrhythmia Agents; D018076:DNA, Complementary; D007093:Imidazoles; D009842:Oligopeptides; D011725:Pyridines; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D011993:Recombinant Fusion Proteins; C060894:CGP 42112A; C073402:PD 123319; D007293:Inosine Triphosphate; D019808:Losartan