Purification and characterization of angiotensin II AT2 receptors from neonatal rat kidney.
Angiotensin II (Ang II) AT2 receptors were purified 40,000-fold to a nearly homogeneous state after solubilization from neonatal rat kidney membranes with 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane-sulfonic acid. Comparable IC50 values for the soluble extract (0.32 nM) and membranes (0.31 nM) were obtained by competition curves with 125I-labeled CGP42112, a selective AT2 ligand. Binding to AT2 receptors in the soluble extract was not sensitive to dithiothreitol. AT2 receptors were further purified by gel filtration and a CGP42112 Sepharose affinity column. Ang II AT2 receptors were selectively eluted with 5 microM CGP42112 at 4 degrees C, and a single band with an apparent molecular mass of 71 kDa was obtained after SDS/PAGE. Two-dimensional electrophoresis confirmed the purity of the protein and an isoelectric point of 5.3-5.5 was obtained. A highly selective elution of the AT2 receptors from the affinity column was performed with 5 nM 125I-labeled CGP42112 at room temperature after the column was treated with 1 microM losartan in the presence of high salt. After cross-linking, a major labeled protein with similar molecular mass and isoelectric point was obtained. Dissociation of the radiolabeled protein was insensitive to losartan but was enhanced by CGP42112, PD123177, Ang II, and [Sar1]Ang II. In summary, Ang II AT2 receptors were purified by CGP42112 affinity chromatography and selective elution and retain the pharmacological specificity of particulate receptors.
Ciuffo
G M
GM
Section on Pharmacology, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892.
Heemskerk
F M
FM
Saavedra
J M
JM
D000818:Animals; D000831:Animals, Newborn; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D007668:Kidney / Q000737:chemistry; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D011945:Receptors, Angiotensin / Q000737:chemistry / Q000302:isolation & purification*; D012995:Solubility
D011945:Receptors, Angiotensin