PURIFICATION AND CHARACTERIZATION OF ANGIOTENSIN-II AT(2)-RECEPTORS FROM NEONATAL RAT-KIDNEY

Angiotensin II (Ang II) AT2 receptors were purified 40,000-fold to a n early homogeneous state after solubilization from neonatal rat kidney membranes with opyl)dimethylammonio]-2-hydroxy-1-propane-sulfonic acid . Comparable IC50 values for the soluble extract (0.32 nM) and membran es (0.31 nM) were obtained by competition curves with I-125-labeled CG P42112, a selective AT2 ligand. Binding to AT2 receptors in the solubl e extract was not sensitive to dithiothreitol. AT2 receptors were furt her purified by gel filtration and a CGP42112 Sepharose affinity colum n. Ang II AT2 receptors were selectively eluted with 5 muM 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 confi rmed 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 af finity column was performed with 5 nM I-125-labeled CGP42112 at room t emperature after the column was treated with 1 muM losartan in the pre sence of high salt. After cross-linking, a major labeled protein with similar molecular mass and isoelectric point was obtained. Dissociatio n of the radiolabeled protein was insensitive to losartan but was enha nced 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 parti culate receptors.