Characterization and Purification of the Bovine Adrenal Angiotensin IV Receptor (AT4) Using [125I]Benzoylphenylalanine-Angiotensin IV as a Specific Photolabel

The Ang IV receptor, AT4, has been shown to play important roles in various mammalian tissues. In this study, structural properties of the AT4 receptor from bovine adrenals are described using a novel photoactive analog of Ang IV, [125I]Benzoylphenylalanine-Ang IV (BP-Ang IV), recently developed in our laboratory. [125I]BP-Ang IV is identical to Ang IV with regards to binding specificity and affinity and is easily cross-linked to the AT4 receptor under UV light, thus greatly facilitating the structural analysis of the AT4 receptor by SDS-PAGE. Comparisons between the native, reduced and nonreduced forms of the AT4 receptors by SDS-PAGE revealed that this receptor consists of multiple subunits. The subunit containing the Ang IV binding site (designated as the alpha subunit) has a molecular weight of ∼165 kDa and contained ∼20% N-linked carbohydrates. A subunit similar to the adrenal alpha subunit of the AT4 receptor was identified in all of the bovine tissues examined. Hippocampus and aorta contained additional [125I]BP-Ang IV bound protein bands with molecular weights of 150 and 125 kDa, respectively. Further, the alpha subunit was purified to homogeneity using a method that integrates electrofractionation with conventional protein purification techniques.