Abstract

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 thealpha 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.