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 approximately 165 kDa and contained approximately 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.

The angiotensin IV system: functional implications.

The brain renin-angiotensin system has been implicated in the central regulation of the cardiovascular system, body water balance, and cyclic regulation of reproductive hormones and behaviors. It also exerts some influence over the secretion of pituitary hormones. This system appears to be complete with the necessary precursors and enzymes for the formation and degradation of biologically active forms of angiotensins and several binding subtypes that are presumed to mediate these and other functions. Much information is now available on the AT1 site which preferentially binds angiotensin II (AngII), but also binds angiotensin III (AngIII), and appears to be responsible for mediating the above described classic angiotensin physiologies and behaviors. Less is known about the functional importance of the AT2 site which also binds AngII but preferentially binds AngIII. This site has been implicated in vascular growth and cerebral blood flow. Recently, an AT4 site has been discovered and characterized that preferentially binds AngII (3-8), a fragment of AngII referred to as angiotensin IV (AngIV). This AT4 site is prominent in cerebral cortex, hippocampus, basal ganglia, cerebellum, and spinal cord, as well as several peripheral tissues including kidney, bladder, heart, spleen, prostate, adrenals, and colon. The AT4 site may mediate memory acquisition and recall and the regulation of blood flow. The function(s) of the AT4 receptor subtype in peripheral tissues is currently unknown, although it does appear to be involved in kidney blood flow.

Discovery of a distinct binding site for angiotensin II (3-8), a putative angiotensin IV receptor.

We report here the discovery of a unique and novel angiotensin binding site and peptide system based upon the C-terminal 3-8 hexapeptide fragment of angiotensin II (NH3(+)-Val-Tyr-Ile-His-Pro-Phe-COO-) (AII(3-8) (AIV)). This fragment binds saturably, reversibly, specifically, and with high affinity to membrane-binding sites in a variety of tissues and from many species. The binding site is pharmacologically distinct from the classic angiotensin receptors (AT1 or AT2) displaying low affinity for the known agonists (AII and AIII) and antagonist (Sar1,Ile8-AII). Although a definitive function has not been assigned to this system in many of the tissues in which it resides, AIV's interaction with endothelial cells may involve a role in endothelial cell-dependent vasodilation. Consequent to this action, AIV is a potent stimulator of renal cortical blood flow.

Identification of an AII(3-8) [AIV] binding site in guinea pig hippocampus.

A unique angiotensin binding site specific for the hexapeptide, AII(3-8), has been identified in guinea pig hippocampus. This binding site, which is present in the pyramidal cell layer of CA1, CA2, CA3 of the hippocampus and dentate gyrus, binds AII(3-8) with high affinity (KD = 1.29 +/- 0.18 nM) in a saturable manner (Bmax = 449 +/- 62 fmol/mg protein). The N-terminal structure of the binding ligand is paramount in determining the binding affinity. The C-terminal requirements seem less stringent as evidenced by the binding affinity of AII(3-7) (KD = 20.9 +/- 2.1 nM). Neither AII, AIII,Sar1, Ile8-AII, Dup 753 nor CGP42112A appear to bind, indicating that this binding site is neither the AT1 nor AT2 sites described for AII/AIII. Autoradiographic analysis of hippocampus binding confirms the inability of Sar1,Ile8-AII to compete for [125I]AII(3-8) binding. Conversely AII(3-8) was unable to displace [125I]Sar1,Ile8-AII binding.