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Radioligand binding studies revealed that Ang IV binds to insulin-regulated aminopeptidase (IRAP)/'AT4 receptors' with high affinity. Yet, as these experiments were routinely carried out in the presence of chelators, only the catalytic zinc-depleted apo-form of IRAP was labelled. While the chelators remove the catalytic zinc from IRAP and protect Ang IV from proteolytic degradation, the aminopeptidase N selective inhibitor '7B' only exerts the latter effect. By using 7B along with the new stable Ang IV-analog [3H]AL-11, we here show that the native enzyme is only a low-affinity target for Ang IV. [ABSTRACT FROM AUTHOR]

Abstract: The hexapeptide angiotensin IV (Ang IV) induces diverse biological effects such as memory enhancement and protection against ischemic stroke. Studies on the mechanism of Ang IV however are hampered by its instability and its lack of selectivity. The high-affinity binding site for Ang IV is the insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3), but Ang IV also acts as a weak agonist for the Ang II-receptor (AT1), implying the need for stable and highly selective Ang IV-analogues. Here we present the screening of novel Ang IV-analogues, selected on basis of high affinity for IRAP, high selectivity (compared to aminopeptidase N and the AT1 receptor) and resistance against proteases. The selected compound IVDE77 possesses a number of advantages compared to Ang IV: (i) it has a 40 times higher affinity for IRAP (K i 1.71nM), (ii) it does not activate the AT1 receptor, (iii) it is easily radiolabeled with tritium and (iv) it is resistant to proteolysis, even in human plasma. In addition, pre-treatment of intact CHO-K1 cells with IVDE77 led to a virtually complete inhibition of subsequent intracellular accumulation of [3H]IVDE77-IRAP complexes. IVDE77 thus represents the first Ang IV-analogue able to abolish IRAP-availability completely at the cell surface in vitro. In summary, IVDE77 is a useful tool for the detection of IRAP under physiological conditions, and may contribute to elucidating the mechanism of Ang IV to ascertain which functions are IRAP-dependent. [Copyright &y& Elsevier]

Abstract: Insulin regulated aminopeptidase (IRAP) recognises “AT4-receptor” ligands like angiotensin IV (Ang IV) and peptidomimetics like AL-11. The metabolic stability and high affinity of [3H]AL-11 for catalytically active IRAP allowed its detection in Chinese hamster ovary (CHO-K1) cell membranes in the absence of chelators (). Here, we show that, contrary to [3H]Ang IV, [3H]AL-11 displays high affinity and specificity for IRAP in intact CHO-K1 cells as well. After binding to IRAP at the surface, [3H]AL-11 is effectively internalized by an endocytotic process. Unexpectedly, surface binding and internalization of [3H]AL-11 was not affected by pretreating the cells with Ang IV but declined with AL-11. In the latter case surface expression of IRAP even increased. After elimination of simpler explanations, it is proposed that metabolically stable “AT4-receptor” ligands undergo semi-continuous cycling between the cell surface and endosomal compartments. The in vivo efficacy of stable and unstable “AT4-receptor” ligands could therefore differ. [Copyright &y& Elsevier]

Abstract: ‘AT4 receptors’ through which Angiotensin IV (Ang IV) improves memory acquisition, were recently identified as insulin regulated aminopeptidase (IRAP). Radioligand binding studies have hitherto been performed with iodinated Ang IV in the presence of divalent cation chelators EDTA and 1,10-phenanthrolin. Hence, they referred to the apo-form of IRAP. Presently, binding of [3H]Ang IV and [3H]AL-11, a stable Ang IV analog, was compared on Chinese hamster ovary (CHO-K1) and mouse hippocampal (P40H1) cell membranes. With chelators, their high affinity sites showed the same pharmacological profile as for [125I]Ang IV binding. Without chelators, only high affinity binding was perceived for [3H]AL-11. The same pharmacological profile was recorded in both membrane preparations; it was different from the one in the presence of chelators and corresponded to catalytically active IRAP (despite the concurrent presence of aminopeptidase N (APN) in P40H1 cell membranes). This confirms that the active and apo-forms of IRAP have a distinct pharmacological profile. [Copyright &y& Elsevier]