'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 take place at the non-physiological apo-form of IRAP. Presently, binding of [³H]AL-11, a stable Ang IV analog, was characterized on Chinese hamster ovary (CHO-K1) cell membranes. In the presence of chelators, its high affinity sites showed the same pharmacological profile as for radiolabeled Ang IV binding. Without chelators, only for [³H]AL-11 high affinity binding could be perceived. This pharmacological profile corresponded to catalytically active IRAP and was different from the one in presence of chelators. This confirms that the active and apo-forms of IRAP have a distinct pharmacological profile and that [³H]AL-11 can specifically label both forms.

Moreover, we show that [³H]AL-11 can also be used to detect IRAP in intact cells. While [³H]AL-11 only binds with high affinity to IRAP, [³H]Ang IV fails to do so. As an application, we also show that in adipocytes the translocation of IRAP to the cell surface, mediated by insulin, can be measured by the use of [³H]AL-11.