Angiotensin AT4 ligands are potent, competitive inhibitors of insulin regulated aminopeptidase (IRAP).

Angiotensin IV (Ang IV) exerts profound effects on memory and learning, a phenomenon ascribed to its binding to a specific AT4 receptor. However the AT4 receptor has recently been identified as the insulin-regulated aminopeptidase (IRAP). In this study, we demonstrate that AT4 receptor ligands, including Ang IV, Nle1-Ang IV, divalinal-Ang IV, and the structurally unrelated LVV-hemorphin-7, are all potent inhibitors of IRAP catalytic activity, as assessed by cleavage of leu-beta-naphthylamide by recombinant human IRAP. Both Ang IV and divalinal-Ang IV display competitive kinetics, indicating that AT4 ligands mediate their effects by binding to the catalytic site of IRAP. The AT4 ligands also displaced [125I]-Nle1-Ang IV or [125I]-divalinal1-Ang IV from IRAP-HEK293T membranes with high affinity, which was up to 200-fold greater than in the catalytic assay; this difference was not consistent among the peptides, and could not be ascribed to ligand degradation. Although some AT4 ligands were subject to minor cleavage by HEK293T membranes, none were substrates for IRAP. Of a range of peptides tested, only vasopressin, oxytocin, and met-enkephalin were rapidly cleaved by IRAP. We propose that the physiological effects of AT4 ligands result, in part, from inhibition of IRAP cleavage of neuropeptides involved in memory processing.

Defective importin beta recognition and nuclear import of the sex-determining factor SRY are associated with XY sex-reversing mutations.

The architectural transcription factor SRY (sex-determining region of the Y chromosome) plays a key role in sex determination as indicated by the fact that mutations in SRY are responsible for XY gonadal dysgenesis in humans. Although many SRY mutations reduce DNA-binding/bending activity, it is not clear how SRY mutations that do not affect interaction with DNA contribute to disease. The SRY high-mobility group domain harbors two nuclear localization signals (NLSs), and here we examine SRY from four XY females with missense mutations in these signals. In all cases, mutant SRY protein is partly localized to the cytoplasm, whereas wild-type SRY is strictly nuclear. Each NLS can independently direct nuclear transport of a carrier protein in vitro and in vivo, with mutations in either affecting the rate and extent of nuclear accumulation. The N-terminal NLS function is independent of the conventional NLS-binding importins (IMPs) and requires unidentified cytoplasmic transport factors, whereas the C-terminal NLS is recognized by IMPbeta. The SRY-R133W mutant shows reduced IMPbeta binding as a direct consequence of the sex-reversing C-terminal NLS mutation. Of the N-terminal NLS mutants examined, SRY-R62G unexpectedly shows a marked reduction in IMPbeta binding, whereas SRY-R75N and SRY-R76P show normal IMPbeta binding, suggesting defects in the IMP-independent pathway. We conclude that SRY normally requires the two distinct NLS-dependent nuclear import pathways to reach sufficient levels in the nucleus for sex determination. This study documents cases of human disease being explained, at a molecular level, by the impaired ability of a protein to accumulate in the nucleus.

Structure-activity study of LVV-hemorphin-7: angiotensin AT4 receptor ligand and inhibitor of insulin-regulated aminopeptidase.

The decapeptide LVV-hemorphin-7 binds with high affinity to the angiotensin IV (Ang IV) receptor (AT(4) receptor), eliciting a number of physiological effects, including cellular proliferation and memory enhancement. We have recently shown that the AT(4) receptor is identical to insulin-regulated aminopeptidase (IRAP) and that both LVV-hemorphin-7 and Ang IV inhibit the catalytic activity of IRAP. In the current study, a series of alanine-substituted and N- or C-terminally modified analogs of LVV-hemorphin-7 were evaluated for their abilities to compete for (125)I-Ang IV binding in sheep adrenal and cerebellar membranes. Selected analogs were also analyzed for binding to recombinant human IRAP and inhibition of IRAP aminopeptidase activity. C-Terminal deletions of LVV-hemorphin-7 resulted in modest changes in affinity for IRAP, whereas deletion of the first three N-terminal residues abolished binding. Monosubstitutions of Tyr(4) and Trp(6) with alanine resulted in a 10-fold reduction in affinity. Competition binding studies using recombinant human IRAP demonstrated the same rank order of affinity as obtained for the ovine tissues. All LVV-hemorphin-7 analogs tested, except for Leu-Val-Val-Tyr, inhibit the cleavage of the synthetic substrate, leucine beta-naphthylamide, by IRAP, with K(i) values between 56 and 620 nM. We find that the Val(3) residue is crucial for LVV-hemorphin-7 binding to IRAP, whereas the C-terminal domain seems to play a minor role. The current study highlights the minimal residues necessary for binding and inhibition of IRAP and provides a basis to design peptidomimetic analogs for experimental and potentially clinical use.

AT4 receptor is insulin-regulated membrane aminopeptidase: potential mechanisms of memory enhancement.

Although angiotensin IV (Ang IV) was thought initially to be an inactive product of Ang II degradation, it was subsequently shown that the hexapeptide markedly enhances learning and memory in normal rodents and reverses the memory deficits seen in animal models of amnesia. These central nervous system effects of Ang IV are mediated by binding to a specific site, known as the AT(4) receptor, which is found in appreciable levels throughout the brain and is concentrated particularly in regions involved in cognition. This field of research was redefined by the identification of the AT(4) receptor as the transmembrane enzyme, insulin-regulated membrane aminopeptidase (IRAP). Here, we explore the potential mechanisms by which Ang IV binding to IRAP leads to the facilitation of learning and memory.