Abstract
The decapeptide LVV-hemorphin-7 binds with high affinity to the angiotensin IV (Ang IV) receptor (AT4 receptor), eliciting a number of physiological effects, including cellular proliferation and memory enhancement. We have recently shown that the AT4receptor 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 125I-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 Tyr4 and Trp6 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 β-naphthylamide, by IRAP, withKi values between 56 and 620 nM. We find that the Val3 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.
Footnotes
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↵1 J.L. and T.M. contributed equally to the work.
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This work was supported by a National Health and Medical Research Council of Australia Block Grant 983 001.
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DOI: 10.1124/jpet.102.045492
- Abbreviations:
- Ang IV
- angiotensin IV
- AT4
- angiotensin IV receptor
- IRAP
- insulin-regulated aminopeptidase
- Leu-β-Na
- Leu-β-naphthylamide
- HEK
- human embryonic kidney
- Received October 10, 2002.
- Accepted November 19, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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