title: Structure-activity study of LVV-hemorphin-7: angiotensin AT4 receptor ligand and inhibitor of insulin-regulated aminopeptidase.
abstract: 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.
authors.affiliation: Howard Florey Institute, University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Joohyung
authors.initials: J
authors.lastname: Lee
authors.forename: Tomris
authors.initials: T
authors.lastname: Mustafa
authors.forename: Sharon G
authors.initials: SG
authors.lastname: McDowall
authors.forename: Frederick A O
authors.initials: FA
authors.lastname: Mendelsohn
authors.forename: Michelle
authors.initials: M
authors.lastname: Brennan
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Lew
authors.forename: Anthony L
authors.initials: AL
authors.lastname: Albiston
authors.forename: Siew Yeen
authors.initials: SY
authors.lastname: Chai
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000626:Aminopeptidases / Q000378:metabolism*; D057911:Angiotensin Receptor Antagonists; D002478:Cells, Cultured; D004347:Drug Interactions; D017353:Gene Deletion; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D053590:Interleukin 1 Receptor Antagonist Protein; D008024:Ligands; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D010455:Peptides / Q000494:pharmacology; D011945:Receptors, Angiotensin; D011994:Recombinant Proteins / Q000378:metabolism; D012795:Sialoglycoproteins / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: C090830:AT4 receptor; D057911:Angiotensin Receptor Antagonists; D006454:Hemoglobins; C507763:IL1RN protein, human; D007328:Insulin; D053590:Interleukin 1 Receptor Antagonist Protein; D008024:Ligands; D010446:Peptide Fragments; D010455:Peptides; D011945:Receptors, Angiotensin; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; C074309:LVV-hemorphin-7; D000626:Aminopeptidases; D000409:Alanine