Abstract
Human adrenomedullin (AM) is a 52-amino acid peptide belonging to the calcitonin peptide family, which also includes calcitonin gene-related peptide (CGRP) and AM2. The two AM receptors, AM1 and AM2, are calcitonin receptor-like receptor (CL)/receptor activity-modifying protein (RAMP) (RAMP2 and RAMP3, respectively) heterodimers. CGRP receptors comprise CL/RAMP1. The only human AM receptor antagonist (AM22–52) is a truncated form of AM; it has low affinity and is only weakly selective for AM1 over AM2 receptors. To develop novel AM receptor antagonists, we explored the importance of different regions of AM in interactions with AM1, AM2, and CGRP receptors. AM22–52 was the framework for generating further AM fragments (AM26–52 and AM30–52), novel AM/αCGRP chimeras (C1–C5 and C9), and AM/AM2 chimeras (C6–C8). cAMP assays were used to screen the antagonists at all receptors to determine their affinity and selectivity. Circular dichroism spectroscopy was used to investigate the secondary structures of AM and its related peptides. The data indicate that the structures of AM, AM2, and αCGRP differ from one another. Our chimeric approach enabled the identification of two nonselective high-affinity antagonists of AM1, AM2, and CGRP receptors (C2 and C6), one high-affinity antagonist of AM2 receptors (C7), and a weak antagonist selective for the CGRP receptor (C5). By use of receptor mutagenesis, we also determined that the C-terminal nine amino acids of AM seem to be responsible for its interaction with Glu74 of RAMP3. We provide new information on the structure-activity relationship of AM, αCGRP, and AM2 and how AM interacts with CGRP and AM2 receptors.
Footnotes
This work was supported by grants from the University of Auckland Faculty Research Development Fund and the Health Research Council of New Zealand [Grant 06/314].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.156448
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ABBREVIATIONS:
- AM
- adrenomedullin
- CGRP
- calcitonin gene-related peptide
- CL
- calcitonin receptor-like receptor
- RAMP
- receptor activity-modifying protein
- dH2O
- distilled water
- TFE
- trifluoroethanol
- CD
- circular dichroism
- WT
- wild type.
- Received May 26, 2009.
- Accepted July 29, 2009.
- © 2009 by The American Society for Pharmacology and Experimental Therapeutics
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