Novel peptide antagonists of adrenomedullin and calcitonin gene-related peptide receptors: identification, pharmacological characterization, and interactions with position 74 in receptor activity-modifying protein 1/3.
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, AM(1) and AM(2), 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 (AM(22-52)) is a truncated form of AM; it has low affinity and is only weakly selective for AM(1) over AM(2) receptors. To develop novel AM receptor antagonists, we explored the importance of different regions of AM in interactions with AM(1), AM(2), and CGRP receptors. AM(22-52) was the framework for generating further AM fragments (AM(26-52) and AM(30-52)), novel AM/alphaCGRP chimeras (C1-C5 and C9), and AM/AM(2) 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 alphaCGRP differ from one another. Our chimeric approach enabled the identification of two nonselective high-affinity antagonists of AM(1), AM(2), and CGRP receptors (C2 and C6), one high-affinity antagonist of AM(2) 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, alphaCGRP, and AM2 and how AM interacts with CGRP and AM(2) receptors.
Robinson
Samuel D
SD
School of Biological Sciences, University of Auckland, Auckland, New Zealand.
Aitken
Jacqueline F
JF
Bailey
Richard J
RJ
Poyner
David R
DR
Hay
Debbie L
DL
D053607:Adrenomedullin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D015740:Calcitonin Gene-Related Peptide / Q000037:antagonists & inhibitors / Q000378:metabolism; D000077221:Calcitonin Gene-Related Peptide Receptor Antagonists; D002522:Chlorocebus aethiops; D002942:Circular Dichroism; D000242:Cyclic AMP / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000187:drug effects / Q000235:genetics*; D008565:Membrane Proteins / Q000187:drug effects / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010455:Peptides / Q000494:pharmacology*; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000737:chemistry / Q000235:genetics; D014162:Transfection
C507862:ADM protein, human; D000077221:Calcitonin Gene-Related Peptide Receptor Antagonists; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010455:Peptides; C545502:RAMP1 protein, human; C545503:RAMP2 protein, human; C545504:RAMP3 protein, human; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D018015:Receptors, Calcitonin Gene-Related Peptide; D053607:Adrenomedullin; D000242:Cyclic AMP; D015740:Calcitonin Gene-Related Peptide