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

Samuel D Robinson, Jacqueline F Aitken, Richard J Bailey, David R Poyner, Debbie L Hay

Research output: Contribution to journalArticle

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, 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.
Original languageEnglish
Pages (from-to)513-521
Number of pages9
JournalJournal of Pharmacology and Experimental Therapeutics
Volume331
Issue number2
Early online date30 Jul 2009
DOIs
Publication statusPublished - Nov 2009

Fingerprint

Receptor Activity-Modifying Protein 3
Receptor Activity-Modifying Protein 1
Calcitonin Gene-Related Peptide Receptors
Adrenomedullin
Pharmacology
Peptides
Adrenomedullin Receptors
Calcitonin Gene-Related Peptide

Keywords

  • adrenomedullin
  • amino acid sequence
  • animals
  • COS cells
  • calcitonin gene-related peptide
  • cercopithecus aethiops
  • circular dichroism
  • cyclic AMP
  • gene expression
  • humans
  • intracellular signaling peptides and proteins
  • membrane proteins
  • molecular sequence data
  • mutagenesis
  • peptides
  • receptor activity-modifying protein 1
  • Receptor Activity-Modifying Protein 2
  • receptor activity-modifying protein 3
  • receptor activity-modifying proteins
  • calcitonin gene-related peptide receptors
  • transfection

Cite this

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title = "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",
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, 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.",
keywords = "adrenomedullin, amino acid sequence, animals, COS cells, calcitonin gene-related peptide, cercopithecus aethiops, circular dichroism, cyclic AMP, gene expression, humans, intracellular signaling peptides and proteins, membrane proteins, molecular sequence data, mutagenesis, peptides, receptor activity-modifying protein 1, Receptor Activity-Modifying Protein 2, receptor activity-modifying protein 3, receptor activity-modifying proteins, calcitonin gene-related peptide receptors, transfection",
author = "Robinson, {Samuel D} and Aitken, {Jacqueline F} and Bailey, {Richard J} and Poyner, {David R} and Hay, {Debbie L}",
year = "2009",
month = "11",
doi = "10.1124/jpet.109.156448",
language = "English",
volume = "331",
pages = "513--521",
journal = "Journal of Pharmacology and Experimental Therapeutics",
issn = "0022-3565",
publisher = "American Society for Pharmacology and Experimental Therapeutics",
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TY - JOUR

T1 - Novel peptide antagonists of adrenomedullin and calcitonin gene-related peptide receptors

T2 - identification, pharmacological characterization, and interactions with position 74 in receptor activity-modifying protein 1/3

AU - Robinson, Samuel D

AU - Aitken, Jacqueline F

AU - Bailey, Richard J

AU - Poyner, David R

AU - Hay, Debbie L

PY - 2009/11

Y1 - 2009/11

N2 - 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.

AB - 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.

KW - adrenomedullin

KW - amino acid sequence

KW - animals

KW - COS cells

KW - calcitonin gene-related peptide

KW - cercopithecus aethiops

KW - circular dichroism

KW - cyclic AMP

KW - gene expression

KW - humans

KW - intracellular signaling peptides and proteins

KW - membrane proteins

KW - molecular sequence data

KW - mutagenesis

KW - peptides

KW - receptor activity-modifying protein 1

KW - Receptor Activity-Modifying Protein 2

KW - receptor activity-modifying protein 3

KW - receptor activity-modifying proteins

KW - calcitonin gene-related peptide receptors

KW - transfection

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UR - http://jpet.aspetjournals.org/content/331/2/513

U2 - 10.1124/jpet.109.156448

DO - 10.1124/jpet.109.156448

M3 - Article

C2 - 19644039

VL - 331

SP - 513

EP - 521

JO - Journal of Pharmacology and Experimental Therapeutics

JF - Journal of Pharmacology and Experimental Therapeutics

SN - 0022-3565

IS - 2

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