Structure-function analysis of amino acid 74 of human RAMP1 and RAMP3 and its role in peptide interactions with adrenomedullin and calcitonin gene-related peptide receptors

Tao Qi, Kien Ly, David R. Poyner, George Christopoulos, Patrick M. Sexton, Debbie L. Hay

Research output: Contribution to journalArticle

Abstract

The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM(1) and AM(2), respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM(2)/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM(2) potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM(2) and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM(2) and CGRP receptors.
LanguageEnglish
Pages1060-1067
Number of pages8
JournalPeptides
Volume32
Issue number5
Early online date12 Mar 2011
DOIs
Publication statusPublished - May 2011

Fingerprint

Calcitonin Gene-Related Peptide Receptors
Adrenomedullin
Amino Acids
Peptides
Calcitonin Receptor-Like Protein
Calcitonin Gene-Related Peptide
Receptor Activity-Modifying Proteins
Substitution reactions
Adrenomedullin Receptors

Keywords

  • adrenomedullin
  • animals
  • COS Cells
  • cercopithecus aethiops
  • cyclic AMP
  • enzyme-linked immunosorbent assay
  • humans
  • protein binding
  • receptor activity-modifying protein 1
  • receptor activity-modifying protein 3
  • calcitonin gene-related peptide receptors
  • structure-activity relationship

Cite this

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title = "Structure-function analysis of amino acid 74 of human RAMP1 and RAMP3 and its role in peptide interactions with adrenomedullin and calcitonin gene-related peptide receptors",
abstract = "The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM(1) and AM(2), respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM(2)/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM(2) potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM(2) and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM(2) and CGRP receptors.",
keywords = "adrenomedullin, animals, COS Cells, cercopithecus aethiops, cyclic AMP, enzyme-linked immunosorbent assay, humans, protein binding, receptor activity-modifying protein 1, receptor activity-modifying protein 3, calcitonin gene-related peptide receptors, structure-activity relationship",
author = "Tao Qi and Kien Ly and Poyner, {David R.} and George Christopoulos and Sexton, {Patrick M.} and Hay, {Debbie L.}",
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Structure-function analysis of amino acid 74 of human RAMP1 and RAMP3 and its role in peptide interactions with adrenomedullin and calcitonin gene-related peptide receptors. / Qi, Tao; Ly, Kien; Poyner, David R.; Christopoulos, George; Sexton, Patrick M.; Hay, Debbie L.

In: Peptides, Vol. 32, No. 5, 05.2011, p. 1060-1067.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Structure-function analysis of amino acid 74 of human RAMP1 and RAMP3 and its role in peptide interactions with adrenomedullin and calcitonin gene-related peptide receptors

AU - Qi, Tao

AU - Ly, Kien

AU - Poyner, David R.

AU - Christopoulos, George

AU - Sexton, Patrick M.

AU - Hay, Debbie L.

N1 - Copyright © 2011. Published by Elsevier Inc.

PY - 2011/5

Y1 - 2011/5

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AB - The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM(1) and AM(2), respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM(2)/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM(2) potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM(2) and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM(2) and CGRP receptors.

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KW - humans

KW - protein binding

KW - receptor activity-modifying protein 1

KW - receptor activity-modifying protein 3

KW - calcitonin gene-related peptide receptors

KW - structure-activity relationship

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