Mapping interaction sites within the N-terminus of the calcitonin gene-related peptide receptor; the role of residues 23-60 of the calcitonin receptor-like receptor

James Barwell, Philip S. Miller, Dan Donnelly, David R. Poyner

Research output: Contribution to journalArticle

Abstract

The calcitonin receptor-like receptor (CLR) acts as a receptor for the calcitonin gene-related peptide (CGRP) but in order to recognize CGRP, it must form a complex with an accessory protein, receptor activity modifying protein 1 (RAMP1). Identifying the protein/protein and protein/ligand interfaces in this unusual complex would aid drug design. The role of the extreme N-terminus of CLR (Glu23-Ala60) was examined by an alanine scan and the results were interpreted with the help of a molecular model. The potency of CGRP at stimulating cAMP production was reduced at Leu41Ala, Gln45Ala, Cys48Ala and Tyr49Ala; furthermore, CGRP-induced receptor internalization at all of these receptors was also impaired. Ile32Ala, Gly35Ala and Thr37Ala all increased CGRP potency. CGRP specific binding was abolished at Leu41Ala, Ala44Leu, Cys48Ala and Tyr49Ala. There was significant impairment of cell surface expression of Gln45Ala, Cys48Ala and Tyr49Ala. Cys48 takes part in a highly conserved disulfide bond and is probably needed for correct folding of CLR. The model suggests that Gln45 and Tyr49 mediate their effects by interacting with RAMP1 whereas Leu41 and Ala44 are likely to be involved in binding CGRP. Ile32, Gly35 and Thr37 form a separate cluster of residues which modulate CGRP binding. The results from this study may be applicable to other family B GPCRs which can associate with RAMPs.
LanguageEnglish
Pages170-176
Number of pages7
JournalPeptides
Volume31
Issue number1
Early online date11 Nov 2009
DOIs
Publication statusPublished - Jan 2010

Fingerprint

Calcitonin Receptor-Like Protein
Calcitonin Gene-Related Peptide Receptors
Calcitonin Gene-Related Peptide
Receptor Activity-Modifying Protein 1
Proteins
Molecular Models
Drug Design
Disulfides
Alanine
Accessories
Ligands

Bibliographical note

Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported (CC BY-NC-ND 3.0)

Keywords

  • animals
  • binding sites
  • COS cells
  • calcitonin gene-related peptide
  • calcitonin receptor-like protein
  • cercopithecus aethiops
  • cyclic AMP
  • humans
  • intracellular signaling peptides and proteins
  • membrane proteins
  • molecular models
  • molecular sequence data
  • site-directed mutagenesis
  • protein binding
  • protein conformation
  • radioligand assay
  • receptor activity-modifying protein 1
  • receptor activity-modifying proteins
  • calcitonin receptors
  • CLR
  • CGRP
  • alanine scan
  • molecular modelling
  • family B GPCRs
  • RAMP1

Cite this

@article{0b86986d6d5542c9914ea483ebdcb03e,
title = "Mapping interaction sites within the N-terminus of the calcitonin gene-related peptide receptor; the role of residues 23-60 of the calcitonin receptor-like receptor",
abstract = "The calcitonin receptor-like receptor (CLR) acts as a receptor for the calcitonin gene-related peptide (CGRP) but in order to recognize CGRP, it must form a complex with an accessory protein, receptor activity modifying protein 1 (RAMP1). Identifying the protein/protein and protein/ligand interfaces in this unusual complex would aid drug design. The role of the extreme N-terminus of CLR (Glu23-Ala60) was examined by an alanine scan and the results were interpreted with the help of a molecular model. The potency of CGRP at stimulating cAMP production was reduced at Leu41Ala, Gln45Ala, Cys48Ala and Tyr49Ala; furthermore, CGRP-induced receptor internalization at all of these receptors was also impaired. Ile32Ala, Gly35Ala and Thr37Ala all increased CGRP potency. CGRP specific binding was abolished at Leu41Ala, Ala44Leu, Cys48Ala and Tyr49Ala. There was significant impairment of cell surface expression of Gln45Ala, Cys48Ala and Tyr49Ala. Cys48 takes part in a highly conserved disulfide bond and is probably needed for correct folding of CLR. The model suggests that Gln45 and Tyr49 mediate their effects by interacting with RAMP1 whereas Leu41 and Ala44 are likely to be involved in binding CGRP. Ile32, Gly35 and Thr37 form a separate cluster of residues which modulate CGRP binding. The results from this study may be applicable to other family B GPCRs which can associate with RAMPs.",
keywords = "animals, binding sites, COS cells, calcitonin gene-related peptide, calcitonin receptor-like protein, cercopithecus aethiops, cyclic AMP, humans, intracellular signaling peptides and proteins, membrane proteins, molecular models, molecular sequence data, site-directed mutagenesis, protein binding, protein conformation, radioligand assay, receptor activity-modifying protein 1, receptor activity-modifying proteins, calcitonin receptors, CLR, CGRP, alanine scan, molecular modelling, family B GPCRs, RAMP1",
author = "James Barwell and Miller, {Philip S.} and Dan Donnelly and Poyner, {David R.}",
note = "Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported (CC BY-NC-ND 3.0)",
year = "2010",
month = "1",
doi = "10.1016/j.peptides.2009.10.021",
language = "English",
volume = "31",
pages = "170--176",
journal = "Peptides",
issn = "0196-9781",
publisher = "Elsevier",
number = "1",

}

Mapping interaction sites within the N-terminus of the calcitonin gene-related peptide receptor; the role of residues 23-60 of the calcitonin receptor-like receptor. / Barwell, James; Miller, Philip S.; Donnelly, Dan; Poyner, David R.

In: Peptides, Vol. 31, No. 1, 01.2010, p. 170-176.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Mapping interaction sites within the N-terminus of the calcitonin gene-related peptide receptor; the role of residues 23-60 of the calcitonin receptor-like receptor

AU - Barwell, James

AU - Miller, Philip S.

AU - Donnelly, Dan

AU - Poyner, David R.

N1 - Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported (CC BY-NC-ND 3.0)

PY - 2010/1

Y1 - 2010/1

N2 - The calcitonin receptor-like receptor (CLR) acts as a receptor for the calcitonin gene-related peptide (CGRP) but in order to recognize CGRP, it must form a complex with an accessory protein, receptor activity modifying protein 1 (RAMP1). Identifying the protein/protein and protein/ligand interfaces in this unusual complex would aid drug design. The role of the extreme N-terminus of CLR (Glu23-Ala60) was examined by an alanine scan and the results were interpreted with the help of a molecular model. The potency of CGRP at stimulating cAMP production was reduced at Leu41Ala, Gln45Ala, Cys48Ala and Tyr49Ala; furthermore, CGRP-induced receptor internalization at all of these receptors was also impaired. Ile32Ala, Gly35Ala and Thr37Ala all increased CGRP potency. CGRP specific binding was abolished at Leu41Ala, Ala44Leu, Cys48Ala and Tyr49Ala. There was significant impairment of cell surface expression of Gln45Ala, Cys48Ala and Tyr49Ala. Cys48 takes part in a highly conserved disulfide bond and is probably needed for correct folding of CLR. The model suggests that Gln45 and Tyr49 mediate their effects by interacting with RAMP1 whereas Leu41 and Ala44 are likely to be involved in binding CGRP. Ile32, Gly35 and Thr37 form a separate cluster of residues which modulate CGRP binding. The results from this study may be applicable to other family B GPCRs which can associate with RAMPs.

AB - The calcitonin receptor-like receptor (CLR) acts as a receptor for the calcitonin gene-related peptide (CGRP) but in order to recognize CGRP, it must form a complex with an accessory protein, receptor activity modifying protein 1 (RAMP1). Identifying the protein/protein and protein/ligand interfaces in this unusual complex would aid drug design. The role of the extreme N-terminus of CLR (Glu23-Ala60) was examined by an alanine scan and the results were interpreted with the help of a molecular model. The potency of CGRP at stimulating cAMP production was reduced at Leu41Ala, Gln45Ala, Cys48Ala and Tyr49Ala; furthermore, CGRP-induced receptor internalization at all of these receptors was also impaired. Ile32Ala, Gly35Ala and Thr37Ala all increased CGRP potency. CGRP specific binding was abolished at Leu41Ala, Ala44Leu, Cys48Ala and Tyr49Ala. There was significant impairment of cell surface expression of Gln45Ala, Cys48Ala and Tyr49Ala. Cys48 takes part in a highly conserved disulfide bond and is probably needed for correct folding of CLR. The model suggests that Gln45 and Tyr49 mediate their effects by interacting with RAMP1 whereas Leu41 and Ala44 are likely to be involved in binding CGRP. Ile32, Gly35 and Thr37 form a separate cluster of residues which modulate CGRP binding. The results from this study may be applicable to other family B GPCRs which can associate with RAMPs.

KW - animals

KW - binding sites

KW - COS cells

KW - calcitonin gene-related peptide

KW - calcitonin receptor-like protein

KW - cercopithecus aethiops

KW - cyclic AMP

KW - humans

KW - intracellular signaling peptides and proteins

KW - membrane proteins

KW - molecular models

KW - molecular sequence data

KW - site-directed mutagenesis

KW - protein binding

KW - protein conformation

KW - radioligand assay

KW - receptor activity-modifying protein 1

KW - receptor activity-modifying proteins

KW - calcitonin receptors

KW - CLR

KW - CGRP

KW - alanine scan

KW - molecular modelling

KW - family B GPCRs

KW - RAMP1

UR - http://www.scopus.com/inward/record.url?scp=72749105799&partnerID=8YFLogxK

U2 - 10.1016/j.peptides.2009.10.021

DO - 10.1016/j.peptides.2009.10.021

M3 - Article

VL - 31

SP - 170

EP - 176

JO - Peptides

T2 - Peptides

JF - Peptides

SN - 0196-9781

IS - 1

ER -