Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function

James Barwell, Alex C. Conner, David R Poyner

Research output: Contribution to journalArticle

Abstract

The first and third extracellular loops (ECL) of G protein-coupled receptors (GPCRs) have been implicated in ligand binding and receptor function. This study describes the results of an alanine/leucine scan of ECLs 1 and 3 and loop-associated transmembrane (TM) domains of the secretin-like GPCR calcitonin receptor-like receptor which associates with receptor activity modifying protein 1 to form the CGRP receptor. Leu195Ala, Val198Ala and Ala199Leu at the top of TM2 all reduced aCGRP-mediated cAMP production and internalization; Leu195Ala and Ala199Leu also reduced aCGRP binding. These residues form a hydrophobic cluster within an area defined as the "minor groove" of rhodopsin-like GPCRs. Within ECL1, Ala203Leu and Ala206Leu influenced the ability of aCGRP to stimulate adenylate cyclase. In TM3, His219Ala, Leu220Ala and Leu222Ala have influences on aCGRP binding and cAMP production; they are likely to indirectly influence the binding site for aCGRP as well as having an involvement in signal transduction. On the exofacial surfaces of TMs 6 and 7, a number of residues were identified that reduced cell surface receptor expression, most noticeably Leu351Ala and Glu357Ala in TM6. The residues may contribute to the RAMP1 binding interface. Ile360Ala impaired aCGRP-mediated cAMP production. Ile360 is predicted to be located close to ECL2 and may facilitate receptor activation. Identification of several crucial functional loci gives further insight into the activation mechanism of this complex receptor system and may aid rational drug design.
LanguageEnglish
Pages1906-1916
Number of pages11
JournalBBA - Molecular Cell Research
Volume1813
Issue number10
Early online date16 Jun 2011
DOIs
Publication statusPublished - Oct 2011

Fingerprint

Calcitonin Receptor-Like Protein
Calcitonin Gene-Related Peptide Receptors
G-Protein-Coupled Receptors
Receptor Activity-Modifying Protein 1
Secretin
Rhodopsin
Drug Design
Cell Surface Receptors
Adenylyl Cyclases
Leucine
Alanine
Signal Transduction
Binding Sites
Ligands

Bibliographical note

Open access under CC BY-NC-ND license

Keywords

  • amino acid sequence
  • amino acid substitution
  • animals
  • COS cells
  • calcitonin gene-related peptide
  • calcitonin receptor-like protein
  • cattle
  • cell membrane
  • cercopithecus aethiops
  • cyclic AMP
  • humans
  • biological models
  • molecular models
  • molecular sequence data
  • site-directed mutagenesis
  • mutant proteins
  • protein binding
  • protein interaction domains and motifs
  • secondary protein structure
  • receptor activity-modifying protein 1

Cite this

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title = "Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function",
abstract = "The first and third extracellular loops (ECL) of G protein-coupled receptors (GPCRs) have been implicated in ligand binding and receptor function. This study describes the results of an alanine/leucine scan of ECLs 1 and 3 and loop-associated transmembrane (TM) domains of the secretin-like GPCR calcitonin receptor-like receptor which associates with receptor activity modifying protein 1 to form the CGRP receptor. Leu195Ala, Val198Ala and Ala199Leu at the top of TM2 all reduced aCGRP-mediated cAMP production and internalization; Leu195Ala and Ala199Leu also reduced aCGRP binding. These residues form a hydrophobic cluster within an area defined as the {"}minor groove{"} of rhodopsin-like GPCRs. Within ECL1, Ala203Leu and Ala206Leu influenced the ability of aCGRP to stimulate adenylate cyclase. In TM3, His219Ala, Leu220Ala and Leu222Ala have influences on aCGRP binding and cAMP production; they are likely to indirectly influence the binding site for aCGRP as well as having an involvement in signal transduction. On the exofacial surfaces of TMs 6 and 7, a number of residues were identified that reduced cell surface receptor expression, most noticeably Leu351Ala and Glu357Ala in TM6. The residues may contribute to the RAMP1 binding interface. Ile360Ala impaired aCGRP-mediated cAMP production. Ile360 is predicted to be located close to ECL2 and may facilitate receptor activation. Identification of several crucial functional loci gives further insight into the activation mechanism of this complex receptor system and may aid rational drug design.",
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Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function. / Barwell, James; Conner, Alex C.; Poyner, David R.

In: BBA - Molecular Cell Research, Vol. 1813, No. 10, 10.2011, p. 1906-1916.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function

AU - Barwell, James

AU - Conner, Alex C.

AU - Poyner, David R

N1 - Open access under CC BY-NC-ND license

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AB - The first and third extracellular loops (ECL) of G protein-coupled receptors (GPCRs) have been implicated in ligand binding and receptor function. This study describes the results of an alanine/leucine scan of ECLs 1 and 3 and loop-associated transmembrane (TM) domains of the secretin-like GPCR calcitonin receptor-like receptor which associates with receptor activity modifying protein 1 to form the CGRP receptor. Leu195Ala, Val198Ala and Ala199Leu at the top of TM2 all reduced aCGRP-mediated cAMP production and internalization; Leu195Ala and Ala199Leu also reduced aCGRP binding. These residues form a hydrophobic cluster within an area defined as the "minor groove" of rhodopsin-like GPCRs. Within ECL1, Ala203Leu and Ala206Leu influenced the ability of aCGRP to stimulate adenylate cyclase. In TM3, His219Ala, Leu220Ala and Leu222Ala have influences on aCGRP binding and cAMP production; they are likely to indirectly influence the binding site for aCGRP as well as having an involvement in signal transduction. On the exofacial surfaces of TMs 6 and 7, a number of residues were identified that reduced cell surface receptor expression, most noticeably Leu351Ala and Glu357Ala in TM6. The residues may contribute to the RAMP1 binding interface. Ile360Ala impaired aCGRP-mediated cAMP production. Ile360 is predicted to be located close to ECL2 and may facilitate receptor activation. Identification of several crucial functional loci gives further insight into the activation mechanism of this complex receptor system and may aid rational drug design.

KW - amino acid sequence

KW - amino acid substitution

KW - animals

KW - COS cells

KW - calcitonin gene-related peptide

KW - calcitonin receptor-like protein

KW - cattle

KW - cell membrane

KW - cercopithecus aethiops

KW - cyclic AMP

KW - humans

KW - biological models

KW - molecular models

KW - molecular sequence data

KW - site-directed mutagenesis

KW - mutant proteins

KW - protein binding

KW - protein interaction domains and motifs

KW - secondary protein structure

KW - receptor activity-modifying protein 1

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U2 - 10.1016/j.bbamcr.2011.06.005

DO - 10.1016/j.bbamcr.2011.06.005

M3 - Article

VL - 1813

SP - 1906

EP - 1916

JO - BBA - Molecular Cell Research

T2 - BBA - Molecular Cell Research

JF - BBA - Molecular Cell Research

SN - 0167-4889

IS - 10

ER -