Understanding the molecular functions of the second extracellular loop (ECL2) of the calcitonin gene-related peptide (CGRP) receptor using a comprehensive mutagenesis approach

Michael J. Woolley, John Simms, Juan Carlos Mobarec, Christopher A. Reynolds*, David R. Poyner, Alex C. Conner

*Corresponding author for this work

Research output: Contribution to journalArticle

Abstract

The extracellular loop 2 (ECL2) region is the most conserved of the three ECL domains in family B G protein-coupled receptors (GPCRs) and has a fundamental role in ligand binding and activation across the receptor super-family. ECL2 is fundamental for ligand-induced activation of the calcitonin gene related peptide (CGRP) receptor, a family B GPCR implicated in migraine and heart disease. In this study we apply a comprehensive targeted non-alanine substitution analysis method and molecular modelling to the functionally important residues of ECL2 to reveal key molecular interactions. We identified an interaction network between R274/Y278/D280/W283. These amino acids had the biggest reduction in signalling following alanine substitution analysis and comprise a group of basic, acidic and aromatic residues conserved in the wider calcitonin family of class B GPCRs. This study identifies key and varied constraints at each locus, including diverse biochemical requirements for neighbouring tyrosine residues and a W283H substitution that recovered wild-type (WT) signalling, despite the strictly conserved nature of the central ECL2 tryptophan and the catastrophic effects on signalling of W283A substitution. In contrast, while the distal end of ECL2 requires strict conservation of hydrophobicity or polarity in each position, mutation of these residues never has a large effect. This approach has revealed linked networks of amino acids, consistent with structural models of ECL2 and likely to represent a shared structural framework at an important ligand-receptor interface that is present across the family B GPCRs.
Original languageEnglish
JournalMolecular and Cellular Endocrinology
Volumein press
Early online date30 May 2017
DOIs
Publication statusE-pub ahead of print - 30 May 2017

Fingerprint

Calcitonin Gene-Related Peptide Receptors
Mutagenesis
G-Protein-Coupled Receptors
Substitution reactions
Ligands
Chemical activation
Amino Acids
Emitter coupled logic circuits
Molecular modeling
Molecular interactions
Structural Models
Calcitonin
Hydrophobicity
Migraine Disorders
Hydrophobic and Hydrophilic Interactions
Tryptophan
Alanine
Tyrosine
Heart Diseases
Conservation

Bibliographical note

© 2017, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/

Funding: British Heart Foundation (PG/12/59/29795); BBSRC (BB/M00015X/1, BB/M000176/1 and BB/M006883/1).

Keywords

  • GPCR
  • family B
  • cAMP signaling

Cite this

Woolley, Michael J. ; Simms, John ; Mobarec, Juan Carlos ; Reynolds, Christopher A. ; Poyner, David R. ; Conner, Alex C. / Understanding the molecular functions of the second extracellular loop (ECL2) of the calcitonin gene-related peptide (CGRP) receptor using a comprehensive mutagenesis approach. In: Molecular and Cellular Endocrinology. 2017 ; Vol. in press.
@article{077b854325f243948b722c4ad3e4f6ba,
title = "Understanding the molecular functions of the second extracellular loop (ECL2) of the calcitonin gene-related peptide (CGRP) receptor using a comprehensive mutagenesis approach",
abstract = "The extracellular loop 2 (ECL2) region is the most conserved of the three ECL domains in family B G protein-coupled receptors (GPCRs) and has a fundamental role in ligand binding and activation across the receptor super-family. ECL2 is fundamental for ligand-induced activation of the calcitonin gene related peptide (CGRP) receptor, a family B GPCR implicated in migraine and heart disease. In this study we apply a comprehensive targeted non-alanine substitution analysis method and molecular modelling to the functionally important residues of ECL2 to reveal key molecular interactions. We identified an interaction network between R274/Y278/D280/W283. These amino acids had the biggest reduction in signalling following alanine substitution analysis and comprise a group of basic, acidic and aromatic residues conserved in the wider calcitonin family of class B GPCRs. This study identifies key and varied constraints at each locus, including diverse biochemical requirements for neighbouring tyrosine residues and a W283H substitution that recovered wild-type (WT) signalling, despite the strictly conserved nature of the central ECL2 tryptophan and the catastrophic effects on signalling of W283A substitution. In contrast, while the distal end of ECL2 requires strict conservation of hydrophobicity or polarity in each position, mutation of these residues never has a large effect. This approach has revealed linked networks of amino acids, consistent with structural models of ECL2 and likely to represent a shared structural framework at an important ligand-receptor interface that is present across the family B GPCRs.",
keywords = "GPCR, family B, cAMP signaling",
author = "Woolley, {Michael J.} and John Simms and Mobarec, {Juan Carlos} and Reynolds, {Christopher A.} and Poyner, {David R.} and Conner, {Alex C.}",
note = "{\circledC} 2017, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ Funding: British Heart Foundation (PG/12/59/29795); BBSRC (BB/M00015X/1, BB/M000176/1 and BB/M006883/1).",
year = "2017",
month = "5",
day = "30",
doi = "10.1016/j.mce.2017.05.034",
language = "English",
volume = "in press",

}

Understanding the molecular functions of the second extracellular loop (ECL2) of the calcitonin gene-related peptide (CGRP) receptor using a comprehensive mutagenesis approach. / Woolley, Michael J.; Simms, John; Mobarec, Juan Carlos; Reynolds, Christopher A.; Poyner, David R.; Conner, Alex C.

In: Molecular and Cellular Endocrinology, Vol. in press, 30.05.2017.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Understanding the molecular functions of the second extracellular loop (ECL2) of the calcitonin gene-related peptide (CGRP) receptor using a comprehensive mutagenesis approach

AU - Woolley, Michael J.

AU - Simms, John

AU - Mobarec, Juan Carlos

AU - Reynolds, Christopher A.

AU - Poyner, David R.

AU - Conner, Alex C.

N1 - © 2017, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ Funding: British Heart Foundation (PG/12/59/29795); BBSRC (BB/M00015X/1, BB/M000176/1 and BB/M006883/1).

PY - 2017/5/30

Y1 - 2017/5/30

N2 - The extracellular loop 2 (ECL2) region is the most conserved of the three ECL domains in family B G protein-coupled receptors (GPCRs) and has a fundamental role in ligand binding and activation across the receptor super-family. ECL2 is fundamental for ligand-induced activation of the calcitonin gene related peptide (CGRP) receptor, a family B GPCR implicated in migraine and heart disease. In this study we apply a comprehensive targeted non-alanine substitution analysis method and molecular modelling to the functionally important residues of ECL2 to reveal key molecular interactions. We identified an interaction network between R274/Y278/D280/W283. These amino acids had the biggest reduction in signalling following alanine substitution analysis and comprise a group of basic, acidic and aromatic residues conserved in the wider calcitonin family of class B GPCRs. This study identifies key and varied constraints at each locus, including diverse biochemical requirements for neighbouring tyrosine residues and a W283H substitution that recovered wild-type (WT) signalling, despite the strictly conserved nature of the central ECL2 tryptophan and the catastrophic effects on signalling of W283A substitution. In contrast, while the distal end of ECL2 requires strict conservation of hydrophobicity or polarity in each position, mutation of these residues never has a large effect. This approach has revealed linked networks of amino acids, consistent with structural models of ECL2 and likely to represent a shared structural framework at an important ligand-receptor interface that is present across the family B GPCRs.

AB - The extracellular loop 2 (ECL2) region is the most conserved of the three ECL domains in family B G protein-coupled receptors (GPCRs) and has a fundamental role in ligand binding and activation across the receptor super-family. ECL2 is fundamental for ligand-induced activation of the calcitonin gene related peptide (CGRP) receptor, a family B GPCR implicated in migraine and heart disease. In this study we apply a comprehensive targeted non-alanine substitution analysis method and molecular modelling to the functionally important residues of ECL2 to reveal key molecular interactions. We identified an interaction network between R274/Y278/D280/W283. These amino acids had the biggest reduction in signalling following alanine substitution analysis and comprise a group of basic, acidic and aromatic residues conserved in the wider calcitonin family of class B GPCRs. This study identifies key and varied constraints at each locus, including diverse biochemical requirements for neighbouring tyrosine residues and a W283H substitution that recovered wild-type (WT) signalling, despite the strictly conserved nature of the central ECL2 tryptophan and the catastrophic effects on signalling of W283A substitution. In contrast, while the distal end of ECL2 requires strict conservation of hydrophobicity or polarity in each position, mutation of these residues never has a large effect. This approach has revealed linked networks of amino acids, consistent with structural models of ECL2 and likely to represent a shared structural framework at an important ligand-receptor interface that is present across the family B GPCRs.

KW - GPCR

KW - family B

KW - cAMP signaling

UR - http://www.scopus.com/inward/record.url?scp=85020437392&partnerID=8YFLogxK

U2 - 10.1016/j.mce.2017.05.034

DO - 10.1016/j.mce.2017.05.034

M3 - Article

VL - in press

ER -