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


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.

Publication DOI: https://doi.org/10.1016/j.mce.2017.05.034
Divisions: College of Health & Life Sciences > School of Biosciences
College of Health & Life Sciences
College of Health & Life Sciences > Chronic and Communicable Conditions
College of Health & Life Sciences > Aston Pharmacy School
College of Health & Life Sciences > School of Biosciences > Cellular and Molecular Biomedicine
Additional Information: © 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).
Uncontrolled Keywords: GPCR,family B,cAMP signaling,Biochemistry,Molecular Biology,Endocrinology
Publication ISSN: 0303-7207
Last Modified: 10 Jun 2024 07:20
Date Deposited: 06 Jun 2017 12:40
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Related URLs: http://www.scop ... tnerID=8YFLogxK (Scopus URL)
PURE Output Type: Article
Published Date: 2017-10-15
Published Online Date: 2017-05-30
Accepted Date: 2017-05-28
Submitted Date: 2017-04-07
Authors: Woolley, Michael J.
Simms, John (ORCID Profile 0000-0002-4675-0902)
Mobarec, Juan Carlos
Reynolds, Christopher A.
Poyner, David R. (ORCID Profile 0000-0003-1590-112X)
Conner, Alex C.

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