The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach

Michael J Woolley,David R Poyner,Alex C Conner,Debbie L Hay,Harriet A Watkins,Kevin J Smith,Christopher A Reynolds,James Barwell,Z Gamze Karakullukcu,Bruck Taddese

doi:10.1098/rsif.2013.0589

Abstract

The calcitonin gene-related peptide (CGRP) receptor is a complex of a calcitonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271–294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed.

Highlights

The extracellular loops of G protein-coupled receptors (GPCRs) are important for receptor function
Twenty-four individual residues of calcitonin receptor-like receptor (CLR) ECL2 from A271 to I294 were mutated to alanine and their ability to respond to calcitonin gene-related peptide (CGRP) and stimulate cAMP production was investigated when coexpressed with human receptor activitymodifying protein 1 (RAMP1)
We have evaluated the role of ECL2 in the binding of both CGRP and AM to the CGRP receptor and have interpreted the results by means of molecular modelling

Summary

Introduction

The extracellular loops of G protein-coupled receptors (GPCRs) are important for receptor function. They contribute to protein folding, provide structure to the extracellular region and mediate movement of the transmembrane (TM) helices on activation. The second extracellular loop (ECL2) is of significance for ligand binding and receptor activation [1,2,3]. In family B (or secretin-like) GPCRs, it is the most conserved and often the longest of all the ECLs, and so is in a good position to interact with the endogenous peptide agonists for these receptors and is in a prominent central position to mediate conformational changes [1]. 100 amino acids), which interacts with the C-termini of their cognate peptide ligands [4].

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Conclusion