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

Abstract

Understanding the molecular functions of the second extracellular loop (ECL2) of the calcitonin gene-related peptide (CGRP) receptor using a comprehensive mutagenesis approach, Molecular and Cellular Endocrinology (2017), doi: 10.1016/j.mce.2017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 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.

Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor

“…D113 R in the membrane proximal segment of RAMP1 formed H-bonds with ECL2 residues proximal to CLR TM4 (Y278 ECL2 ), and TM5 (T288 5.33 , H289 5.34 ) (Figure 2B). Alanine mutagenesis studies of CLR residues28–33 revealed decreased CGRP potency for the Y278 ECL2 , T288 5.33/ECL2 and W254 4.44 mutants with no impact on H289 5.34/ECL2 , I293 5.38 , T239 3.56 , V243 ICL2 and Y255 4.45 mutants32,33, consistent with important but weak interactions between RAMP1 and CLR. Likewise, there was a small decrease in CGRP potency with D113 R A mutation indicating an indirect impact on CGRP peptide binding34.…”

“…There are only limited H-bonds in the static structure between the peptide N-terminus and the CLR core; these include interactions between Y292 5.37 and the backbone of D3 P , between H295 5.40 and T6 P , and S286 ECL2 and the backbone of H10 P (Figure 3C, 3D). Of these, only the interaction between H295 5.40 and T6 P is functionally important, with H295 5.40 A reported to cause ~30-fold loss of CGRP potency in cAMP accumulation28. This amino acid is equivalent to H302 5.40 of the CTR that is predicted to form a H-bond with T6 P of sCT14.…”

“…Data Figure 8B). With the exception of S286 ECL2 , alanine mutation of these residues caused marked impairment in CGRP signalling28,30–32 (Ext. Data Figure 8B), with I284 ECL2 and L195 2.68 forming a hydrophobic barrier that coincides with the exit of the peptide from the receptor core (Ext.…”

“…Alanine substitution of T9 P causes a 15-fold loss of CGRP potency29, consistent with the importance of interaction of this side chain. While mutations to amino acids in the distal segment of ECL2 (S286 ECL2 , D287 ECL2 , H289 ECL2 , L291 5.36 ) had relatively limited effects on CGRP potency28 (Ext. Data Figure 8B), ECL2 conformation is critical to CGRP activation of its receptor, with R274 4.64 and, in particular, W283 ECL2 mutation to alanine highly detrimental to CGRP signaling32 (Ext.…”

Differential GLP-1R binding and activation by peptide and non-peptide agonists

“…T11 GLP-1 and D15 GLP-1 form polar and hydrophobic interactions with D372 ECL3 , R380 ECL3/7.35 and L384 7.39 (Figures 3 & S4). Residues at these positions within ECL2 and 3 are crucial for peptide affinity and receptor activation in numerous class B1 GPCRs (Coin et al, 2013;Koole et al, 2012a;Woolley et al, 2017). Other notable interactions include multiple hydrophobic contacts of the peptide with residues in TM1 (L141 1.36 , L144 1.39 , Y148 1.43 ), hydrogen bonding between K197 2.67 and T13 and Y205 2.75 with both S17 GLP-1 and E21 GLP-1 .…”