Understanding the common themes and diverse roles of the second extracellular loop (ECL2) of the GPCR super-family

Woolley, Michael J.; Conner, Alex C.

doi:10.1016/j.mce.2016.11.023

Cited by 73 publications

(77 citation statements)

References 83 publications

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“…This allows the latter section of ECL2 to adopt a lower position between the extracellular ends of TM3 and TM7. However, in the other two replicas, ECL2 broadly maintains its original crystal structure position (average RMSD across replicas #1 and #4 of 2.4 Å (0.7 SD) (S4 Fig), which demonstrates ECL2 variability as previously discussed in class A GPCRs, especially in the apo state [102]. Despite the differences in observed ECL2 dynamics across replicas, little conformational change is observed in the receptor as a whole with an average RMSD of 2.2 Å (0.…”

Section: Apo A2ar Embedded In Dopc Membrane Remains In Inactive Statesupporting

confidence: 61%

Insights into adenosine A2A receptor activation through cooperative modulation of agonist and allosteric lipid interactions

2020

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The activation process of G protein-coupled receptors (GPCRs) has been extensively studied, both experimentally and computationally. In particular, Molecular Dynamics (MD) simulations have proven useful in exploring GPCR conformational space. The typical behaviour of class A GPCRs, when subjected to unbiased MD simulations from their crystallized inactive state, is to fluctuate between inactive and intermediate(s) conformations, even with bound agonist. Fully active conformation(s) are rarely stabilized unless a G protein is also bound. Despite several crystal structures of the adenosine A2a receptor (A2aR) having been resolved in complex with co-crystallized agonists and G s protein, its agonist-mediated activation process is still not completely understood. In order to thoroughly examine the conformational landscape of A2aR activation, we performed unbiased microsecond-length MD simulations in quadruplicate, starting from the inactive conformation either in apo or with bound agonists: endogenous adenosine or synthetic NECA, embedded in two homogeneous phospholipid membranes: 1,2-dioleoyl-sn-glycerol-3-phosphoglycerol (DOPG) or 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC). In DOPC with bound adenosine or NECA, we observe transition to an intermediate receptor conformation consistent with the known adenosine-bound crystal state. In apo state in DOPG, two different intermediate conformations are obtained. One is similar to that observed with bound adenosine in DOPC, while the other is closer to the active state but not yet fully active. Exclusively, in DOPG with bound adenosine or NECA, we reproducibly identify receptor conformations with fully active features, which are able to dock G s protein. These different receptor conformations can be attributed to the action/absence of agonist and phospholipid-mediated allosteric effects on the intracellular side of the receptor.

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Section: Apo A2ar Embedded In Dopc Membrane Remains In Inactive Statesupporting

confidence: 61%

Insights into adenosine A2A receptor activation through cooperative modulation of agonist and allosteric lipid interactions

2020

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“…Of the three ECLs, ECL2 is the most structurally diverse, reflecting its functional importance (Cherezov et al, 2007;Haga et al, 2012;Palczewski et al, 2000;Woolley and Conner, 2016). This has been shown through biochemical techniques and has been supported by the many crystal structures of GPCRs bound to both agonists and antagonists.…”

Section: Introductionmentioning

confidence: 88%

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

Woolley

Simms

Mobarec

et al. 2017

Molecular and Cellular Endocrinology

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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.

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“…When considering ECLs, ECL2 is usually the longest and most flexible loop in GPCRs, and therefore its resolution is often lower. However, ECL2 can exhibit different secondary structural elements, including a-helical or b-sheet elements (Woolley and Conner, 2017). In chemokine receptors, ECL2 has an organized secondary structure of two antiparallel b-sheets (Arimont et al, 2017) and is solved in all structures except CCR9.…”

Section: Challenges and Pitfallsmentioning

confidence: 99%

Chemokine Receptor Crystal Structures: What Can Be Learned from Them?

et al. 2019

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Chemokine receptors belong to the class A of G protein-coupled receptors (GPCRs) and are implicated in a wide variety of physiologic functions, mostly related to the homeostasis of the immune system. Chemokine receptors are also involved in multiple pathologic processes, including immune and autoimmune diseases, as well as cancer. Hence, several members of this GPCR subfamily are considered to be very relevant therapeutic targets. Since drug discovery efforts can be significantly reinforced by the availability of crystal structures, substantial efforts in the area of chemokine receptor structural biology could dramatically increase the outcome of drug discovery campaigns. This short review summarizes the available data on chemokine receptor crystal structures, discusses the numerous applications from chemokine receptor structures that can enhance the daily work of molecular pharmacologists, and describes the challenges and pitfalls to consider when relying on crystal structures for further research applications. SIGNIFICANCE STATEMENT This short review summarizes the available data on chemokine receptor crystal structures, discusses the numerous applications from chemokine receptor structures that can enhance the daily work of molecular pharmacologists, and describes the challenges and pitfalls to consider when relying on crystal structures for further research applications.

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Understanding the common themes and diverse roles of the second extracellular loop (ECL2) of the GPCR super-family

Cited by 73 publications

References 83 publications

Insights into adenosine A2A receptor activation through cooperative modulation of agonist and allosteric lipid interactions

Insights into adenosine A2A receptor activation through cooperative modulation of agonist and allosteric lipid interactions

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

Chemokine Receptor Crystal Structures: What Can Be Learned from Them?

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