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

The extracellular loops (ECLs) of G protein-coupled receptors (GPCRs) can bind directly to docked orthosteric or allosteric ligands, they can contain transient contact points for ligand entry into the transmembrane (TM) bundle and they can regulate the activation of the receptor signalling pathways. Of the three ECLs, ECL2 is the largest and most structurally diverse reflecting its functional importance. This has been shown through biochemical techniques and has been supported by the many subsequent crystal structures of GPCRs bound to both agonists and antagonists. ECL2 shares common structural features between (and sometimes across) receptor sub-families and can facilitate ligand entry to the TM core or act directly as a surface of the ligand-binding pocket. Structural similarities seem to underpin common binding mechanisms; however, where these exist, variations in primary sequence ensure ligand-binding specificity. This review will compare current understanding of the structural themes and main functional roles of ECL2 in ligand binding, activation and regulation of the major families of GPCRs.

[1]  A. Shukla,et al.  Emerging structural insights into biased GPCR signaling. , 2014, Trends in biochemical sciences.

[2]  P. Sexton,et al.  Second Extracellular Loop of Human Glucagon-like Peptide-1 Receptor (GLP-1R) Has a Critical Role in GLP-1 Peptide Binding and Receptor Activation* , 2011, The Journal of Biological Chemistry.

[3]  Jianping Hu,et al.  Difference and Influence of Inactive and Active States of Cannabinoid Receptor Subtype CB2: From Conformation to Drug Discovery , 2016, J. Chem. Inf. Model..

[4]  Hualiang Jiang,et al.  Structural Basis for Molecular Recognition at Serotonin Receptors , 2013, Science.

[5]  Krzysztof Palczewski,et al.  The Significance of G Protein-Coupled Receptor Crystallography for Drug Discovery , 2011, Pharmacological Reviews.

[6]  Aashish Manglik,et al.  Propagation of conformational changes during μ-opioid receptor activation , 2015, Nature.

[7]  F. Leu,et al.  GPCR Somatostatin Receptor Extracellular Loop 2 Is a Key Ectodomain for Making Subtype-Selective Antibodies With Agonist-Like Activities in the Pancreatic Neuroendocrine Tumor BON Cell Line , 2010, Pancreas.

[8]  Stephen M. Husbands,et al.  Structural insights into μ-opioid receptor activation , 2015, Nature.

[9]  Aashish Manglik,et al.  Structure of the δ-opioid receptor bound to naltrindole , 2012, Nature.

[10]  K. Ahn,et al.  Dual Role of the Second Extracellular Loop of the Cannabinoid Receptor 1: Ligand Binding and Receptor Localization , 2009, Molecular Pharmacology.

[11]  J. Wess,et al.  Glutamate residues in the second extracellular loop of the human A2a adenosine receptor are required for ligand recognition. , 1996, Molecular pharmacology.

[12]  R. Abagyan,et al.  Structures of the CXCR4 Chemokine GPCR with Small-Molecule and Cyclic Peptide Antagonists , 2010, Science.

[13]  M. Babu,et al.  Molecular signatures of G-protein-coupled receptors , 2013, Nature.

[14]  Shane M. Devine,et al.  Molecular Mechanisms of Bitopic Ligand Engagement with the M1 Muscarinic Acetylcholine Receptor* , 2014, The Journal of Biological Chemistry.

[15]  K. Jacobson,et al.  Role of the second extracellular loop of adenosine receptors in agonist and antagonist binding. Analysis of chimeric A1/A3 adenosine receptors. , 1994, The Journal of biological chemistry.

[16]  G. Ladds,et al.  Receptor activity-modifying protein-directed G protein signaling specificity for the calcitonin gene-related peptide family of receptors. , 2016, The Journal of Biological Chemistry.

[17]  S. Karnik,et al.  Ligand-specific Conformation of Extracellular Loop-2 in the Angiotensin II Type 1 Receptor* , 2010, The Journal of Biological Chemistry.

[18]  T. Schwartz,et al.  Molecular Mechanism of Agonism and Inverse Agonism in the Melanocortin Receptors , 2003, Annals of the New York Academy of Sciences.

[19]  Scot Mente,et al.  Dopamine D1 receptor-agonist interactions: A mutagenesis and homology modeling study. , 2015, Bioorganic & medicinal chemistry letters.

[20]  G. Liapakis,et al.  Alanine Scanning Mutagenesis of the Second Extracellular Loop of Type 1 Corticotropin-Releasing Factor Receptor Revealed Residues Critical for Peptide Binding , 2009, Molecular Pharmacology.

[21]  S. P. Andrews,et al.  Extra-helical binding site of a glucagon receptor antagonist , 2016, Nature.

[22]  C. Reynolds,et al.  The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach , 2013, Journal of The Royal Society Interface.

[23]  A. Kruse,et al.  Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist , 2011, Nature.

[24]  D. Donnelly,et al.  Functional coupling of Cys-226 and Cys-296 in the glucagon-like peptide-1 (GLP-1) receptor indicates a disulfide bond that is close to the activation pocket , 2010, Peptides.

[25]  A. Abou-Samra,et al.  Sauvagine Cross-links to the Second Extracellular Loop of the Corticotropin-releasing Factor Type 1 Receptor* , 2002, The Journal of Biological Chemistry.

[26]  Anthony Ivetac,et al.  High-resolution structure of the human GPR40 receptor bound to allosteric agonist TAK-875 , 2014, Nature.

[27]  Arthur Christopoulos,et al.  Crystal structures of the M1 and M4 muscarinic acetylcholine receptors , 2016, Nature.

[28]  Bryan L. Roth,et al.  Structure of the human kappa opioid receptor in complex with JDTic , 2012, Nature.

[29]  Arthur Christopoulos,et al.  Critical Role for the Second Extracellular Loop in the Binding of Both Orthosteric and Allosteric G Protein-coupled Receptor Ligands* , 2007, Journal of Biological Chemistry.

[30]  M. Peitsch,et al.  Acidic residues in extracellular loops of the human Y1 neuropeptide Y receptor are essential for ligand binding. , 1994, The Journal of biological chemistry.

[31]  Gebhard F. X. Schertler,et al.  Structure of a β1-adrenergic G-protein-coupled receptor , 2008, Nature.

[32]  T. Watanabe,et al.  Human endothelin receptor ETB. Amino acid sequence requirements for super stable complex formation with its ligand. , 1994, The Journal of biological chemistry.

[33]  Ali Jazayeri,et al.  Structure of class B GPCR corticotropin-releasing factor receptor 1 , 2013, Nature.

[34]  M. Clozel,et al.  Specific receptors for endothelin on membranes from human placenta. Characterization and use in a binding assay. , 1989, Life sciences.

[35]  J. Ramachandran,et al.  Structure and Function of G Protein Coupled Receptors , 1990, Pharmaceutical Research.

[36]  T. Frimurer,et al.  Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding* , 2016, The Journal of Biological Chemistry.

[37]  R. Stevens,et al.  High-Resolution Crystal Structure of an Engineered Human β2-Adrenergic G Protein–Coupled Receptor , 2007, Science.

[38]  R. Stevens,et al.  Structural Features for Functional Selectivity at Serotonin Receptors , 2013, Science.

[39]  S. Rasmussen,et al.  Crystal Structure of the β2Adrenergic Receptor-Gs protein complex , 2011, Nature.

[40]  Ruben Abagyan,et al.  Mapping spatial approximations between the amino terminus of secretin and each of the extracellular loops of its receptor using cysteine trapping , 2012, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[41]  A. Leslie,et al.  Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation , 2011, Nature.

[42]  R. Osman,et al.  Role of the extracellular loops of the thyrotropin-releasing hormone receptor: evidence for an initial interaction with thyrotropin-releasing hormone. , 1997, Biochemistry.

[43]  M. Clozel,et al.  Specific binding of endothelin on human vascular smooth muscle cells in culture. , 1989, The Journal of clinical investigation.

[44]  S. Opella,et al.  Structure of the Chemokine Receptor CXCR1 in Phospholipid Bilayers , 2012, Nature.

[45]  Hualiang Jiang,et al.  Structure of the CCR5 Chemokine Receptor–HIV Entry Inhibitor Maraviroc Complex , 2013, Science.

[46]  J. Wess,et al.  Activation and allosteric modulation of a muscarinic acetylcholine receptor , 2013, Nature.

[47]  Jens Meiler,et al.  Structure of a Class C GPCR Metabotropic Glutamate Receptor 1 Bound to an Allosteric Modulator , 2014, Science.

[48]  Chris de Graaf,et al.  Structure of the human glucagon class B G-protein-coupled receptor , 2013, Nature.

[49]  Jonathan A. Javitch,et al.  Structure of the Human Dopamine D3 Receptor in Complex with a D2/D3 Selective Antagonist , 2010, Science.

[50]  R. Vandlen,et al.  6agonist selectivity determinants in somatostatin receptor subtypes I and II. , 1994, The Journal of biological chemistry.

[51]  Thomas H. Smith,et al.  N-linked glycosylation of protease-activated receptor-1 at extracellular loop 2 regulates G-protein signaling bias , 2015, Proceedings of the National Academy of Sciences.

[52]  J. Simms,et al.  Lifting the lid on GPCRs: the role of extracellular loops , 2011, British journal of pharmacology.

[53]  Hugh Rosen,et al.  Crystal Structure of a Lipid G Protein–Coupled Receptor , 2012, Science.

[54]  R. Abagyan,et al.  Use of Cysteine Trapping to Map Spatial Approximations between Residues Contributing to the Helix N-capping Motif of Secretin and Distinct Residues within Each of the Extracellular Loops of Its Receptor* , 2016, The Journal of Biological Chemistry.

[55]  S. Kortagere,et al.  An amino acid residue in the second extracellular loop determines the agonist-dependent tolerance property of the human D3 dopamine receptor. , 2013, ACS chemical neuroscience.

[56]  J. Simms,et al.  Systematic analysis of the entire second extracellular loop of the V(1a) vasopressin receptor: key residues, conserved throughout a G-protein-coupled receptor family, identified. , 2007, The Journal of biological chemistry.

[57]  Tomohiro Nishizawa,et al.  Activation mechanism of endothelin ETB receptor by endothelin-1 , 2016, Nature.

[58]  H. Schiöth,et al.  The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. , 2003, Molecular pharmacology.

[59]  A. Doré,et al.  Structure of class C GPCR metabotropic glutamate receptor 5 transmembrane domain , 2014, Nature.

[60]  T. Crook,et al.  Electronic Sculpting of Ligand-GPCR Subtype Selectivity: The Case of Angiotensin II , 2014, ACS chemical biology.

[61]  C. Bissantz,et al.  Structural determinants of allosteric antagonism at metabotropic glutamate receptor 2: mechanistic studies with new potent negative allosteric modulators , 2011, British journal of pharmacology.

[62]  A. Buschauer,et al.  The Extracellular Loop 2 (ECL2) of the Human Histamine H4 Receptor Substantially Contributes to Ligand Binding and Constitutive Activity , 2015, PloS one.

[63]  R. Stevens,et al.  The 2.6 Angstrom Crystal Structure of a Human A2A Adenosine Receptor Bound to an Antagonist , 2008, Science.

[64]  K. Palczewski,et al.  Crystal Structure of Rhodopsin: A G‐Protein‐Coupled Receptor , 2002, Chembiochem : a European journal of chemical biology.

[65]  A. Strasser,et al.  Molecular determinants for the high constitutive activity of the human histamine H4 receptor: functional studies on orthologues and mutants , 2015, British journal of pharmacology.

[66]  J. Klco,et al.  Essential role for the second extracellular loop in C5a receptor activation , 2005, Nature Structural &Molecular Biology.

[67]  Albert C. Pan,et al.  Structure and Dynamics of the M3 Muscarinic Acetylcholine Receptor , 2012, Nature.

[68]  J. Bockaert,et al.  Molecular tinkering of G protein‐coupled receptors: an evolutionary success , 1999, The EMBO journal.

[69]  G Vriend,et al.  The role of the second and third extracellular loops of the adenosine A1 receptor in activation and allosteric modulation. , 2012, Biochemical pharmacology.

[70]  P. Sexton,et al.  The Extracellular Surface of the GLP-1 Receptor Is a Molecular Trigger for Biased Agonism , 2016, Cell.

[71]  L. Pardo,et al.  Crystal structure of the μ-opioid receptor bound to a morphinan antagonist , 2012, Nature.

[72]  Cheng Zhang,et al.  High-resolution Crystal Structure of Human Protease-activated Receptor 1 Bound to the Antagonist Vorapaxar Hhs Public Access , 2022 .

[73]  S. Moro,et al.  Role of the extracellular loops of G protein-coupled receptors in ligand recognition: a molecular modeling study of the human P2Y1 receptor. , 1999, Biochemistry.

[74]  L. Luttrell.  Minireview: More than just a hammer: ligand "bias" and pharmaceutical discovery. , 2014, Molecular endocrinology.

[75]  A. Smolyar,et al.  Static and dynamic roles of extracellular loops in G-protein-coupled receptors: a mechanism for sequential binding of thyrotropin-releasing hormone to its receptor. , 1998, Biophysical journal.

[76]  S. Rasmussen,et al.  Structure of a nanobody-stabilized active state of the β2 adrenoceptor , 2010, Nature.

[77]  Arthur Christopoulos,et al.  Molecular Determinants of Allosteric Modulation at the M1 Muscarinic Acetylcholine Receptor* , 2014, The Journal of Biological Chemistry.

[78]  C. Wenk,et al.  Conserved disulfide bond is not essential for the adenosine A2A receptor: Extracellular cysteines influence receptor distribution within the cell and ligand-binding recognition. , 2015, Biochimica et biophysica acta.

[79]  Ruben Abagyan,et al.  The Second Extracellular Loop of the Adenosine A1 Receptor Mediates Activity of Allosteric Enhancers , 2014, Molecular Pharmacology.

[80]  Ruben Abagyan,et al.  Structure of the human histamine H1 receptor complex with doxepin , 2011, Nature.

[81]  Bryan L. Roth,et al.  Structure of the Nociceptin/Orphanin FQ Receptor in Complex with a Peptide Mimetic , 2012, Nature.

[82]  Ali Jazayeri,et al.  From G Protein-coupled Receptor Structure Resolution to Rational Drug Design* , 2015, The Journal of Biological Chemistry.

[83]  Arthur Christopoulos,et al.  Identification of Orthosteric and Allosteric Site Mutations in M2 Muscarinic Acetylcholine Receptors That Contribute to Ligand-selective Signaling Bias* , 2010, The Journal of Biological Chemistry.