Structure of the human glucagon class B G-protein-coupled receptor

Binding of the glucagon peptide to the glucagon receptor (GCGR) triggers the release of glucose from the liver during fasting; thus GCGR plays an important role in glucose homeostasis. Here we report the crystal structure of the seven transmembrane helical domain of human GCGR at 3.4 Å resolution, complemented by extensive site-specific mutagenesis, and a hybrid model of glucagon bound to GCGR to understand the molecular recognition of the receptor for its native ligand. Beyond the shared seven transmembrane fold, the GCGR transmembrane domain deviates from class A G-protein-coupled receptors with a large ligand-binding pocket and the first transmembrane helix having a ‘stalk’ region that extends three alpha-helical turns above the plane of the membrane. The stalk positions the extracellular domain (∼12 kilodaltons) relative to the membrane to form the glucagon-binding site that captures the peptide and facilitates the insertion of glucagon’s amino terminus into the seven transmembrane domain.

[1]  A. Couvineau,et al.  Spatial proximity between the VPAC1 receptor and the amino terminus of agonist and antagonist peptides reveals distinct sites of interaction , 2012, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[2]  M. Caffrey,et al.  Detergents destabilize the cubic phase of monoolein: implications for membrane protein crystallization. , 2003, Biophysical journal.

[3]  Arthur Christopoulos,et al.  Polar transmembrane interactions drive formation of ligand-specific and signal pathway-biased family B G protein-coupled receptor conformations , 2013, Proceedings of the National Academy of Sciences.

[4]  V. Hruby,et al.  Development of potent truncated glucagon antagonists. , 2001, Journal of medicinal chemistry.

[5]  P. Conn Methods in neurosciences , 1991 .

[6]  Lotte Bjerre Knudsen,et al.  Crystal Structure of Glucagon-like Peptide-1 in Complex with the Extracellular Domain of the Glucagon-like Peptide-1 Receptor* , 2009, The Journal of Biological Chemistry.

[7]  R. Stevens,et al.  Rastering strategy for screening and centring of microcrystal samples of human membrane proteins with a sub-10 µm size X-ray synchrotron beam , 2009, Journal of The Royal Society Interface.

[8]  V. Cherezov,et al.  Crystallizing membrane proteins using lipidic mesophases , 2009, Nature Protocols.

[9]  S. Hoare,et al.  Mechanisms of peptide and nonpeptide ligand binding to Class B G-protein-coupled receptors. , 2005, Drug discovery today.

[10]  Martine Prévost,et al.  Mutational and Cysteine Scanning Analysis of the Glucagon Receptor N-terminal Domain* , 2010, The Journal of Biological Chemistry.

[11]  T. Yaqub,et al.  Identification of Determinants of Glucose-Dependent Insulinotropic Polypeptide Receptor That Interact with N-Terminal Biologically Active Region of the Natural Ligand , 2010, Molecular Pharmacology.

[12]  Avinash Peddi,et al.  Electronic Reprint Biological Crystallography a Robotic System for Crystallizing Membrane and Soluble Proteins in Lipidic Mesophases Biological Crystallography a Robotic System for Crystallizing Membrane and Soluble Proteins in Lipidic Mesophases , 2022 .

[13]  I. Hötzel,et al.  Molecular basis for negative regulation of the glucagon receptor , 2012, Proceedings of the National Academy of Sciences.

[14]  Randy J. Read,et al.  phenix.mr_rosetta: molecular replacement and model rebuilding with Phenix and Rosetta , 2012, Journal of Structural and Functional Genomics.

[15]  A. Couvineau,et al.  Diffuse Pharmacophoric Domains of Vasoactive Intestinal Peptide (VIP) and Further Insights into the Interaction of VIP with the N-terminal Ectodomain of Human VPAC1 Receptor by Photoaffinity Labeling with [Bpa6]-VIP* , 2004, Journal of Biological Chemistry.

[16]  R. Stevens,et al.  Development of an Automated High Throughput LCP-FRAP Assay to Guide Membrane Protein Crystallization in Lipid Mesophases. , 2011, Crystal growth & design.

[17]  T. Ikegami,et al.  Conformation of a peptide ligand bound to its G-protein coupled receptor , 2001, Nature Structural Biology.

[18]  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.

[19]  Maxim Totrov,et al.  Development of a new physics‐based internal coordinate mechanics force field and its application to protein loop modeling , 2011, Proteins.

[20]  Ruben Abagyan,et al.  Refinement of Glucagon-like Peptide 1 Docking to Its Intact Receptor Using Mid-region Photolabile Probes and Molecular Modeling* , 2011, The Journal of Biological Chemistry.

[21]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[22]  T. Kieffer,et al.  Targeting the glucagon receptor family for diabetes and obesity therapy. , 2012, Pharmacology & therapeutics.

[23]  Talapady N. Bhat,et al.  Calculation of an OMIT map , 1988 .

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

[25]  L. Lin,et al.  A point mutation in the glucose-dependent insulinotropic peptide receptor confers constitutive activity. , 1997, Biochemical and biophysical research communications.

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

[27]  L. Miller,et al.  Structural basis of natural ligand binding and activation of the Class II G-protein-coupled secretin receptor. , 2007, Biochemical Society transactions.

[28]  Adam Godzik,et al.  The JCSG MR pipeline: optimized alignments, multiple models and parallel searches , 2007, Acta crystallographica. Section D, Biological crystallography.

[29]  Ruben Abagyan,et al.  Molecular Basis of Secretin Docking to Its Intact Receptor Using Multiple Photolabile Probes Distributed throughout the Pharmacophore* , 2011, The Journal of Biological Chemistry.

[30]  Hyeon Joo,et al.  OPM database and PPM web server: resources for positioning of proteins in membranes , 2011, Nucleic Acids Res..

[31]  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.

[32]  A. Couvineau,et al.  Class-B GPCR activation: is ligand helix-capping the key? , 2008, Trends in biochemical sciences.

[33]  P. de Neef,et al.  Mutations of aromatic residues in the first transmembrane helix impair signalling by the secretin receptor. , 1999, Receptors & channels.

[34]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

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

[36]  P. de Neef,et al.  Contribution of the second transmembrane helix of the secretin receptor to the positioning of secretin , 1998, FEBS letters.

[37]  P. Emsley,et al.  Features and development of Coot , 2010, Acta crystallographica. Section D, Biological crystallography.

[38]  Joshua M. Kunken,et al.  Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors. , 2012, Structure.

[39]  R. Rudolph,et al.  Passing the baton in class B GPCRs: peptide hormone activation via helix induction? , 2009, Trends in biochemical sciences.

[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]  Kuntal Pal,et al.  Structure and mechanism for recognition of peptide hormones by Class B G-protein-coupled receptors , 2012, Acta Pharmacologica Sinica.

[42]  L. Miller,et al.  Protean effects of a natural peptide agonist of the G protein-coupled secretin receptor demonstrated by receptor mutagenesis. , 1998, The Journal of pharmacology and experimental therapeutics.

[43]  R. Stevens,et al.  Profiling of membrane protein variants in a baculovirus system by coupling cell-surface detection with small-scale parallel expression. , 2007, Protein expression and purification.

[44]  Randy J. Read,et al.  Electronic Reprint Biological Crystallography Decision-making in Structure Solution Using Bayesian Estimates of Map Quality: the Phenix Autosol Wizard Biological Crystallography Decision-making in Structure Solution Using Bayesian Estimates of Map Quality: the Phenix Autosol Wizard , 2022 .

[45]  Analysis of the glucagon receptor first extracellular loop by the substituted cysteine accessibility method , 2011, Peptides.

[46]  J. Vilardaga,et al.  Role of charged amino acids conserved in the vasoactive intestinal polypeptide/secretin family of receptors on the secretin receptor functionality☆ , 1999, Peptides.

[47]  R. Stevens,et al.  Structure-function of the G protein-coupled receptor superfamily. , 2013, Annual review of pharmacology and toxicology.

[48]  B. Wulff,et al.  Three Distinct Epitopes on the Extracellular Face of the Glucagon Receptor Determine Specificity for the Glucagon Amino Terminus* , 2003, Journal of Biological Chemistry.

[49]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[50]  Randy J. Read,et al.  Dauter Iterative model building , structure refinement and density modification with the PHENIX AutoBuild wizard , 2007 .

[51]  R. B. Merrifield,et al.  Roles of specific extracellular domains of the glucagon receptor in ligand binding and signaling. , 2002, Biochemistry.

[52]  F. Grégoire,et al.  Mutational analysis of the glucagon receptor: similarities with the vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide (PACAP)/secretin receptors for recognition of the ligand's third residue. , 2002, The Biochemical journal.

[53]  K. Coopman,et al.  Residues within the transmembrane domain of the glucagon-like peptide-1 receptor involved in ligand binding and receptor activation: modelling the ligand-bound receptor. , 2011, Molecular endocrinology.

[54]  Didier Rognan,et al.  Structure‐Based Discovery of Allosteric Modulators of Two Related Class B G‐Protein‐Coupled Receptors , 2011, ChemMedChem.

[55]  M. Wheeler,et al.  Characterization of glucagon-like peptide-1 receptor-binding determinants. , 2000, Journal of molecular endocrinology.

[56]  M. Maccoss,et al.  Characterization of a Novel, Non-peptidyl Antagonist of the Human Glucagon Receptor* , 1999, The Journal of Biological Chemistry.

[57]  P. Robberecht,et al.  Two Basic Residues of the h-VPAC1 Receptor Second Transmembrane Helix Are Essential for Ligand Binding and Signal Transduction* , 2001, The Journal of Biological Chemistry.

[58]  T. Gardella,et al.  Identification of a contact site for residue 19 of parathyroid hormone (PTH) and PTH-related protein analogs in transmembrane domain two of the type 1 PTH receptor. , 2003, Molecular endocrinology.

[59]  R. Stevens,et al.  Structure of the human k-opioid receptor in complex with JDTic , 2012 .