Solution structure and mutational analysis of pituitary adenylate cyclase-activating polypeptide binding to the extracellular domain of PAC1-R
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Sun, Chaohong; Song, Danying; Davis-Taber, Rachel; Barrett, Leo W.; Scott, Victoria E.; Richardson, Paul L.; Pereda‐Lopez, Ana; Uchic, Marie E.; Solomon, Larry R.; Lake, Marc; Walter, Karl A.; Hajduk, Philip J.; Olejniczak, Edward T.

doi:10.1073/pnas.0611397104

Cited by 132 publications

(159 citation statements)

References 37 publications

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“…This interaction induces a structural change in the transmembrane and intracellular face of the receptor that enables G protein coupling, likely similar to that described for the activated form of the β-adrenergic receptor (15). Recent structural studies of several class B GPCR ECDs and ECD-ligand complexes support this model (16)(17)(18)(19)(20)(21). Glucagon likely interacts with GCGR in a similar fashion to the interaction of other peptide ligands with class B GPCRs, although currently undefined differences would ensure receptor specificity.…”

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confidence: 93%

“…The GCGR ECD structure resembles the α-β-β-α fold common to other class B GPCR ECD structures (16)(17)(18)(19)(20)(21)(22)(23) and is most closely related to the glucagon-like peptide-1 receptor (GLP-1R). These receptors share 46% sequence identity within their ECDs, and their overall structures superimpose well, with an rmsd of 1.5 Å (Fig.…”

Section: Antagonist and Inverse Agonist Antibodies Targeting The Gcgrmentioning

confidence: 99%

“…An individual homozygous for a P86S mutation has hallmarks of loss of glucagon action, and this receptor variant was unable to bind glucagon in vitro (10). Residues at the base of L4 adjacent to P86, including the invariant P82 and conserved Y84 and L85, form part of an extended hydrophobic surface in the canonical hormone-binding pocket (16)(17)(18)(19)(20)(21)(22)(23). Residues in L2 have also been shown to be critical for glucagon binding and/or receptor structure (24,25), including D63, which forms a salt bridge with the sidechains of K98 and R116 and is within H-bond distance of W68 and the backbone amide of S66 (Fig.…”

Section: Antagonist and Inverse Agonist Antibodies Targeting The Gcgrmentioning

confidence: 99%

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Molecular basis for negative regulation of the glucagon receptor

Koth¹,

Murray²,

Mukund³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

123

179

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Members of the class B family of G protein-coupled receptors (GPCRs) bind peptide hormones and have causal roles in many diseases, ranging from diabetes and osteoporosis to anxiety. Although peptide, small-molecule, and antibody inhibitors of these GPCRs have been identified, structure-based descriptions of receptor antagonism are scarce. Here we report the mechanisms of glucagon receptor inhibition by blocking antibodies targeting the receptor's extracellular domain (ECD). These studies uncovered a role for the ECD as an intrinsic negative regulator of receptor activity. The crystal structure of the ECD in complex with the Fab fragment of one antibody, mAb1, reveals that this antibody inhibits glucagon receptor by occluding a surface extending across the entire hormone-binding cleft. A second antibody, mAb23, blocks glucagon binding and inhibits basal receptor activity, indicating that it is an inverse agonist and that the ECD can negatively regulate receptor activity independent of ligand binding. Biochemical analyses of receptor mutants in the context of a high-resolution ECD structure show that this previously unrecognized inhibitory activity of the ECD involves an interaction with the third extracellular loop of the receptor and suggest that glucagon-mediated structural changes in the ECD accompany receptor activation. These studies have implications for the design of drugs to treat class B GPCR-related diseases, including the potential for developing novel allosteric regulators that target the ECDs of these receptors.T he glucagon receptor (GCGR) is a member of the class B G protein-coupled receptor (GPCR) family (1) that mediates the activity of glucagon, a pancreatic islet-derived peptide hormone that plays a central role in the pathophysiology of diabetes (2). Several GCGR antagonists that improve glycemic control in animal models of diabetes and diabetic patients have been described (3-8). Although biochemical studies of glucagon and GCGR mutants have facilitated the mapping of some elements that contribute to glucagon binding (4, 9-12), the molecular mechanisms of GCGR activation and inhibition remain largely unknown because there are currently no high-resolution structures of GCGR. The current model for activation class B GPCRs proposes a tethering mechanism whereby the C-terminal half of the peptide ligand first binds a large extracellular domain (ECD), thereby enabling a high-affinity interaction of the N-terminal half of the ligand with a cleft formed by the transmembrane α-helical bundle (13,14), termed the juxtamembrane (JM) domain. This interaction induces a structural change in the transmembrane and intracellular face of the receptor that enables G protein coupling, likely similar to that described for the activated form of the β-adrenergic receptor (15). Recent structural studies of several class B GPCR ECDs and ECD-ligand complexes support this model (16)(17)(18)(19)(20)(21). Glucagon likely interacts with GCGR in a similar fashion to the interaction of other peptide ligands with class B GPC...

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mentioning

confidence: 93%

Section: Antagonist and Inverse Agonist Antibodies Targeting The Gcgrmentioning

confidence: 99%

Section: Antagonist and Inverse Agonist Antibodies Targeting The Gcgrmentioning

confidence: 99%

See 1 more Smart Citation

Molecular basis for negative regulation of the glucagon receptor

Koth¹,

Murray²,

Mukund³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

123

179

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“…Because the anionic and cationic amino acid residues as well as the tryptophans are absolutely conserved within the B1 subfamily, it was proposed that all B1 ECD1s include Sushi domain folds (17,18). The two subsequent reports of Sushi domain folds for the ECD1s of a pituitary adenylate cyclaseactivating polypeptide receptor (20) (derived from a solution NMR data) and of an incretin receptor (21) (derived from a crystal structure data) provide strong support for our proposal of the Sushi module in the ECD1 of all B1 receptors. The conserved amino acid residues relevant to the studies presented here are the anionic and cationic residues, Asp 65 and Arg…”

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confidence: 99%

Distinct Structural and Functional Roles of Conserved Residues in the First Extracellular Domain of Receptors for Corticotropin-releasing Factor and Related G-protein-coupled Receptors

Perrin

Grace

DiGruccio

et al. 2007

Journal of Biological Chemistry

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The G-protein-coupled receptor B1 family includes corticotropin-releasing factor (CRF), growth hormone-releasing hormone, incretin, and pituitary adenylate cyclase-activating polypeptide receptors. The three-dimensional NMR structure of the first extracellular domain (ECD1) of CRF receptor 2␤ (CRF-R2␤), free and complexed with astressin, comprises a Sushi domain. This domain is stabilized in part by a salt bridge between Asp 65 and Arg 101 . Analogous residues are conserved in other members of the B1 family. To address the importance of the salt bridge residues within this receptor family, we studied the effects of mutating the residues in full-length CRF-R2␤ and isolated ECD1. Mutation D65A or D65R/R101D resulted in loss of the canonical disulfide arrangement, whereas R101A retained the The B1 family of G-protein-coupled receptors (GPCRs) 4 comprises 15 genes in human, including receptors for corticotropin-releasing factor (CRF), growth hormone-releasing hormone (GHRH), vasoactive intestinal peptide (VIP), parathyroid hormone, pituitary adenylate cyclase-activating polypeptide, incretins (GIP and GLP), secretin, calcitonin, and glucagon and are characterized by relatively large first extracellular domains (ECD1s). In particular, the CRF system includes two distinct receptors, CRF-R1 and CRF-R2, as well as the peptide ligands CRF, urocortins (Ucns) 1, 2, and 3 in mammals, and the related peptides, (fish) urotensin and (frog) sauvagine. In addition, high affinity antagonist peptides have been synthesized, most notably astressin, which binds with equally high affinity to both receptors (1). Many studies have shown that the ECD1s of the CRF receptors constitute major ligand-binding sites. Chimeric receptors expressing the ECD1s bind CRF family ligands with high affinity, as do soluble proteins corresponding to the ECD1s (2-9). Studies of other B1 receptors report similar data (10 -16).A significant advance in understanding the structural determinants of ligand recognition for this receptor subfamily follows upon the determination of the three-dimensional NMR structure of the ECD1 of the type 2␤ CRF receptor, both free (17) and in complex with a peptide antagonist, astressin (18). In both forms, the ECD1 folds into a short consensus repeat (19) or Sushi domain, which contains two antiparallel ␤-sheets, three disulfide bonds, and a salt bridge between aspartic acid 65

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“…Insights into the structure of the predominant ligand binding domain, the amino terminus of the Family B GPCRs, have substantially advanced with the solution of NMR and crystal structures of the isolated ligand-bound amino terminus of the receptors for corticotrophin-releasing factor (11)(12)(13), pituitary adenylate cyclase-activating peptide (14), gastric inhibitory polypeptide (GIP) (15), glucagon-like peptide 1 (GLP1) (16), and parathyroid hormone (PTH) (17). Most of these ligands represented amino-terminally truncated hormones or analogues of these hormones.…”

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confidence: 99%

Molecular Basis of Glucagon-like Peptide 1 Docking to Its Intact Receptor Studied with Carboxyl-terminal Photolabile Probes

Chen

Pinon

Miller

et al. 2009

Journal of Biological Chemistry

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The glucagon-like peptide 1 (GLP1) receptor is a member of Family B G protein-coupled receptors and represents an important drug target for type 2 diabetes. Despite recent solution of the structure of the amino-terminal domain of this receptor and that of several close family members, understanding of the molecular basis of natural ligand GLP1 binding to its intact receptor remains limited. The goal of this study was to explore spatial approximations between specific receptor residues within the carboxyl terminus of GLP1 and its receptor as normally docked. Therefore, we developed and characterized two high affinity, full-agonist photolabile GLP1 probes having sites for covalent attachment in positions 24 and 35. Both probes labeled the receptor specifically and saturably. Subsequent peptide mapping using chemical and proteinase cleavages of purified wild-type and mutant GLP1 receptor identified that the Arg 131 -Lys 136 segment at the juxtamembrane region of the receptor amino terminus contained the site of labeling for the position 24 probe, and the specific receptor residue labeled by this probe was identified as Glu 133 by radiochemical sequencing. Similarly, nearby residue Glu 125 within the same region of the receptor amino-terminal domain was identified as the site of labeling by the position 35 probe. These data represent the first direct demonstration of spatial approximation between GLP1 and its intact receptor as docked, providing two important constraints for the modeling of this interaction. This should expand our understanding of the molecular basis of natural agonist ligand binding to the GLP1 receptor and may be relevant to other family members.G protein-coupled receptors (GPCRs) 2 are the largest group of membrane receptors with seven transmembrane domains and represent targets for over 30% of approved drugs. Understanding of the molecular basis of ligand binding and activation of these receptors will facilitate the development and refinement of drugs acting at these targets. Currently, the molecular basis of ligand binding and activation of Family B GPCRs is less well understood than that of the larger Family A, where high resolution crystal structures have recently been described for several members (1-4).A characteristic structural feature of this family is a long and structurally complex extracellular amino-terminal domain containing six conserved cysteine residues that form disulfide bonds important for stabilizing the folded structure. This domain has been suggested to interact with the carboxyl-terminal region of the natural peptide ligands, based on structure-activity relationship, site-directed mutagenesis, and chimeric receptor studies (5-10), and this is a consistent theme throughout their family.Insights into the structure of the predominant ligand binding domain, the amino terminus of the Family B GPCRs, have substantially advanced with the solution of NMR and crystal structures of the isolated ligand-bound amino terminus of the receptors for corticotrophin-releasing factor (11-13...

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Solution structure and mutational analysis of pituitary adenylate cyclase-activating polypeptide binding to the extracellular domain of PAC1-R _S

Cited by 132 publications

References 37 publications

Molecular basis for negative regulation of the glucagon receptor

Molecular basis for negative regulation of the glucagon receptor

Distinct Structural and Functional Roles of Conserved Residues in the First Extracellular Domain of Receptors for Corticotropin-releasing Factor and Related G-protein-coupled Receptors

Molecular Basis of Glucagon-like Peptide 1 Docking to Its Intact Receptor Studied with Carboxyl-terminal Photolabile Probes

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Solution structure and mutational analysis of pituitary adenylate cyclase-activating polypeptide binding to the extracellular domain of PAC1-R S

Cited by 132 publications

References 37 publications

Molecular basis for negative regulation of the glucagon receptor

Molecular basis for negative regulation of the glucagon receptor

Distinct Structural and Functional Roles of Conserved Residues in the First Extracellular Domain of Receptors for Corticotropin-releasing Factor and Related G-protein-coupled Receptors

Molecular Basis of Glucagon-like Peptide 1 Docking to Its Intact Receptor Studied with Carboxyl-terminal Photolabile Probes

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Solution structure and mutational analysis of pituitary adenylate cyclase-activating polypeptide binding to the extracellular domain of PAC1-R _S