Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Coupled Receptors

Kumar, Sanjiv; Pioszak, Augen A.; Zhang, Chenghai; Swaminathan, Kunchithapadam; Xu, H. Eric

doi:10.1371/journal.pone.0019682

Cited by 59 publications

(61 citation statements)

References 44 publications

(66 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The encoded fragments were digested with BamHI and NotI restriction endonucleases and inserted into a modified pcDNA6 expression vector to encode a fusion protein consisting of an N-terminal human IgG leader (MGWSCIILFL-VATATGVHSE) for targeting into the cell membrane and a FLAG tag (DYKDDDD) at the C terminus for detection by immunoblotting. In addition to the full-length receptors, we generated the same set of constructs with (i) the TMDs of GCGR (residues 123-431), GLP-1R (residues 140 -463), PTH1R (residues 182-484), CRF 1 R (residues 110 -384), and PAC1R (residues 148 -421), (ii) fusions between the N-terminal peptide hormones (glucagon (1-15), GLP-1(7-21), PTH(1-15), UNC (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), PACAP (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)) and their corresponding TMDs, (iii) fusions of the same N-terminal peptide fragments as above to the BRIL-TMD constructs, and (iv) fusions of the full-length peptide hormones to the N terminus of their corresponding full-length receptors with five copies of Gly-Ser-Ala (GSA 5 ) linker. All these constructs contain the same IgG leader as above.…”

Section: Methodsmentioning

confidence: 99%

“…Signaling-To examine the requirement of the GCGR ECD for signaling, we transiently transfected four different FLAGtagged constructs into HEK293 cells: the full-length receptor, TMD, TMD-interacting N terminus of glucagon (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) fused to the TMD, and a fusion in which glucagon (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) and the TMD are separated by a thermostabilized cytochrome b562 variant, BRIL, which has been used for facilitating GPCR crystallization (16) (Fig. 1, B-D).…”

Section: Class B Gpcrs Differ In Their Ecd Requirements Formentioning

confidence: 99%

See 1 more Smart Citation

Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-coupled Receptors

Zhao

Yin

Yang

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

G protein-coupled receptors (GPCRs) from the secretin-like (class B) family are key players in hormonal homeostasis and are important drug targets for the treatment of metabolic disorders and neuronal diseases. They consist of a large N-terminal extracellular domain (ECD) and a transmembrane domain (TMD) with the GPCR signature of seven transmembrane helices. Class B GPCRs are activated by peptide hormones with their C termini bound to the receptor ECD and their N termini bound to the TMD. It is thought that the ECD functions as an affinity trap to bind and localize the hormone to the receptor. This in turn would allow the hormone N terminus to insert into the TMD and induce conformational changes of the TMD to activate downstream signaling. In contrast to this prevailing model, we demonstrate that human class B GPCRs vary widely in their requirement of the ECD for activation. In one group, represented by corticotrophin-releasing factor receptor 1 (CRF 1 R), parathyroid hormone receptor (PTH1R), and pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R), the ECD requirement for high affinity hormone binding can be bypassed by induced proximity and mass action effects, whereas in the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the ECD is required for signaling even when the hormone is covalently linked to the TMD. Furthermore, the activation of GLP-1R by small molecules that interact with the intracellular side of the receptor is dependent on the presence of its ECD, suggesting a direct role of the ECD in GLP-1R activation.The class B or secretin family of GPCRs consists of 15 receptors for peptide hormones that include glucagon, glucagon-like peptides, parathyroid hormone, and calcitonin (Fig. 1A). These receptors are important drug targets for many human diseases including diabetes, neurodegeneration, cardiovascular diseases, and psychiatric disorders. The full-length receptors consist of two modular domains: a globular extracellular domain (ECD) 3 defined by three conserved disulfide bonds and a TMD that contains seven transmembrane helices (1-4). The ECD is responsible for the high affinity and specificity of hormone binding, and the TMD is required for receptor activation and signal coupling to downstream G proteins and other signaling effectors (4). Peptide hormone binding is thought to proceed through fast binding of its C terminus to the ECD followed by a slower association of the peptide N terminus with the receptor TMD (5), which leads to conformational changes in the receptor TMD and the receptor activation. The activated receptors are coupled primarily to stimulatory G proteins, resulting in elevation of the intracellular cAMP level.Structures of the ECDs of class B GPCRs in complex with their peptide ligands have been determined by x-ray crystallography (1, 6 -9) and NMR (10) and have provided useful information about structural mechanisms of ligand recognition and selectivity (2, 11). Only recently, crystal structures of t...

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Class B Gpcrs Differ In Their Ecd Requirements Formentioning

confidence: 99%

Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-coupled Receptors

Zhao

Yin

Yang

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…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: 97%

“…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%

Molecular basis for negative regulation of the glucagon receptor

Koth¹,

Murray²,

Mukund³

et al. 2012

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

123

179

View full text Add to dashboard Cite

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...

show abstract

“…The glucagon receptor (GCGR) 3 is one of the 15 members of the secretin-like family of class B G protein-coupled receptors (GPCRs) (1). Upon activation by binding to the 29-amino-acid hormonal peptide glucagon (GCG), GCGR stimulates both glycogenolysis and gluconeogenesis to maintain normal blood glucose levels (2).…”

mentioning

confidence: 99%

Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors

Yin

Waal

et al. 2017

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical ␤ 2 -adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation.

show abstract

Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Coupled Receptors

Cited by 59 publications

References 44 publications

Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-coupled Receptors

Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-coupled Receptors

Molecular basis for negative regulation of the glucagon receptor

Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors

Contact Info

Product

Resources

About