NMR Structure of the First Extracellular Domain of Corticotropin-releasing Factor Receptor 1 (ECD1-CRF-R1) Complexed with a High Affinity Agonist

Grace, Christy R.; Perrin, Marilyn H.; Gulyás, József; Rivier, Jean; Vale, Wylie; Riek, Roland

doi:10.1074/jbc.m110.121897

Cited by 41 publications

(37 citation statements)

References 56 publications

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“…Similar 3D NMR and cocrystal structures of the CRHR 1 -ECD1 in complex with the peptide antagonist α-helical CRH 9-41 or CRH were recently disclosed (Grace et al 2010;Pioszak et al 2008). Interestingly, these studies show that complex formation between a peptide ligand and the CRHR 1 -ECD1 promotes the ligands helical conformation, thereby enhancing the ligands affinity.…”

Section: Peptide Ligands Binding Mechanismmentioning

confidence: 62%

“…Recently, a considerable insight into the ligand binding mechanisms of class B GPCRs has been gained from several reports of ECD-peptide complex structures determined by NMR and X-ray methods (Grace et al 2007;Grace et al 2010;Pioszak et al 2008). The NMR solution structure of astressin (a peptide antagonist analogue based on human CRH, table 2) bound to the mouse CRHR 2 -ECD1 showed that the ECD1 consists of two antiparallel β-sheets, each with two β-strands that are held together by three conserved disulfide bonds.…”

Section: Peptide Ligands Binding Mechanismmentioning

confidence: 99%

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Biomimetic Screening of Class-B G Protein-Coupled Receptors

et al. 2011

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The 41-amino acid peptide corticotropin releasing factor (CRF) is a major modulator of the mammalian stress response. Upon stressful stimuli, it binds to the corticotropin releasing factor receptor 1 (CRF1R), a typical member of the class-B G-protein-coupled receptors (GPCRs) and a prime target in the treatment of mood disorders. To chemically probe the molecular interaction of CRF with the transmembrane domain of its cognate receptor, we developed a high-throughput conjugation approach that mimics the natural activation mechanism of class-B GPCRs. An acetylene-tagged peptide library was synthesized and conjugated to an azide-modified high-affinity carrier peptide derived from the CRF C-terminus using copper-catalyzed dipolar cycloaddition. The resulting conjugates reconstituted potent agonists and were tested in situ for activation of the CRF1 receptor in a cell-based assay. By use of this approach we (i) defined the minimal sequence motif that is required for full receptor activation, (ii) identified the critical functional groups and structure–activity relationships, (iii) developed an optimized, highly modified peptide probe with high potency (EC50 = 4 nM) that is specific for the activation domain of the receptor, and (iv) probed the behavioral role of CRF receptors in living mice. The membrane recruitment by a high-affinity carrier enhanced the potency of the tethered peptides by >4 orders of magnitude and thus allowed the testing of very weak initial fragments that otherwise would have been inactive on their own. As no chromatography purification of the test peptides was necessary, a substantial increase in screening throughput was achieved. Importantly, the peptide conjugates can be used to probe the endogenous receptor in its native environment in vivo.

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Section: Peptide Ligands Binding Mechanismmentioning

confidence: 62%

Section: Peptide Ligands Binding Mechanismmentioning

confidence: 99%

Biomimetic Screening of Class-B G Protein-Coupled Receptors

et al. 2011

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“…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%

“…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 the iso-lated TMDs of two class B receptors in an inactive conformation have been published, i.e.…”

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confidence: 99%

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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

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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...

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