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Inooka, Hiroshi; Ohtaki, Tetsuya; Kitahara, Osamu; Ikegami, Takahisa; Endo, Satoshi; Kitada, Chieko; Ogi, Kazuhiro; Onda, Haruo; Fujino, Masahiko; Shirakawa, Masahiro

doi:10.1038/84159

Cited by 164 publications

(102 citation statements)

References 26 publications

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“…Typically the N-terminal segment of the ligand is responsible for receptor signaling, whereas the C-terminal segment is an important determinant for the receptor binding (45,46) and may interact with the ECD1 of the receptor as evidenced by cross-linking (47). Furthermore, the bioactive conformations of various peptide hormones are proposed to be of amphipathic helical nature (48), similar to the conformation observed for astressin. The 3D structure of ECD1 of CRF-R2␤ in complex with astressin elucidates the major determinants and the physical basis for the ligand binding affinity and specificity for CRF receptors, which are crucial components toward an understanding of diverse biological functions of the CRF ligand/receptor system.…”

Section: Crf Ligand-receptor Interactions Have a Common Binding Modementioning

confidence: 94%

Structure of the N-terminal domain of a type B1 G protein-coupled receptor in complex with a peptide ligand

Grace¹,

Perrin

Gulyás

et al. 2007

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

126

175

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The corticotropin releasing factor (CRF) family of ligands and their receptors coordinate endocrine, behavioral, autonomic, and metabolic responses to stress and play additional roles within the cardiovascular, gastrointestinal, and other systems. The actions of CRF and the related urocortins are mediated by activation of two receptors, CRF-R1 and CRF-R2, belonging to the B1 family of G protein-coupled receptors. The short-consensus-repeat fold (SCR) within the first extracellular domain (ECD1) of the CRF receptor(s) comprises the major ligand binding site and serves to dock a peptide ligand via its C-terminal segment, thus positioning the N-terminal segment to interact with the receptor's juxtamembrane domains to activate the receptor. Here we present the 3D NMR structure of ECD1 of CRF-R2␤ in complex with astressin, a peptide antagonist. In the structure of the complex the C-terminal segment of astressin forms an amphipathic helix, whose entire hydrophobic face interacts with the short-consensus-repeat motif, covering a large intermolecular interface. In addition, the complex is characterized by intermolecular hydrogen bonds and a salt bridge. These interactions are quantitatively weighted by an analysis of the effects on the full-length receptor affinities using an Ala scan of CRF. These structural studies identify the major determinants for CRF ligand specificity and selectivity and support a two-step model for receptor activation. Furthermore, because of a proposed conservation of the fold for both the ECD1s and ligands, this structure can serve as a model for ligand recognition for the entire B1 receptor family.3D structure ͉ astressin ͉ corticotropin releasing factor ͉ NMR T he ability of the body to adapt to stressful stimuli and the role of stress maladaptation in human diseases has been intensively investigated. Corticotropin releasing factor (CRF) (1), a 41-residue peptide, and its three paralogous peptides, urocortin (Ucn) 1, 2, and 3, play important and diverse roles in coordinating endocrine, autonomic, metabolic, and behavioral responses to stress (2, 3). CRF family peptides and their receptors are also implicated in the modulation of additional central nervous system functions including appetite, addiction, hearing, and neurogenesis and act peripherally within the endocrine, cardiovascular, reproductive, gastrointestinal, and immune systems (4, 5). CRF and related ligands initially act by binding to their G protein-coupled receptors (GPCRs). These belong to the peptide hormone B1 family (family B1 GPCRs), comprising receptors for growth hormone releasing factor, secretin, calcitonin, vasoactive intestinal peptide, glucagon, glucagon-like peptide-1, and parathyroid hormone. Two CRF receptors, CRF-R1 and CRF-R2, have been cloned in mammals (6, 7).Structure activity studies of CRF showed that the first eight N-terminal residues of the hormone are necessary for GPCR signaling (1, 8), whereas the C-terminal (Ϸ15) residues are important for binding (9,10). A two-domain behavior for ligand binding was a...

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Section: Crf Ligand-receptor Interactions Have a Common Binding Modementioning

confidence: 94%

Structure of the N-terminal domain of a type B1 G protein-coupled receptor in complex with a peptide ligand

Grace¹,

Perrin

Gulyás

et al. 2007

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

126

175

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“…In contrast, mutational mapping of ligand-binding sites in peptide receptors indicates that extracellular domains are also involved in ligand recognition (2). To date, direct structural information regarding ligand binding and GPCR activation is very limited (6). Targeted drug design is restricted to computational methods and other ligand design approaches to find and optimize lead compounds (7).…”

mentioning

confidence: 99%

“…The high affinity of the ligand to its receptor has precluded the elucidation of the receptor-bound ligand conformation by solution-state NMR (see, for example, ref. 6). A recent NMR study in the presence of detergent reported NT(8-13) chemical shift changes upon receptor interaction (18,19).…”

mentioning

confidence: 99%

The conformation of neurotensin bound to its G protein-coupled receptor

Luca

White

Sohal

et al. 2003

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

224

210

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G protein-coupled receptors (GPCRs) mediate the perception of smell, light, taste, and pain. They are involved in signal recognition and cell communication and are some of the most important targets for drug development. Because currently no direct structural information on high-affinity ligands bound to GPCRs is available, rational drug design is limited to computational prediction combined with mutagenesis experiments. Here, we present the conformation of a high-affinity peptide agonist (neurotensin, NT) bound to its GPCR NTS-1, determined by direct structural methods. Functional receptors were expressed in Escherichia coli, purified in milligram amounts by using optimized procedures, and subsequently reconstituted into lipid vesicles. Solid-state NMR experiments were tailored to allow for the unequivocal detection of microgram quantities of 13 C, 15 N-labeled NT(8 -13) in complex with functional NTS-1. The NMR data are consistent with a disordered state of the ligand in the absence of receptor. Upon receptor binding, the peptide undergoes a linear rearrangement, adopting a ␤-strand conformation. Our results provide a viable structural template for further pharmacological investigations.G protein-coupled receptors (GPCRs) are integral membrane proteins involved in a number of important physiological processes, including sensory transduction, mediation of hormonal activity, and cell-to-cell communication (for reviews, see refs. 1 and 2). More than 1,000 different GPCRs have been identified, and many of them have been implicated as major therapeutic routes to the treatment of human diseases (3). Despite the striking clinical relevance of GPCRs, only one high-resolution structure (rhodopsin) is available (4, 5). The diversity among endogenous GPCR ligands is exceptional. Small molecule ligands such as biogenic amines have been proposed to bind within the hydrophobic core of their respective receptors. In contrast, mutational mapping of ligand-binding sites in peptide receptors indicates that extracellular domains are also involved in ligand recognition (2). To date, direct structural information regarding ligand binding and GPCR activation is very limited (6). Targeted drug design is restricted to computational methods and other ligand design approaches to find and optimize lead compounds (7). The precise knowledge of receptor-bound ligand structures would substantially aid the development of tailor-made medicines.Neurotensin (NT) is a 13-aa peptide (8) that is involved in a variety of neuromodulatory functions in the central and peripheral nervous system (9). NT binds to its GPCRs, NT type I receptor (NTS-1) and NTS-2 (10 -13). The levocabastineinsensitive NTS-1 (10-12) interacts with the agonist NT with high (i.e., subnanomolar) affinity. Similar observations were made for the N-terminally truncated form of rat NTS-1 when expressed as a maltose-binding protein (MBP) fusion in Escherichia coli (14, 15) and purified in the presence of detergents (14, 16). Notably, not only the full-length peptide, but also the C-...

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“…Therefore, it has been suggested that the conformation, location, and orientation of the ligand in the membrane are critical in determining its ability to reach and interact productively with its site of action (3)(4)(5). Exploring the conformational and dynamic properties of these ligands in the membrane can lead to a better understanding of the molecular features involved in their interactions with the target proteins (6).…”

mentioning

confidence: 99%

The Conformation, Location, and Dynamic Properties of the Endocannabinoid Ligand Anandamide in a Membrane Bilayer

Tian

Guo

Yao

et al. 2005

Journal of Biological Chemistry

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The endogenous cannabinoid ligand anandamide is biosynthesized from membrane phospholipid precursors and is believed to reach its sites of action on the CB1 and CB2 receptors through fast lateral diffusion within the cell membrane. To gain a better insight on the stereochemical features of its association with the cell membrane and its interaction with the cannabinoid receptors, we have studied its conformation, location, and dynamic properties in a dipalmitoylphosphatidylcholine multilamellar model membrane bilayer system. By exploiting the bilayer lattice as an internal threedimensional reference grid, the conformation and location of anandamide were determined by measuring selected inter-and intramolecular distances between strategically introduced isotopic labels using the rotational echo double resonance (REDOR) NMR method. A molecular model was proposed to represent the structural features of our anandamide/lipid system and was subsequently used in calculating the multispin dephasing curves. Our results demonstrate that anandamide adopts an extended conformation within the membrane with its headgroup at the level of the phospholipid polar group and its terminal methyl group near the bilayer center. Parallel static 2 H NMR experiments further confirmed these findings and provided evidence that anandamide experiences dynamic properties similar to those of the membrane phospholipids and produces no perturbation to the bilayer. Our results are congruent with a hypothesis that anandamide approaches its binding site by laterally diffusing within one membrane leaflet in an extended conformation and interacts with a hydrophobic groove formed by helices 3 and 6 of CB1, where its terminal carbon is positioned close to a key cysteine residue in helix 6 leading to receptor activation.The membrane lipid bilayer is a ubiquitous molecular assembly into which are embedded a variety of proteins, natural hormones, and neurotransmitters. Accumulated evidence indicates that many fatty acid-derived lipophilic neurotransmitters are synthesized, stored, and degraded and also exert their functions within the lipid membrane (1, 2). Therefore, it has been suggested that the conformation, location, and orientation of the ligand in the membrane are critical in determining its ability to reach and interact productively with its site of action (3-5). Exploring the conformational and dynamic properties of these ligands in the membrane can lead to a better understanding of the molecular features involved in their interactions with the target proteins (6).N-Arachidonoylethanolamine (anandamide), initially isolated from mammalian brain, has been identified as an endogenous ligand for the two known G protein-coupled cannabinoid receptors (CB1 and CB2) (7). This endocannabinoid exerts its activity by modulating several physiological functions such as pain, cognition, and memory. Site-directed mutagenesis evidence has shown that the anandamide binding sites are embedded in the trans-membrane helices of the receptor (8, 9). This correlates wel...

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Cited by 164 publications

References 26 publications

Structure of the N-terminal domain of a type B1 G protein-coupled receptor in complex with a peptide ligand

Structure of the N-terminal domain of a type B1 G protein-coupled receptor in complex with a peptide ligand

The conformation of neurotensin bound to its G protein-coupled receptor

The Conformation, Location, and Dynamic Properties of the Endocannabinoid Ligand Anandamide in a Membrane Bilayer

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