Orphan G‐protein‐coupled receptors: the next generation of drug targets?

Wilson, Shelagh; Bergsma, Derk J.; Chambers, Jon; Muir, Alison I.; Fantom, Kenneth; Ellis, Catherine; Murdock, Paul R.; Herrity, Nicole C.; Stadel, Jeffrey M.

doi:10.1038/sj.bjp.0702238

Cited by 110 publications

(67 citation statements)

References 28 publications

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“…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).…”

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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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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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“…SP1999 is an orphan G protein-coupled receptor cloned from a human hypothalamus cDNA library (7). Phylogenetic analysis shows that SP1999 shares homology with a group of G protein-coupled receptors, most of which are orphans as well.…”

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confidence: 99%

ADP Is the Cognate Ligand for the Orphan G Protein-coupled Receptor SP1999

Zhang¹,

Luo²,

Gustafson³

et al. 2001

Journal of Biological Chemistry

208

152

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P2Y receptors are a class of G protein-coupled receptors activated primarily by ATP, UTP, and UDP. Five mammalian P2Y receptors have been cloned so far including P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11. P2Y1, P2Y2, and P2Y6 couple to the activation of phospholipase C, whereas P2Y4 and P2Y11 couple to the activation of both phospholipase C and the adenylyl cyclase pathways. Additional ADP receptors linked to G␣ i have been described but have not yet been cloned. SP1999 is an orphan G protein-coupled receptor, which is highly expressed in brain, spinal cord, and blood platelets. In the present study, we demonstrate that SP1999 is a G␣ icoupled receptor that is potently activated by ADP. In an effort to identify ligands for SP1999, fractionated rat spinal cord extracts were assayed for Ca 2؉ mobilization activity against Chinese hamster ovary cells transiently transfected with SP1999 and chimeric G␣ subunits (G␣ q/i ). A substance that selectively activated SP1999-transfected cells was identified and purified through a series of chromatographic steps. Mass spectral analysis of the purified material definitively identified it as ADP. ADP was subsequently shown to inhibit forskolin-stimulated adenylyl cyclase activity through selective activation of SP1999 with an EC 50 of 60 nM. Other nucleotides were able to activate SP1999 with a rank order of potency 2-MeS-ATP ‫؍‬ 2-MeS-ADP > ADP ‫؍‬ adenosine 5-O-2-(thio-)diphosphate > 2-Cl-ATP > adenosine 5-O-(thiotriphosphate). Thus, SP1999 is a novel, G␣ i -linked receptor for ADP.Purine and pyrimidine nucleotides are known to modulate a variety of physiological functions by interaction with two types of cell surface receptors: P2X and P2Y receptors (1, 2). P2X receptors are ligand-gated ion-channels, whereas P2Y receptors are G protein-coupled receptors (GPCRs).

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“…Agonist binding to these receptors activates intracellular signaling events, mediated by G-proteins, such as modulation of adenylate cyclase or Ca 2ϩ mobilization (1,2). Completion of the human genome sequencing project has identified ϳ140 "orphan" GPCRs for which the ligand and function are unknown (3)(4)(5). We have used bioinformatic and tissue distribution analysis to prioritize those orphans with potential therapeutic relevance followed by a "reverse pharmacology" approach to identify cognate and surrogate ligands (6,7).…”

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confidence: 99%

MrgX2 Is a High Potency Cortistatin Receptor Expressed in Dorsal Root Ganglion

Robas

Mead

Fidock

2003

Journal of Biological Chemistry

220

187

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MrgX2 is a recently identified orphan G-protein-coupled receptor whose ligand and physiological function were unknown. Here we describe cortistatin, a neuropeptide for which no specific receptor has been identified previously, as a high potency ligand at MrgX2. Cortistatin has several biological functions including roles in sleep regulation, locomotor activity, and cortical function. Using a "reverse pharmacology" approach, we have identified a number of additional cyclic peptide agonists for MrgX2, determined their rank order of potency, and demonstrated that this receptor has a pharmacological profile distinct from the other characterized members of the Mrg (Mas-related genes) family. In MrgX2-expressing cells, cortistatin-stimulated increases in intracellular Ca 2؉ but had no effect on basal or forskolin-stimulated cAMP levels, suggesting that this receptor is G q -coupled. Immunohistochemical and quantitative PCR studies show MrgX2 to have a limited expression profile, both peripheral and within the central nervous system, with highest levels in dorsal root ganglion.

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Orphan G‐protein‐coupled receptors: the next generation of drug targets?

Cited by 110 publications

References 28 publications

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

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

ADP Is the Cognate Ligand for the Orphan G Protein-coupled Receptor SP1999

MrgX2 Is a High Potency Cortistatin Receptor Expressed in Dorsal Root Ganglion

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