A Bioluminescent Assay for Agonist Activity at Potentially Any G-Protein-Coupled Receptor

Stables, J.M.; Green, Andrew; Marshall, Fiona H.; Fraser, Neil; Knight, Emma; Sautel, Martine; Milligan, Graeme; Lee, Melanie; Rees, Stephen Edward

doi:10.1006/abio.1997.2308

Cited by 198 publications

(156 citation statements)

References 28 publications

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“…The junction is formed by three localized interactions between AQP0 molecules in adjoining membranes, mainly mediated by proline residues conserved in AQP0s from different species but not present in most other aquaporins. Whereas all previously determined aquaporin structures show the pore in an open conformation [4][5][6][7][8][9] , the water pore is closed in AQP0 junctions. The water pathway in AQP0 also contains an additional pore constriction, not seen in other known aquaporin structures [4][5][6][7][8][9] , which may be responsible for pore gating.…”

Section: In Vivo Experimentsmentioning

confidence: 99%

“…Whereas all previously determined aquaporin structures show the pore in an open conformation [4][5][6][7][8][9] , the water pore is closed in AQP0 junctions. The water pathway in AQP0 also contains an additional pore constriction, not seen in other known aquaporin structures [4][5][6][7][8][9] , which may be responsible for pore gating. AQP0 is a member of the aquaporin family, members of which form pores that are either highly selective for water (aquaporins) or also permeable to other small neutral solutes such as glycerol (aquaglyceroporins) (reviewed in ref.…”

Section: In Vivo Experimentsmentioning

confidence: 99%

“…10). To date, the atomic structures of three aquaporins have been determined (AQP1 [4][5][6] , GlpF 7,8 and AQPZ 9 ). Sequence alignment shows AQP0 to be closely related to the pure water channel AQP1 (43.6% identity, 62.6% similarity).…”

Section: In Vivo Experimentsmentioning

confidence: 99%

“…In a search for natural ligands for orphan GPCRs, we tested extracts from various animal tissues for their ability to evoke an increase in intracellular Ca 2þ concentration ([Ca 2þ ] i ) using the aequorin assay 7 . We found that fractions from pig kidney extracts specifically activated cells expressing GPR91 (Fig.…”

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confidence: 99%

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Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors

Miao²,

Lin³

et al. 2004

Nature

774

832

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The citric acid cycle is central to the regulation of energy homeostasis and cell metabolism 1 . Mutations in enzymes that catalyse steps in the citric acid cycle result in human diseases with various clinical presentations 2 . The intermediates of the citric acid cycle are present at micromolar concentration in blood and are regulated by respiration, metabolism and renal reabsorption/ extrusion. Here we show that GPR91 (ref.3), a previously orphan G-protein-coupled receptor (GPCR), functions as a receptor for the citric acid cycle intermediate succinate. We also report that GPR99 (ref. 4), a close relative of GPR91, responds to a-ketoglutarate, another intermediate in the citric acid cycle. Thus by acting as ligands for GPCRs, succinate and a-ketoglutarate are found to have unexpected signalling functions beyond their traditional roles. Furthermore, we show that succinate increases blood pressure in animals. The succinate-induced hypertensive effect involves the renin-angiotensin system and is abolished in GPR91-deficient mice. Our results indicate a possible role for GPR91 in renovascular hypertension, a disease closely linked to atherosclerosis, diabetes and renal failure 5,6 .In a search for natural ligands for orphan GPCRs, we tested extracts from various animal tissues for their ability to evoke an increase in intracellular Ca 2þ concentration ([Ca 2þ ] i ) using the aequorin assay 7 . We found that fractions from pig kidney extracts specifically activated cells expressing GPR91 (Fig. 1a). GPR91 is an orphan GPCR highly expressed in the kidney and shares 33% amino acid identity with GPR99/GPR80 (refs 4, 8). On the basis of their homology with the purinergic receptor P2Y1, nucleotide ligands were predicted for GPR91 and GPR99 (ref. 4). However, the GPR91 ligand activity in pig kidney extracts was resistant to various stringent treatments including alkaline phosphatase, peptidase, and hydrolysis in 6 M HCl at 100 8C. Accordingly, the supposition that GPR91 might be activated by a nucleotide or peptide ligand was unlikely. We purified the natural ligand for GPR91 by ion-exchange, size-exclusion and reversed-phase fast performance liquid chromatography/high-performance liquid chromatography (Fig. 1a).A major molecular ion [M þ H] þ at m/z 119.2 was observed by mass spectrometry (Fig. 1b). 1 H NMR analysis revealed a single type of proton in the highly purified GPR91 ligand (Fig. 1c). 13 C NMR analysis further suggested the presence of -CH 2 -(methylene) and ¼C¼O (carbonyl) groups (Fig. 1d). Combined with mass spectrometry results and the biochemical properties of the ligand, the purified GPR91 ligand was predicted and confirmed to be succinic acid (Fig. 1c, d).Commercially obtained succinate (the physiological form of succinic acid) increased [Ca 2þ ] i dose-dependently in the aequorin assay (Fig. 2a). Succinate also activated mouse and rat orthologues of GPR91 (Fig. 2a). The succinate-induced increase in [Ca 2þ ] i was further confirmed with a fluorimetric imaging plate reader (FLIPR) system in the 293-hGP...

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Section: In Vivo Experimentsmentioning

confidence: 99%

Section: In Vivo Experimentsmentioning

confidence: 99%

Section: In Vivo Experimentsmentioning

confidence: 99%

mentioning

confidence: 99%

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Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors

Miao²,

Lin³

et al. 2004

Nature

774

832

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“…Intracellular Ca 2ϩ release was measured as described (27) by a functional assay based on the luminescence of mitochondrial aequorin (29).…”

Section: Aequorin-based Functional Assaymentioning

confidence: 99%

Urokinase Plasminogen Activator and Plasmin Efficiently Convert Hemofiltrate CC Chemokine 1 into Its Active [9–74] Processed Variant

Vakili

Ständker

Detheux

et al. 2001

The Journal of Immunology

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We have previously isolated from human hemofiltrate an N-terminally truncated form of the hemofiltrate CC chemokine 1 (HCC-1), and characterized HCC-1[9–74] as a strong agonist of CCR1, CCR5, and to a lower extent CCR3. In this study, we show that conditioned media from human tumor cell lines PC-3 and 143B contain proteolytic activities that convert HCC-1 into the [9–74] form. This activity was fully inhibited by inhibitors of urokinase-type plasminogen activator (uPA), including PA inhibitor-1, an anti-uPA mAb, and amiloride. Pure preparations of uPA processed HCC-1 with high efficiency, without further degrading HCC-1[9–74]. Plasmin could also generate HCC-1[9–74], but degraded the active product as well. The kinetics of HCC-1 cleavage by uPA and plasmin (Michaelis constant, Km, of 0.76 ± 0.4 μM for uPA, and 0.096 ± 0.05 μM for plasmin; catalytic rate constant, kcat: 3.36 ± 0.96 s−1 for uPA and 6 ± 3.6 s−1 for plasmin) are fully compatible with a role in vivo. The activation of an abundant inactive precursor into a broad-spectrum chemokine by uPA and plasmin directly links the production of uPA by numerous tumors and their ability to recruit mononuclear leukocytes, without the need for the transcriptional activation of chemokine genes.

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Triple‐Addition Assay Protocols for Detecting and Characterizing Modulators of Seven‐Transmembrane Receptors

Weaver

2011

CP Chemical Biology

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The detection and characterization of seven‐transmembrane‐receptor modulators (orthosteric binding site agonists, antagonists, and more recently allosteric modulators) is an area of intense interest for both drug discovery and basic research. Traditionally, assays used to detect and characterize these different modes of modulation have been executed as separate, discrete protocols focused on a particular mode of action (e.g., agonism). In recent years, investigators have begun to combine aspects of these separate protocols to produce methods that detect multiple modes of modulation simultaneously. The power of such approaches is revealed not only in conservation of time and resources, but more importantly in a superior ability to discover and characterize novel modulators of the targets of interest. The protocols in this article describe a general procedure for developing, validating, and utilizing triple‐addition assays to enable the simultaneous detection and characterization of multiple modes of seven‐transmembrane‐receptor modulation. Curr. Protoc. Chem. Biol. 3:119‐140 © 2011 by John Wiley & Sons, Inc.

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A Bioluminescent Assay for Agonist Activity at Potentially Any G-Protein-Coupled Receptor

Cited by 198 publications

References 28 publications

Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors

Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors

Urokinase Plasminogen Activator and Plasmin Efficiently Convert Hemofiltrate CC Chemokine 1 into Its Active [9–74] Processed Variant

Triple‐Addition Assay Protocols for Detecting and Characterizing Modulators of Seven‐Transmembrane Receptors

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