Abstract
The group III metabotropic glutamate receptors (mGluRs) represent a family of presynaptically expressed G-protein-coupled receptors (GPCRs) with enormous therapeutic potential; however, robust cellular assays to study their function have been difficult to develop. We present here a new assay, compatible with traditional high-throughput screening platforms, to detect activity of pharmacological ligands interacting with Gi/o-coupled GPCRs, including the group III mGluRs 4, 7, and 8. The assay takes advantage of the ability of the Gβγ subunits of Gi and Go heterotrimers to interact with G-protein regulated inwardly rectifying potassium channels (GIRKs), and we show here that we are able to detect the activity of multiple types of pharmacophores including agonists, antagonists, and allosteric modulators of several distinct GPCRs. Using GIRK-mediated thallium flux, we perform a side-by-side comparison of the activity of a number of commercially available compounds, some of which have not been extensively evaluated because of the previous lack of robust assays at each of the three major group III mGluRs. It is noteworthy that several compounds previously considered to be general group III mGluR antagonists have very weak activity using this assay, suggesting the possibility that these compounds may not effectively inhibit these receptors in native systems. We anticipate that the GIRK-mediated thallium flux strategy will provide a novel tool to advance the study of Gi/o-coupled GPCR biology and promote ligand discovery and characterization.
Footnotes
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This work was supported by National Institutes of Health grants NS053536 and NS051342 and the Michael J. Fox Foundation. Vanderbilt University Medical Center is the Molecular Libraries Screening Center Network (MLSCN) site for GPCRs, Ion Channels and Transporters.
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ABBREVIATIONS: mGluR, metabotropic glutamate receptor; CNS, central nervous system; GPCR, G-protein-coupled receptor; GIRK, G-protein regulated inwardly rectifying K+ channel; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; HEK, human embryonic kidney; DMSO, dimethyl sulfoxide; UK 14,304, 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline tartrate; AF-DX 116, 11-[[2-[(diethylamino)methyl]-1-piperidinyl]-acetyl]-5,11-dihydro-6H-pyrid[2,3-b][1,4]benzodiazepin-6-one; BRL 44408 maleate, 2-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole maleate; PHCCC, N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide; MPPG, (R,S)-α-methyl-4-phosphonophenylglycine; MSPG, (R,S)-α-methyl-4-sulfonophenylglycine; MSOP, (R,S)-α-methylserine-O-phosphate; CPPG, (R,S)-α-cyclopropyl-4-phosphonophenylglycine; UBP1112, α-methyl-3-methyl-4-phosphonophenylglycine; (S)-MAP4, (S)-2-amino-2-methyl-4-phosphonobutanoic acid; LY341495, (2S)-2-amino-2-[(1S, 2S)-2-carboxyclycloprop-1-yl]-3-(xanth-9-yl) propanoic acid; BTC-AM, benzothiazole coumarin-acetoxymethyl ester; PTX, pertussis toxin; PAM, positive allosteric modulator; HTS, high-throughput screening; l-AP4, l(+)-2-amino-4-phosphonobutyric acid.
- Received August 20, 2007.
- Accepted December 31, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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