Cell-based high-throughput screening assay system for monitoring G protein-coupled receptor activation using beta-galactosidase enzyme complementation technology
Autor: | Bonnie Tillotson, Xiao-Jia Chang, Brian J. D'eon, Corinne E. M. Olesen, Deborah M. Boldt-Houle, Michelle A. J. Palmer, Yu-Xin Yan, Melissa Gee |
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Rok vydání: | 2003 |
Předmět: |
0301 basic medicine
Arrestins High-throughput screening Drug Evaluation Preclinical Biology 01 natural sciences Biochemistry Receptors Interleukin-8B Analytical Chemistry Receptors G-Protein-Coupled 03 medical and health sciences Protein Interaction Mapping Cyclic AMP Combinatorial Chemistry Techniques Humans 5-HT5A receptor Receptor Cells Cultured G protein-coupled receptor Dose-Response Relationship Drug Genes erbB-1 Ligand (biochemistry) Phosphoproteins beta-Galactosidase Fusion protein Recombinant Proteins 0104 chemical sciences Complementation 010404 medicinal & biomolecular chemistry 030104 developmental biology Molecular Medicine Biological Assay Receptors Adrenergic beta-2 Signal transduction Biotechnology |
Zdroj: | Journal of biomolecular screening. 7(5) |
ISSN: | 1087-0571 |
Popis: | A novel cell-based functional assay to directly monitor G protein-coupled receptor (GPCR) activation in a high-throughput format, based on a common GPCR regulation mechanism, the interaction between beta-arrestin and ligand-activated GPCR, is described. A protein-protein interaction technology, the InteraX trade mark system, uses a pair of inactive beta-galactosidase (beta-gal) deletion mutants as fusion partners to the protein targets of interest. To monitor GPCR activation, stable cell lines expressing both GPCR- and beta-arrestin-beta-gal fusion proteins are generated. Following ligand stimulation, beta-arrestin binds to the activated GPCR, and this interaction drives functional complementation of the beta-gal mutant fragments. GPCR activation is measured directly by quantitating restored beta-gal activity. The authors have validated this assay system with two functionally divergent GPCRs: the beta2-adrenergic amine receptor and the CXCR2 chemokine-binding receptor. Both receptors are activated or blocked with known agonists and antagonists in a dose-dependent manner. The beta2-adrenergic receptor cell line was screened with the LOPAC trade mark compound library to identify both agonists and antagonists, validating this system for high-throughput screening performance in a 96-well microplate format. Hit specificity was confirmed by quantitating the level of cAMP. This assay system has also been performed in a high-density (384-well) microplate format. This system provides a specific, sensitive, and robust methodology for studying and screening GPCR-mediated signaling pathways. |
Databáze: | OpenAIRE |
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