| Autor: |
Eric R. Prossnitz, Sean M. Biggs, Wei-Jen Tang, Anna Waller, Larry A. Sklar, Richard R. Neubig, Qing Guo, Daniel F. Cimino, Peter C. Simons, Terry D. Foutz |
| Rok vydání: |
2004 |
| Předmět: |
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| Zdroj: |
Journal of Biological Chemistry. 279:13514-13521 |
| ISSN: |
0021-9258 |
| DOI: |
10.1074/jbc.m310306200 |
| Popis: |
We developed a novel and generalized approach to investigate G protein-coupled receptor molecular assemblies. We solubilized a fusion protein consisting of the β2-adrenergic receptor and green fluorescent protein (GFP) for bead-based flow cytometric analysis. β2-Adrenergic receptor GFP bound to dihydroalprenolol-conjugated beads, providing a Kd for the fusion protein and, in competition with β2-adrenergic receptor ligands, Kd values for agonists and antagonists. Beads displaying chelated nickel bound purified hexahistidine-tagged G protein heterotrimers and, subsequently, the binary complex of agonist with β2-adrenergic receptor GFP. The dose-response curves of ternary complex formation revealed maximal assembly for ligands previously classified as full agonists and reduced assembly for ligands previously classified as partial agonists. Guanosine 5′-3-O-(thio)triphosphate-induced dissociation rates of the ternary complex were the same for full and partial agonists. Soluble G protein, competing with ternary complexes on beads provided an affinity estimate of agonist-receptor complexes to G protein. When performed simultaneously, the two assemblies discriminated between agonist, antagonist or inactive molecule in a manner appropriate for high throughput, small volume drug discovery. The assemblies can be further generalized to other G protein coupled receptor protein-protein interactions. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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