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• W2040102227 startingPage "5394" @default.
• W2040102227 abstract "Two strategies are described for detecting constitutive or induced protein-protein interactions in intact mammalian cells; these strategies are based on oligomerization domain-assisted complementation of rationally designed fragments of the murine enzyme dihydrofolate reductase (DHFR; EC 1.5.1.3). We describe a dominant clonal-selection assay of stably transfected cells expressing partner proteins FKBP (FK506 binding protein) and FRAP (FKBP-rapamycin binding protein) fused to DHFR fragments and show a rapamycin dose-dependent survival of clones that requires approximately 25 molecules of reconstituted DHFR per cell. A fluorescence assay also is described, based on stoichiometric binding of fluorescein-methotrexate to reconstituted DHFR in vivo. Formation of the FKBP-rapamycin-FRAP complex is detected in stably and transiently transfected cells. Quantitative rapamycin dose-dependence of this complex is shown to be consistent with in vitro binding and distinguishable from a known constitutive interaction of FKBP and FRAP. We also show that this strategy can be applied to study membrane protein receptors, demonstrating dose-dependent activation of the erythropoietin receptor by ligands. The combination of these clonal-selection and fluorescence assays in intact mammalian cells makes possible selection by simple survival, flow cytometry, or both. High-throughput drug screening and quantitative analysis of induction or disruption of protein-protein interactions are also made possible." @default.
• W2040102227 created "2016-06-24" @default.
• W2040102227 creator A5021912322 @default.
• W2040102227 creator A5082825332 @default.
• W2040102227 date "1999-05-11" @default.
• W2040102227 modified "2023-10-16" @default.
• W2040102227 title "Clonal selection and <i>in vivo</i> quantitation of protein interactions with protein-fragment complementation assays" @default.
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• W2040102227 doi "https://doi.org/10.1073/pnas.96.10.5394" @default.
• W2040102227 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/21870" @default.
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