NDLI: Clonal selection and in vivo quantitation of protein interactions with protein-fragment complementation assays

Content Provider	PubMed Central
Author	Remy, Ingrid Michnick, Stephen W.
Copyright Year	1999
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 ≈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.
Starting Page	5394
File Format	PDF
ISSN	10916490
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	10
Volume Number	96
Language	English
Publisher	The National Academy of Sciences
Publisher Date	1999-05-11
Access Restriction	Open
Rights Holder	The National Academy of Sciences
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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