title: Clonal selection and in vivo quantitation of protein interactions with protein-fragment complementation assays.
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.
mesh_terms: D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins; D002470:Cell Survival; D006224:Cricetinae; D004921:Erythropoietin / Q000494:pharmacology; D005434:Flow Cytometry; D005452:Fluoresceins; D005816:Genetic Complementation Test; D020104:Immunophilins / Q000032:analysis* / Q000235:genetics; D008727:Methotrexate / Q000031:analogs & derivatives; D051379:Mice; D008856:Microscopy, Fluorescence; D010456:Peptides, Cyclic / Q000494:pharmacology; D017853:Phosphotransferases (Alcohol Group Acceptor); D011485:Protein Binding; D017467:Receptors, Erythropoietin / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000032:analysis; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D016559:Tacrolimus / Q000494:pharmacology; D022021:Tacrolimus Binding Proteins; D013762:Tetrahydrofolate Dehydrogenase / Q000032:analysis* / Q000235:genetics; D014162:Transfection