A dual-light reporter system to determine the efficiency of protein-protein interactions in mammalian cells.
Methods for determining protein-protein interactions in mammalian cells typically rely on single reporter functions and are susceptible to variations between samples particularly in regard to levels of transcription, processing and translation. A method has been developed for determining protein-protein interactions in mammalian cells, which bypasses these variables confounding single reporter assays. The approach utilizes two units of gene expression linked to reporter functions that are interposed by a deactivation-activation unit in such a way that the downstream expression unit is switched off. Hence upstream expression occurs regardless of protein-protein interaction, leading to the production of the upstream reporter. In the event of protein-protein interactions, the downstream expression unit is switched on leading to dual reporter read outs. Thus, the ratio of the two reporter activities provides a measure to determine the efficiency of protein-protein interactions. To access the system we screened a mutant of BMPR2 where the interaction between BMPR-II and LIMK is abrogated. BMPR-II is a type II receptor of the TGFbeta superfamily and plays a key role in the pathogenesis of familial pulmonary arterial hypertension. This system has potential for high-throughput screening of libraries (peptide, chemical, cDNA, etc.) to isolate agents that are capable of interfering with highly selective protein-protein interaction.
Nasim
M T
MT
Department of Genetics, University of Leicester, Leicester LE1 7RH, UK. mtn4@le.ac.uk
Trembath
R C
RC
D052006:Bone Morphogenetic Protein Receptors, Type II; D002460:Cell Line; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D008856:Microscopy, Fluorescence; D025941:Protein Interaction Mapping / Q000379:methods*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D017346:Protein Serine-Threonine Kinases; C498218:BMPR2 protein, human; D052006:Bone Morphogenetic Protein Receptors, Type II; D001616:beta-Galactosidase