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Nucleic Acids Research 2005 33(7):e66; doi:10.1093/nar/gni066
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Published online 11 April 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Methods Online

A dual-light reporter system to determine the efficiency of protein–protein interactions in mammalian cells

M. T. Nasim* and R. C. Trembath

Department of Genetics, University of Leicester Leicester LE1 7RH, UK

*To whom correspondence should be addressed. Tel: +44 116 223 1262; Fax: +44 116 223 1779; Email: mtn4{at}le.ac.uk

Received January 11, 2005. Revised March 24, 2005. Accepted March 24, 2005.

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 TGFß 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.


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Home page
 Hum Mol GenetHome page
M. T. Nasim, A. Ghouri, B. Patel, V. James, N. Rudarakanchana, N. W. Morrell, and R. C. Trembath
Stoichiometric imbalance in the receptor complex contributes to dysfunctional BMPR-II mediated signalling in pulmonary arterial hypertension
Hum. Mol. Genet., June 1, 2008; 17(11): 1683 - 1694.
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