Method to study protein protein interaction in mammalian cells

Protein-protein interactions are an essential key in all biological processes, from replication and expression of genes to the morphogenesis of organisms. The standard technology to study protein-protein interactions is the yeast two-hybrid technique. The system is based on the reconstitution of a transcriptional activator. Upon protein-protein interaction of two fusion proteins, a functional activator is obtained, resulting in the activation of a reporter gene. However, the method has several drawbacks: the fusion proteins need to be translocated to the nucleus, which is not always evident. Proteins with intrinsic activation properties may cause false positives. Moreover, interactions that are dependent upon secondary modifications of the proteins, such as protein phosphorylation, cannot easily be detected.
 

Basic concept

Prof. Jan Tavernier and his VIB team (University of Ghent) have developed a novel screening method for protein-protein interaction in mammalian cells, based on the reconstitution of a membrane receptor. The activation site of the receptor has been eliminated, so that no signal can be detected upon ligand binding. However, a fusion protein, carrying an activation site can be recruited to the receptor by protein-protein interaction, and will restore the signaling upon ligand binding (see figure). This signal leads to the activation of a reporter gene.
The method is called MAPPIT, for mammalian protein-protein interaction trap.
 

Advantages

The method allows to study protein-protein interactions in differenttypes of mammalian cells. Mammalian proteins can therefore be analyzed in their natural environment.
 
The method allows to identify and analyse interactions with posttranslationally modified proteins such as phosphorylated proteins.
No translocation to the nucleus is needed; most protein-protein interactions can be studied in their natural environment.
As the signal is dependent both upon the protein-protein interaction and upon the binding of the ligand to the receptor, false positives can be eliminated.
The MAPPIT screening method is sensitive, and has been convincingly validated.
 

Applications

MAPPIT is applicable in all fields where protein-protein interactions are studied, but it is especially useful in the biopharmaceutical sector for the study of signaling pathways or the identification of new drug targets.
 
The technology can be used to:
- unravel signal transduction pathways;
- distinguish interactions with phosphorylated and non-phosphorylated versions of a target protein;
- map the specific domains and amino acids involved in a protein interaction;
- identify compounds interfering with a specific protein-protein interaction.
 
 

Further reading

 
WO01/090188
Tavernier J et al (2001) Nature Cell Biology; 1114-1119: Design and application of a cytokine-receptor-based interaction trap.
 
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