The mapping of protein–protein interactions in mammalian cells is finally on the move thanks to the development of an automated high-throughput, light-based technology by Jeff Wrana and co-workers. The technology improves on present methods for looking at interaction networks, which focus on static analyses in lower organisms, and has been applied to the transforming growth factor-β (TGFβ) pathway by the researchers.

The authors developed LUMIER — luminescence-based mammalian interactome mapping — by fusing the luciferase enzyme from Renilla to proteins of interest (the bait), and then co-expressing them with individual Flag-tagged partners in mammalian cells (the prey). Prey proteins were then immunoprecipitated using an anti-Flag antibody, and their potential interactions were determined using a Renilla luciferase assay.

Wrana's team verified the approach by studying the interaction between the TGFβ-receptor-regulated Smad2 and Smad4 proteins. In the presence of TGFβ, a Smad2–Smad4 complex accumulates in the nucleus where it regulates transcription. In their assay, this was shown by high levels of Renilla luciferase activity in Flag–Smad2 immunoprecipitates (with Smad4 fused to Renilla luciferase).

Having optimized LUMIER in a 96-well format, the authors set about mapping a TGFβ protein–protein interaction network by tagging core members of the pathway with a Renilla luciferase tag and Flag-tagging 518 cDNAs. The results were represented by the intensity of light for each interaction, the LUMIER intensity ratio (LIR). At a specific LIR cutoff, 947 interactions (out of ∼12,000) were revealed, forming an interconnected network that often had highly connected nodes. Because networks are dynamic, the authors also created a movie of the dynamics of the Smad2 and Smad4 interactome with or without TGFβ, which showed that partner switching occurred frequently.

What, then, did Wrana's team uncover in their analysis of TGFβ signalling by LUMIER? Substantial connections were found between the TGFβ pathway and a network mediated by the p21-activated kinase-1 (PAK1), and between TGFβ signalling and the cell polarity network — the TGFβ receptor II (TβRII) associated with the tight-junction component occludin in a TGFβ-dependent manner, whereas there was a constitutive TβRI–occludin interaction. The latter localized TβRI to tight junctions, which is important for the TGFβ-induced dissolution of such junctions during the transition of epithelial cells into a mesenchymal phenotype.

This is therefore a powerful technique for studying protein–protein interactions in mammalian cells, and is likely to prove popular with those involved in basic research and drug discovery alike. Absolute affinities of protein–protein interactions can't be measured at the moment, but hopefully we won't be in the dark about this for long.