Half reporter gene is better than one

In the previous post, I introduced you Resonance Energy Transfer techniques to study protein-protein interaction (briefly, RET technology exploit two reporter genes coupled to two cognate proteins in exam; once proteins interact, one reporter transfer its energy to the second one that start glowing, "reporting" the interaction otherwise invisible). One can wonder «why using two reporters instead of cutting in half a single one?», this is exactly what argued E. Stefan and S. Michnick at Montreal University during their research on the G protein-coupled receptors (GPCRs), a protein superfamily very probed in the pharmacological field (currently targeted by >30% of approved drugs). In a recent volume of PNAS, the authors report a protein-fragment complementation assay (PCA), that is based only on the reporter enzyme Renilla reniformis luciferase (Rluc).

Binding of the two proteins of interest brings the unfolded fragments [of luciferase] into proximity, allowing for folding and reconstitution of measurable activity of the reporter protein.

Although PCAs assay are not so new (they were used also for pionieristic studies on operon lacZ), the innovation of Stefan's work consists in having designed that PCAs to be reversible: it means that not only the assay mirrors protein interactions, but once this interaction gets off, the two half-reporter split once again stopping to report! Accounting for dissociation-association kinetics these assay could be useful for reporting drug-induced dissociations (drived by antagonists in some models or agonists in others). To be honest, reversible characteristic is shared also by RET techniques, but in contrast to RET...

Rluc-PCA is a [direct] readout for absolute values of protein complexes.

So, advantages of the tecnique are clear: there are some limitations? The assay is blind versus inverse agonism: by definition, inverse agonists stabilize the receptor in its inactive conformation (it means that signal will persist although the dimer is not active). To don't forget, all the time you fuse a protein with a second foreign protein, you should be sure that this "tag" doesn't affect significantly both expression and trafficking of the protein in the physiological cellular context. Saved these points, the choice of bioluminescence (high signal-to-backround ratio) and the viability of the assay to be probed both by microscopic bioluminescence imaging (not so trivial) and by high-throughput plate-luminometers, suggests that Rluc-PCA sensor meets several requirements to study cell biological aspects of signal trasmission in living cells.

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Stefan, E., Aquin, S., Berger, N., Landry, C., Nyfeler, B., Bouvier, M., & Michnick, S. (2007). Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo Proceedings of the National Academy of Sciences, 104 (43), 16916-16921 DOI: 10.1073/pnas.0704257104