Technique: Monitoring modification
Bioluminescence resonance energy transfer (BRET) is a new technique that can be used to detect in situ changes in protein ubiquitylation.
Ubiquitylation is a reversible post-translational modification that has important roles in processes such as protein degradation and intracellular signalling. Yet, despite a growing interest in ubiquitylation, a tool for studying the dynamics of this process has been lacking. Now though, in Nature Methods, Bouvier and colleagues describe a technique that can be used to detect in situ changes in protein ubiquitylation.
Their work revolved around bioluminescence resonance energy transfer (BRET), which allows protein–protein interactions to be detected in real time in vivo, and  -arrestin — a protein that is ubiquitylated in response to G-protein-coupled receptor (GPCR) activation.
They made a
Renilla-luciferase–-arrestin construct (Rluc–-arrestin) and a green-fluorescent-protein–ubiquitin construct (GFP–Ub; mutant ubiquitin was used to prevent polyubiquitin-chain formation), and added a specific Rluc substrate (substrate-1) to cells co-expressing these constructs. Substrate-1 hydrolysis by Rluc emits light that overlaps with the excitation spectrum of GFP. So, if GFP–Ub is covalently conjugated to Rluc–-arrestin, BRET occurs and a fluorescent signal that originates from GFP should be detected.
Indeed, a BRET signal was detected, and the signal increased with increasing concentrations of GFP–Ub until it reached a plateau (weaker, linear signals were obtained using GFP alone or a GFP–Ub construct that could not be conjugated to Rluc–-arrestin). Furthermore, no signal was obtained when GFP–Ub and Rluc were co-expressed. These data therefore confirm that the signal reflects the covalent attachment of GFP–Ub to -arrestin in Rluc–-arrestin.
Next, the authors showed that this technique can be used to study receptor-regulated ubiquitylation by monitoring energy transfer between GFP–Ub and Rluc–-arrestin in the presence of GPCRs. When they activated the receptors using selective agonists, the BRET signal increased in a dose-dependent manner.
It has been proposed that GPCR activation might regulate -arrestin ubiquitylation and its recruitment to the GPCR. By using two different substrates for Rluc and a GPCR–yellow-fluorescent-protein construct (V2R–YFP), Bouvier and colleagues were able to monitor these two events simultaneously. They split a cell culture expressing GFP–Ub, Rluc–-arrestin and V2R–YFP into two samples, and added substrate-1 to one sample (substrate-1 hydrolysis produces light that excites GFP–Ub) and substate-2 to the other (substrate-2 hydrolysis emits light that excites V2R–YFP). They found that the activation of V2R–YFP produced BRET signals indicative of the concomitant ubiquitylation and recruitment of -arrestin.
Finally, Bouvier and co-workers followed the real-time kinetics of agonist-promoted -arrestin ubiquitylation in cells co-expressing GPCRs that interact with -arrestin transiently (class A) or stably (class B). They found that class-B-GPCR activation resulted in a more s ubiquitylation of -arrestin than class-A-GPCR activation. The nature of the interaction between the receptor and -arrestin therefore effects the dynamics of -arrestin ubiquitylation.
So, using -arrestin as a model, these authors have shown that BRET can specifically detect basal and regulated ubiquitylation processes in living cells. Their assay will therefore be useful "...for studying the dynamic ubiquitination of proteins and for understanding which cellular functions are regulated by this post-translational event."
Rachel Smallridge
References
- Perroy, J. et al. Real-time monitoring of ubiquitination in living cells by BRET. Nature Meth. 1, 203–208 (2004) | Article |
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