Trends in Pharmacological Sciences
ReviewSpecial Issue: Illuminating GPCRs in Living CellsIntramolecular and Intermolecular FRET Sensors for GPCRs – Monitoring Conformational Changes and Beyond
Section snippets
G Protein-Coupled Receptors
G protein-coupled receptors (GPCRs) recognize extracellular stimuli and translate this encoded information across the plasma membrane to give rise to multiplexed cellular responses. The almost unlimited diversity of interacting partners, ranging from single photons to large protein domains, is reflected by more than 800 expressed genes for GPCRs all over the human body [1]. Understanding these transmembrane (TM) connectors in greater detail has been a major aim of biomedical research for
Labeling Techniques and RET Sensor Design
The first receptor sensors were designed by introducing cyan or yellow derivatives of GFP into the receptor structure, mostly into a truncated third intracellular loop and shortened C terminus [17]. These first sensors can be regarded as milestones in sensor design, but they proved suboptimal because both of these receptor domains have crucial impact on functionality, leading to hampered downstream signaling capability 17, 19. The downstream signaling of these sensors was significantly improved
Dynamic Receptor Movements
For a long period, the GPCR activation process was understood as a two-state model. The origin of this classical point of view was the well-studied activation mechanism of rhodopsin that is triggered by a cis/trans isomerization of retinal. Nowadays, it appears that rhodopsin rather remains the exception in GPCR dynamics than the rule. Recent studies indicate that GPCRs behave as a highly dynamic system, adopting various conformations of different free energy without favoring a distinct
Can Intermolecular FRET Sensor Signals Be Influenced by G-Protein Coupling?
The initially designed FRET sensors, for the α2A-AR, parathyroid (PTH-R), and adenosine A2A receptor, had been intensively studied for factors other than the conformational changes within the 7TM domains that might cause a change in FRET signal and contribute to the signal. A possible contribution of G proteins was investigated by preparing membranes, which were subjected to 6M urea treatment and treatment with Pertussis toxin, to block endogenous Gi proteins 17, 19. Since none of these
Intramolecular RET Sensors to Study Immediate Events in Receptor Signaling
In this section, we will briefly discuss very recent achievements in the design of sensors to study the immediate activation of G proteins or β-arrestin as molecules with direct contact to the receptor. Sivaraj Sivaramakrishnan and his group 50, 51 have published information on promising novel sensors of GPCRs that report upon signaling specific conformational changes induced by a ligand. Such sensors are based on a dual labeling of the respective receptor C terminus with cerulean and citrine
RET Sensor Applications and Receptor Oligomers
Receptor dimerization has recently been reviewed in the light of novel resonance energy-based approaches and other technical features and is certainly a topic of its own right [57]. In the previous sections, we have briefly touched a few reports that discussed the use of FRET-based conformational sensors to study receptor oligomers. Not many reports currently exist on this research topic. Here, we would like to mention a few studies that have particularly addressed issues of receptor
High-Throughput Applications
For screening substance libraries, a single-cell method is not really feasible because of the relatively intense effort and time-consuming experimental setup. High-throughput screening (HTS) assays are performed in at least 96-well plates, but most frequently in a 384- or 1536-well format. At this point we want to mention that the benefits of high-throughput assays are often counterbalanced by losing important spatial and temporal information and we refer to Box 2 for a comparison of HTS versus
Concluding Remarks
Although the principle of these conformational sensors for GPCRs was described almost 15 years ago, we are still witnessing the development of novel areas for their applications. The recent developments of 96-well assay formats for ligand screening and the proof of principle of fragment-based screening at single cells are encouraging news. Allosteric modulators can now be studied in living cells without a radioactive tracer and with different orthosteric probes to investigate probe dependency
Disclaimer Statement
The authors declare that there is no personal conflict of interest. The University of Würzburg does hold a patent on this technology: WO2004057333 A1.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – TRR 166: project C2 (C.H.), the Marie Curie Initial Training Networks (ITN) ‘WntsApp’ grant agreement number 608180 (C.H.), as well as the international doctoral college ‘Receptor Dynamics: Emerging Paradigms for Novel Drugs’ fund within the framework of the Elite Network of Bavaria (C.H.).
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