A Perspective on Studying G-Protein–Coupled Receptor Signaling with Resonance Energy Transfer Biosensors in Living Organisms

Unen, Jakobus van; Woolard, Jeanette; Rinken, Ago; Hoffmann, Carsten; Hill, Stephen J.; Goedhart, Joachim; Bruchas, Michael R.; Bouvier, Michel; Adjobo‐Hermans, Merel J. W.

doi:10.1124/mol.115.098897

Cited by 29 publications

(19 citation statements)

References 86 publications

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“…Resonance energy transfer techniques have several unique properties that possibly allow new insights into GPCR signaling and their pharmacology, both in vitro and in vivo (Lohse et al, 2012;Clister et al, 2015;van Unen et al, 2015b). These techniques can provide quantitative data, on/off kinetics with high temporal resolution, can be measured in real time, and allow fast and straightforward analysis of the data (Marullo and Bouvier, 2007;Lohse et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Quantitative Single-Cell Analysis of Signaling Pathways Activated Immediately Downstream of Histamine Receptor Subtypes

et al. 2016

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Genetically encoded biosensors based on Förster resonance energy transfer (FRET) can visualize responses of individual cells in real time. Here, we evaluated whether FRET-based biosensors provide sufficient contrast and specificity to measure activity of G-protein-coupled receptors. The four histamine receptor subtypes (H 1 R, H 2 R, H 3 R, and H 4 R) respond to the ligand histamine by activating three canonical heterotrimeric G-protein-mediated signaling pathways with a reported high degree of specificity. Using FRET-based biosensors, we demonstrate that H 1 R activates Gaq. We also observed that H 1 R activates Gai, albeit at a 10-fold lower potency. In addition to increasing cAMP levels, most likely via Gas, we found that the H 2 R induces Gaq-mediated calcium release. The H 3 R and H 4 R activated Gai with high specificity and a high potency. We demonstrate that a number of FRET sensors provide sufficient contrast to: 1) analyze the specificity of the histamine receptor subtypes for different heterotrimeric G-protein families with single-cell resolution, 2) probe for antagonist specificity, and 3) allow the measurement of single-cell concentration-response curves.

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Section: Introductionmentioning

confidence: 99%

Quantitative Single-Cell Analysis of Signaling Pathways Activated Immediately Downstream of Histamine Receptor Subtypes

et al. 2016

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“…The two main intracellular messengers modulated through G protein activation are calcium and cAMP. Several FRET-based genetically encoded sensors for both messengers are available, which allow the monitoring of intracellular concentration changes in living cells with high spatial and temporal resolution (van Unen et al, 2015). cAMP sensors based on EPAC (Nikolaev et al, 2004;Ponsioen et al, 2004;Calebiro and Maiellaro, 2014) and genetically encoded calcium indicators D3cpV (Palmer and Tsien, 2006) and GCaMPs (Tian et al, 2009;Akerboom et al, 2013) are popular examples.…”

Section: Tools For In Vivo Gpcr Researchmentioning

confidence: 99%

Model Organisms in G Protein–Coupled Receptor Research

et al. 2015

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The study of G protein-coupled receptors (GPCRs) has benefited greatly from experimental approaches that interrogate their functions in controlled, artificial environments. Working in vitro, GPCR receptorologists discovered the basic biologic mechanisms by which GPCRs operate, including their eponymous capacity to couple to G proteins; their molecular makeup, including the famed serpentine transmembrane unit; and ultimately, their three-dimensional structure. Although the insights gained from working outside the native environments of GPCRs have allowed for the collection of low-noise data, such approaches cannot directly address a receptor's native (in vivo) functions. An in vivo approach can complement the rigor of in vitro approaches: as studied in model organisms, it imposes physiologic constraints on receptor action and thus allows investigators to deduce the most salient features of receptor function. Here, we briefly discuss specific examples in which model organisms have successfully contributed to the elucidation of signals controlled through GPCRs and other surface receptor systems. We list recent examples that have served either in the initial discovery of GPCR signaling concepts or in their fuller definition. Furthermore, we selectively highlight experimental advantages, shortcomings, and tools of each model organism.

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“…Of these methods, the application of resonance energy transfer (RET) and protein complementation assay (PCA) (for review see e.g. Vidi & Watts 2009, Lohse et al 2012, Ng et al 2012, Bonomi & Persani 2013, Ciruela et al 2014, van Unen et al 2015 have been particularly used to detect GPCR homo-or heteromerization.…”

Section: R65mentioning

confidence: 99%