Chapter 15 - Cellular and subcellular context determine outputs from signaling biosensors

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Abstract

The use of biosensors either individually or as part of panels has now become a common technique to capturing signaling events in living cells. Such biosensors have become particularly important for probing biased signaling and allostery in G protein-coupled receptor drug screening efforts. However, assumptions about the portability of such biosensors between cell types may lead to misinterpretation of drug effects on specific signaling pathways in a given cellular context. Further, the output of a particular biosensor may be different depending on where it is localized in a cell. Here, we discuss strategies to mitigate these concerns which should feed into future biosensor design and usage.

Section snippets

Reagents

  • Where reagents are unique or difficult to obtain, we provide a source.

  • HEK 293 cells

  • HeLa cells

  • AC16 cells (obtained from Dr Mercy Davidson, Columbia University)

  • D-MEM

  • DMEM/F12

  • l-Glutamine

  • trypsin-EDTA solution

  • MEM

  • FBS

  • Penicillin and streptomycin

  • Kreb’s/HEPES (146 mM NaCl, 4.2 mM KCl, 0.5 mM MgCl2, 1 mM CaCl2, 5.9 mM glucose, 10 mM HEPES, pH 7.4).

  • Lipofectamine 2000 (Invitrogen)

  • Polyethylenimine 25 kDa, linear (PEI)

  • Tyrode's solution (140 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 12 mM NaHCO3, 5.6 mM d-glucose, 0.49 mM MgCl2,

Tissue Culture and Transfection

HEK 293 and HeLa cells were respectively cultured in DMEM supplemented with 5% FBS and MEM supplemented with 10% FBS, penicillin, and streptomycin at 37 °C in a 5% CO2 atmosphere. The day before transfection, cells were detached using a trypsin–EDTA solution and replated in the same culture media in 6-well plates and cultured for 24 h under the same conditions. The media was then aspirated and replaced with antibiotic-free media and cells were transfected with the ERK-NLS sensor in the presence

Sensor Design

The ERK-NLS sensor is a unimolecular BRET-based MAP kinase activity detecting protein. As shown in the Figure 1(A), it is composed of multiple copies of a specific MAP kinase substrate and a phosphosensor domain linked together by a 300-amino-acid-long disorganized linker and flanked at the N-terminus by the BRET donor RlucII and at the C-terminus by the acceptor GFP10. To allow docking of activated ERK1/2 to the sensor and for localization to the nucleus, amino acid sequences coding for a

Discussion

Here, we show that the ability to detect nuclear ERK1/2 using a BRET-based biosensor can vary between cell types and experimental conditions. The sensor readily detected ERK1/2 nuclear activation promoted by strong activators such as PMA and EGF in the three cell types studied with a pharmacology that matched that known for ERK1/2 activation. However, detection of GPCR-promoted activation was highly dependent on receptor expression level and cell type considered. For instance, muscarinic M3

Acknowledgments

This work was supported by a grant from the Consortium Québécois sur la Découverte du Médicament (CQDM) to MB and TH and by grants from the Canadian Institutes of Health Research (CIHR, MOP-130309, TEH; MOP-10501, MB). MB holds a Canada Research Chair in Signal Transduction and Molecular Pharmacology. NA was supported by a fellowship from the McGill CIHR Drug Development Training Program.

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