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Methods Mol Biol. 2015;1272:107-18. doi: 10.1007/978-1-4939-2336-6_8.

Monitoring G protein-coupled receptor activation using the protein fragment complementation technique split TEV.

Author information

1
Molecular Neurobiology, Department of Psychiatry, Ludwig Maximilian University, Nußbaumstr. 7, 80336, Munich, Germany, Michael.Wehr@med.uni-muenchen.de.

Abstract

G protein-coupled receptors (GPCRs) modulate cellular signaling, often in a ligand-specific manner. Cellular effects regulated include differentiation, proliferation, hormonal regulation, and neuronal activity. Further, they are involved in many disease-relevant processes, such as cancer and neurodevelopmental diseases, and represent the largest class of drug targets. Therefore, monitoring how GPCRs are regulated in their activity is crucial to understand their role in physiological processes and implications for drug development. Split TEV, a method based on TEV protease fragment complementation, can be used to sensitively assay GPCR activities in living cells. The activity of a given GPCR is monitored through its binding to β-arrestin. Split TEV reporters provide at minimum a two-step amplification process facilitating a flexible format and a robust readout. For the initial setup, a GPCR of interest and β-arrestin are fused to the N- and C-terminal fragments of the TEV protease, and occurred interactions are indicated by increased fluorescence or luminescence of TEV cleavage-dependent reporters. The experimental procedure takes 24-72 h to complete, depending on the cell type and complexity of the experimental setup applied.

PMID:
25563180
DOI:
10.1007/978-1-4939-2336-6_8
[Indexed for MEDLINE]

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