Quantitative Determination and Imaging of Gαq Signaling in Live Cells via Split-Luciferase Complementation.

Abstract

G Protein-coupled receptors (GPCRs) transduce signals elicited by bioactive chemical agents (ligands), such as hormones, neurotransmitters, or cytokines, across the cellular membrane. Upon ligand binding, the receptor undergoes structural rearrangements, which cause the activation of G proteins. This triggers the activation of signaling cascades involving amplification, which takes place after every stage of the cascade. Consequently, signals from early stages can be masked when the activation of the signaling cascade is probed remote (distal) from the receptor. This led to the development of several techniques, which probe the activation of such signaling cascades as proximal to the receptor as possible. However, these methods often require specialized equipment or are limited in throughput. By applying split-luciferase complementation to the interaction between the Gαq protein and its effector the phospholipase C-β3 (PLC-β3), we introduce a protocol with a conventional plate reader at high throughput. The method is applicable to live cells and additionally allows imaging of the probe by bioluminescence microscopy.