A FRET-based biosensor for measuring Gα13 activation in single cells

Area of Science:

• Molecular Biology
• Cell Signaling
• Biochemistry

Background:

• G-protein coupled receptors (GPCRs) mediate cellular responses to external stimuli.
• Understanding GPCR activation dynamics is crucial for pharmacology and cell biology.
• Existing methods for observing GPCR activation are often indirect.

Purpose of the Study:

• To develop a Förster Resonance Energy Transfer (FRET) based biosensor for direct observation of heterotrimeric Gα13 protein activation.
• To characterize the functionality and responsiveness of the novel Gα13 biosensor in live cells.
• To utilize the biosensor for dissecting G-protein coupling efficiency and inferring regulatory mechanisms.

Main Methods:

• Insertion of the fluorescent protein mTurquoise2 into human Gα13 subunit at multiple sites.
• Construction of FRET biosensors using Gα13-mTurquoise2 as donor and cp173Venus-Gγ2 as acceptor.
• Expression and functional validation in primary human endothelial cells stimulated with thrombin.

Main Results:

• Three functional Gα13-mTurquoise2 variants were identified, showing correct plasma membrane localization and p115-RhoGEF recruitment.
• The developed Gα13 biosensor exhibited a rapid and robust response to thrombin stimulation via endogenous protease-activated receptors (PARs).
• The biosensor data indicated that RGS domain of p115-RhoGEF inhibits Gα13 activation through GTPase-activating protein (GAP) activity.

Conclusions:

• The novel Gα13 biosensor provides a valuable tool for live-cell measurements of Gα13 activation.
• The biosensor allows for the dissection of heterotrimeric G-protein coupling efficiency in single living cells.
• This FRET-based approach offers insights into the spatiotemporal aspects of Gα13 signaling.