Novel GαGTP Sensors Reveal Endogenous and Subcellular G Protein Signaling Dynamics

Area of Science:

• Cellular Biology
• Molecular Biology
• Biochemistry

Background:

• G protein-coupled receptors (GPCRs) are crucial for cellular communication, responding to diverse stimuli by activating G protein heterotrimers.
• Understanding the spatiotemporal dynamics of endogenous G protein activity is vital for deciphering signaling pathways and identifying pathological mechanisms.
• A significant limitation in studying G protein signaling has been the absence of reliable sensors for endogenous G proteins.

Purpose of the Study:

• To engineer and validate novel biosensors capable of probing endogenous GαiGTP and GαqGTP activity at the subcellular level.
• To overcome the limitations of existing methods that rely on overexpressed and fluorescently tagged G proteins.
• To provide a tool for capturing the native fidelity of endogenous G protein signaling dynamics, including generation, equilibrium, and hydrolysis.

Main Methods:

• Development of translocation-based biosensors for endogenous GαiGTP and GαqGTP.
• Utilizing optogenetic tools to control GPCR-G protein activation asymmetrically.
• Applying sensors to investigate Gγ-subtype dependence in Gi-GPCR signaling and Gαq expression levels.

Main Results:

• The engineered sensors accurately capture the magnitude and kinetics of endogenous GαGTP dynamics with native fidelity.
• Demonstrated that heterotrimer dissociation upon Gi-GPCR activation is dependent on the specific Gγ subunit.
• Confirmed low and tightly regulated expression of Gαq in most cells.
• Showcased the sensors' ability to detect GαGTP generation and hydrolysis during optogenetically controlled asymmetric GPCR activation.

Conclusions:

• The novel engineered GαGTP sensors provide unprecedented capabilities for decoding subcellularly resolved endogenous G protein signaling.
• These tools facilitate a deeper understanding of in vivo signaling paradigms and the identification of disease-related signaling pathways.
• The sensors are particularly valuable for studying signaling in morphologically complex cells like neurons.