Systematic Quantification of GPCR/cAMP-Controlled Protein Kinase A Interactions

Area of Science:

• Cellular Biology
• Molecular Signaling
• Biochemistry

Background:

• Cyclic AMP (cAMP) is a crucial second messenger originating from G protein-coupled receptor (GPCR) cascades.
• cAMP activates protein kinase A (PKA), a key intracellular effector regulating diverse cellular functions.
• Understanding PKA's compartmentalized roles requires analyzing spatio-temporal dynamics of molecular interactions and signaling events within specific cellular contexts.

Purpose of the Study:

• To present a comprehensive overview of PKA biosensors for studying cellular signaling.
• To detail a modular protein-fragment complementation assay (PCA) platform using *Renilla* luciferase for PKA network interaction analysis.
• To demonstrate the application of this PCA platform in dissecting dynamic PKA complexes downstream of GPCR activity.

Main Methods:

• Development and specification of unimolecular and bimolecular PKA biosensors.
• Design of a modular *Renilla* luciferase-based protein-fragment complementation assay (PCA) platform.
• Application of the PCA reporter to quantify PKA network interactions, cAMP signaling, and post-translational modifications.

Main Results:

• A collection of cell-based reporters, including PKA biosensors and a PCA platform, has been developed.
• The PCA platform enables visualization and quantitative measurement of kinase activities and PKA complex dynamics.
• The platform allows systematic quantification of transient PKA network interactions in response to GPCR signaling.

Conclusions:

• The developed PKA biosensors and PCA platform are valuable tools for decoding cell-type and cell-compartment specific PKA functions.
• This approach facilitates a deeper understanding of how GPCR signals are processed through the PKA signalosome.
• Systematic quantification of transient PKA network interactions enhances comprehension of cellular signaling pathways.