ER/K linked GPCR-G protein fusions systematically modulate second messenger response in cells.

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) are established methods for studying G protein-coupled receptor (GPCR) and G protein interactions.
• Current FRET/BRET assays depend on probe stoichiometry and expression levels, while GPCR-G protein fusions show inconsistent signaling properties across different receptors.

Purpose of the Study:

• To develop and characterize novel sensors for detecting ligand-induced GPCR-G protein interactions and downstream signaling.
• To investigate the utility of the Systematic Protein Affinity Strength Modulation (SPASM) technique for creating these sensors.

Main Methods:

• Development of SPASM sensors, comprising a GPCR and G protein tethered by an ER/K linker and flanked by FRET probes.
• Testing SPASM sensors with β2-, α1-, and α2- adrenergic receptors, and adenosine type 1 receptor (A1R), coupled to various G protein subunits (Gαs-XL, Gαi2, Gαq).
• Evaluating agonist-induced FRET signal changes and downstream second messenger signaling.

Main Results:

• Agonist stimulation of β2-AR and α2-AR SPASM sensors showed increased FRET signals comparable to existing FRET/BRET sensors.
• SPASM sensors maintained signaling through endogenous G protein pathways.
• The length of the ER/K linker systematically modulated GPCR-G protein interactions and downstream signaling.

Conclusions:

• SPASM GPCR sensors effectively detect agonist-induced changes in GPCR-G protein interactions.
• These novel sensors link interaction changes to downstream signaling modulation.
• The SPASM technique provides a tunable platform for studying GPCR signaling dynamics.