Probing Arrestin Function Using Intramolecular FlAsH-BRET Biosensors

Area of Science:

• Cellular Biology
• Biochemistry
• Pharmacology

Background:

• G protein-coupled receptors (GPCRs) mediate cellular responses to extracellular signals.
• Ligand binding to GPCRs induces conformational changes, influencing downstream signaling via effectors like arrestins.
• Understanding these conformational dynamics is crucial for deciphering signal transduction pathways.

Purpose of the Study:

• To develop and validate a novel biosensor for measuring stimulus-induced conformational changes in arrestin proteins.
• To investigate how GPCR and ligand structures influence arrestin conformation and activation modes.
• To establish a method for classifying GPCRs and ligands based on their distinct arrestin activation signatures.

Main Methods:

• Utilized intramolecular fluorescein arsenical hairpin (FlAsH) bioluminescence resonance energy transfer (BRET) biosensors.
• Engineered Rluc-arrestin3-FlAsH-BRET constructs with N-terminal Renilla luciferase donors and tetracysteine-tagged arrestin3.
• Measured changes in net BRET in live cells upon GPCR stimulation from multiple vantage points within arrestin3.

Main Results:

• Successfully measured stimulus-induced conformational changes in arrestin3 using the developed BRET biosensors.
• Demonstrated that GPCR and ligand structures imprint distinct conformational signatures onto arrestin3.
• Showcased the ability to differentiate arrestin3 activation modes based on receptor-ligand interactions.

Conclusions:

• The Rluc-arrestin3-FlAsH-BRET biosensor provides a powerful tool for real-time monitoring of arrestin conformation.
• This approach elucidates the intricate mechanisms of information transfer across the plasma membrane mediated by GPCRs.
• The findings enable classification of GPCRs and ligands by their unique effects on arrestin activation, advancing drug discovery and signaling research.