Protocol for quantitative live-cell imaging of early GPCR trafficking using acid-stable afCFP-Venus FRET

Area of Science:

• Cellular and Molecular Biology
• Biophysics
• Pharmacology

Background:

• G protein-coupled receptors (GPCRs) mediate cellular responses to diverse stimuli.
• Beta-arrestin proteins play crucial roles in GPCR desensitization, trafficking, and signaling.
• The angiotensin II type 1 receptor (AT1R) is a key mediator of cardiovascular function.

Purpose of the Study:

• To develop and present a protocol for visualizing and quantifying beta-arrestin-dependent signaling of the AT1R.
• To enable the study of early receptor-beta-arrestin interactions during trafficking events.
• To analyze GPCR trafficking and intracellular signaling dynamics in real-time within living cells.

Main Methods:

• Utilized an enhanced cyan fluorescent protein (eCFP)-Venus Förster Resonance Energy Transfer (FRET) pair for detecting receptor-beta-arrestin interactions.
• Developed imaging conditions to capture early trafficking events.
• Detailed procedures for visualizing intracellular signaling dynamics following AT1R activation.

Main Results:

• Successfully established a protocol to visualize and quantify AT1R beta-arrestin signaling.
• Demonstrated the detection of early receptor-beta-arrestin interactions using FRET.
• Enabled the analysis of intracellular signaling dynamics and GPCR trafficking in living cells.

Conclusions:

• The presented protocol offers a powerful tool for studying beta-arrestin-mediated signaling pathways.
• This method facilitates a deeper understanding of GPCR trafficking and signaling dynamics.
• The protocol is applicable to various GPCRs involved in cellular regulation.