Detection and quantification of beta2AR internalization in living cells using FAP-based biosensor technology.

Area of Science:

• Cell biology
• Biochemistry
• Molecular signaling

Background:

• Ligand-dependent receptor internalization is a critical mechanism in cellular signaling pathways.
• Existing methods for studying receptor internalization can be complex and time-consuming.
• There is a need for sensitive and efficient tools to monitor receptor dynamics in live cells.

Purpose of the Study:

• To develop and validate a novel live-cell biosensor for quantifying receptor internalization.
• To utilize fluorogen-activating protein (FAP) technology for real-time detection of receptor translocation.
• To demonstrate the applicability of this biosensor for studying the beta2 adrenergic receptor (beta2AR).

Main Methods:

• Constructed recombinant genes encoding the human beta2 adrenergic receptor (beta2AR) fused with FAP domains.
• Transduced these genes into mammalian cells for expression.
• Utilized membrane-impermeant fluorogens to generate a cell surface fluorescent signal.
• Observed and quantified agonist-dependent receptor internalization using fluorescence microscopy and flow cytometry.

Main Results:

• Successfully generated a robust fluorescent signal from the cell surface upon fluorogen exposure.
• Demonstrated real-time, quantifiable observation of beta2AR internalization upon agonist stimulation.
• Achieved homogeneous assay format without the need for wash or separation steps.

Conclusions:

• The FAP-based biosensor provides a sensitive and efficient method for live-cell monitoring of receptor internalization.
• This technology is generalizable to various cell surface proteins and adaptable for high-throughput screening.
• The developed biosensor simplifies the study of receptor dynamics and drug screening processes.