Cell-based, high-content screen for receptor internalization, recycling and intracellular trafficking.

Area of Science:

• Cell biology
• Biochemistry
• Pharmacology

Background:

• Physiologically important receptors, including transferrin receptors and G-protein coupled receptors (GPCRs), are internalized and recycled via the endocytic recycling compartment.
• GPCR internalization is triggered by agonist stimulation, playing a crucial role in cellular signaling pathways.

Purpose of the Study:

• To develop and validate a cell-based fluorescent imaging assay for detecting and quantifying receptors within the recycling compartment.
• To establish a High Content Screening (HCS) application for assessing compounds that modulate receptor internalization, recycling, and GPCR activation.

Main Methods:

• Utilized a fluorescent imaging assay on the ArrayScan II System.
• Quantified transferrin receptor internalization/recycling using fluorescent ligands.
• Assessed internalization of a GFP-parathyroid hormone receptor chimera (a model GPCR).

Main Results:

• Demonstrated high signal-to-noise ratios in the developed assays.
• Achieved broad dynamic ranges between stimulated and unstimulated receptor activity.
• Observed low variability across multiple screening runs, indicating assay robustness.

Conclusions:

• The Receptor Internalization and Trafficking application, combined with the ArrayScan II System, provides a robust, automated screening platform.
• This system is well-suited for information-rich screening of GPCR activation and related trafficking events.