Imaging GPCR internalization using near-infrared Nebraska red-based reagents.

Area of Science:

• Biochemistry
• Cell Biology
• Chemical Biology

Background:

• G protein-coupled receptor (GPCR) internalization is crucial for receptor downregulation and a target for drug discovery.
• Existing imaging methods for GPCR internalization often lack compatibility with other common cellular assays.
• Near-infrared (NIR) probes are needed for multiplexing with green fluorescent assays.

Purpose of the Study:

• To develop novel NIR probes for imaging membrane protein localization and internalization.
• To create NR-based HaloTag ligands for monitoring human orexin type 2 receptor dynamics.
• To enable multiplexing of GPCR imaging assays.

Main Methods:

• Synthesis of Nebraska Red (NR) dyes with a phosphinate functionality on a xanthene scaffold.
• Development of NR-based HaloTag ligands for live-cell imaging.
• Utilizing NR probes to visualize the human orexin type 2 receptor and its internalization.
• Investigating the effect of fetal bovine serum (FBS) on probe fluorescence.

Main Results:

• NR-based HaloTag ligands were successfully developed for imaging membrane proteins.
• The human orexin type 2 receptor internalization was monitored in real-time.
• Fetal bovine serum (FBS) enhanced NR probe fluorescence by 45-fold, enabling no-wash imaging.
• The probes demonstrated utility for real-time monitoring of receptor internalization upon agonist stimulation.

Conclusions:

• NR-based HaloTag ligands are effective tools for imaging membrane protein localization and internalization.
• These probes facilitate multiplexing with existing GPCR activity assays.
• The developed probes advance the discovery of pharmaceutical agents and ligands for orphan receptors.