Visualizing odorant receptor trafficking in living cells down to the single-molecule level

Area of Science:

• Cell Biology
• Molecular Biology
• Neuroscience

Background:

• G protein-coupled receptor (GPCR) signaling is crucial, yet trafficking of many GPCRs, like odorant receptors, remains poorly understood in live cells.
• Odorant receptors (ORs) are key to our sense of smell, but their dynamic behavior within cells is largely uncharacterized.

Purpose of the Study:

• To investigate the complete life cycle and trafficking dynamics of the human odorant receptor OR17-40 in living cells.
• To understand how expression levels and ligand binding affect OR17-40 trafficking and cell surface presence.

Main Methods:

• Utilized ensemble and single-molecule imaging with a double-labeling strategy (GFP and acyl carrier protein tags).
• Monitored intracellular and cell surface receptor dynamics in real-time.
• Employed single-molecule microscopy to observe early signaling events.

Main Results:

• OR17-40 efficiently reached the plasma membrane at low expression but accumulated intracellularly at higher levels.
• Plasma membrane receptors exhibited high turnover via constitutive clathrin-mediated endocytosis, independent of ligand.
• Ligand binding (agonist or antagonist) promoted receptor partitioning into small domains, likely clathrin-coated pit precursors, modulating internalization.
• Endocytosed receptors were recycled via early endosomes.

Conclusions:

• Ligand binding modulates constitutive internalization of OR17-40, influencing receptor dynamics.
• Continuous internalization and recycling of OR17-40 may enable rapid recovery of odor perception.
• Live-cell imaging provides unprecedented insight into odorant receptor trafficking and signaling.