Quantitative dynamic multicompartmental analysis of cholecystokinin receptor movement in a living cell using dual fluorophores and reconstruction of confocal images

Receptor regulation is a key component of the phenomenon of desensitization in response to agonist stimulation which protects cells from overstimulation. Receptor internalization is one part of this response, often quantified by the portion of saturable ligand binding which becomes resistant to acidic washes. It is now clear that this can include receptor in multiple distinct cellular compartments. We have developed a morphological technique involving dual fluorescent probes to delineate the plasmalemma and the ligand-occupied receptor using confocal microscopy, with analysis involving three-dimensional reconstruction and quantitation of receptor movement through each compartment. When a radioiodinated cholecystokinin (CCK) analogue occupied its receptor on the CHO-CCKR cell line, it became progressively more resistant to dissociation with acidic medium. Quantitation of receptor internalization in these cells over time using this dynamic morphological technique correlated with the acid-resistant receptor fraction, and provided the additional information of the cellular compartments traversed. This technique will have multiple applications to explore the cell-specific handling of this and other ligand-occupied receptors.