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 desensitiz ation in response to agonist stimulation which protects cells from ove rstimulation. Receptor internalization is one part of this response, o ften quantified by the portion of saturable ligand binding which becom es resistant to acidic washes. It is now clear that this can include r eceptor in multiple distinct cellular compartments. We have developed a morphological technique involving dual fluorescent probes to delinea te the plasmalemma and the ligand-occupied receptor using confocal mic roscopy, with analysis involving three-dimensional reconstruction and quantitation of receptor movement through each compartment. When a rad ioiodinated cholecystokinin (CCK) analogue occupied its receptor on th e CHO-CCKR cell line, it became progressively more resistant to dissoc iation with acidic medium. Quantitation of receptor internalization in these cells over time using this dynamic morphological technique corr elated with the acid-resistant receptor fraction, and provided the add itional information of the cellular compartments traversed. This techn ique will have multiple applications to explore the cell-specific hand ling of this and other ligand-occupied receptors. (C) 1997 Academic Pr ess.