Plasma membrane cholesterol level and agonist-induced internalization of δ-opioid receptors; colocalization study with intracellular membrane markers of Rab family

Decrease of cholesterol level in plasma membrane of living HEK293 cells transiently expressing FLAG-delta-OR by beta-cyclodextrin (beta-CDX) resulted in a slight internalization of delta-OR. Massive internalization of delta-OR induced by specific agonist DADLE was diminished in cholesterol-depleted cells. These results suggest that agonist-induced internalization of delta-OR, which has been traditionally attributed exclusively to clathrin-mediated pathway, proceeds at least partially via membrane domains. Identification of internalized pools of FLAG-delta-OR by colocalization studies with proteins of Rab family indicated the decreased presence of receptors in early endosomes (Rab5), late endosomes and lysosomes (Rab7) and fast recycling vesicles (Rab4). Slow type of recycling (Rab11) was unchanged by cholesterol depletion. As expected, agonist-induced internalization of oxytocin receptors was totally suppressed in beta-CDX-treated cells. Determination of average fluorescence lifetime of TMA-DPH, the polar derivative of hydrophobic membrane probe diphenylhexatriene, in live cells by FLIM indicated a significant alteration of the overall PM structure which may be interpreted as an increased "water-accessible space" within PM area. Data obtained by studies of HEK293 cells transiently expressing FLAG-delta-OR by "antibody feeding" method were extended by analysis of the effect of cholesterol depletion on distribution of FLAG-delta-OR in sucrose density gradients prepared from HEK293 cells stably expressing FLAG-delta-OR. Major part of FLAG-delta-OR was co-localized with plasma membrane marker Na,K-ATPase and beta-CDX treatment resulted in shift of PM fragments containing both FLAG-delta-OR and Na,K-ATPase to higher density. Thus, the decrease in content of the major lipid constituent of PM resulted in increased density of resulting PM fragments.