Human oxytocin receptors in choiesterol-rich vs. cholesterol-poor microdomains of the plasma membrane

We analyzed the properties of a G protein-coupled receptor localized in cho lesterol-poor vs. cholesterol-rich microdomains of the plasma membrane. For this purpose, the human oxytocin receptor, which is very sensitive against alterations of the membrane cholesterol level, was stably expressed in HEK 293 cells. To calculate the total number of receptors independent of ligand binding studies, the oxytocin receptor was tagged with an enhanced green f luorescent protein (EGFP) which did not change the functional properties of the receptor. Only 1% of the oxytocin receptors were present in cholestero l-rich detergent-insoluble domains. In contrast, employing a detergent-free fractionation scheme that preserves the functional activity of the recepto r, we detected 10-15% of the receptors in cholesterol-rich low-density memb ranes and therein the high-affinity state receptors were twofold enriched. In cholesterol-poor vs. cholesterol-rich domains, high-affinity oxytocin re ceptors behaved similar with respect to their agonist binding kinetics and GTP sensitivity. However, high-affinity oxytocin receptors localized in cho lesterol-rich low-density membranes showed a markedly enhanced (t 1/2 appro ximate to threefold) stability at 37 degrees C as compared with the oxytoci n receptors localized in the cholesterol-poor high-density membranes. Addit ion of cholesterol to the high-density membranes fully protected the oxytoc in receptors against loss of function. The importance of cholesterol to sta bilize the oxytocin receptor was supported in experiments with solubilized receptors. Cholesterol markedly delayed the inactivation of oxytocin recept ors solubilized with Chapso. In conclusion, the data of this report suggest that functional properties of heptahelical receptor proteins could differ in dependence of their localization in different membrane microdomains.