Human oxytocin receptors in cholesterol-rich vs. cholesterol-poor microdomains of the plasma membrane.
We analyzed the properties of a G protein-coupled receptor localized in cholesterol-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 HEK293 cells. To calculate the total number of receptors independent of ligand binding studies, the oxytocin receptor was tagged with an enhanced green fluorescent protein (EGFP) which did not change the functional properties of the receptor. Only 1% of the oxytocin receptors were present in cholesterol-rich detergent-insoluble domains. In contrast, employing a detergent-free fractionation scheme that preserves the functional activity of the receptor, we detected 10-15% of the receptors in cholesterol-rich low-density membranes and therein the high-affinity state receptors were twofold enriched. In cholesterol-poor vs. cholesterol-rich domains, high-affinity oxytocin receptors behaved similar with respect to their agonist binding kinetics and GTP sensitivity. However, high-affinity oxytocin receptors localized in cholesterol-rich low-density membranes showed a markedly enhanced (t (1/2) approximately threefold) stability at 37 degrees C as compared with the oxytocin receptors localized in the cholesterol-poor high-density membranes. Addition of cholesterol to the high-density membranes fully protected the oxytocin receptors against loss of function. The importance of cholesterol to stabilize the oxytocin receptor was supported in experiments with solubilized receptors. Cholesterol markedly delayed the inactivation of oxytocin receptors 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.
Gimpl
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Institute of Biochemistry, Johannes-Gutenberg-University Mainz, Germany. gimpl@mail.uni-mainz.de
Fahrenholz
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D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D003902:Detergents; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D018045:Receptors, Oxytocin / Q000737:chemistry / Q000378:metabolism*
D003902:Detergents; D008164:Luminescent Proteins; D018045:Receptors, Oxytocin; D049452:Green Fluorescent Proteins; D002784:Cholesterol