Gimpl G, Fahrenholz F
Institute of Biochemistry, Johannes-Gutenberg-University Mainz, Germany.
Eur J Biochem. 2000 May;267(9):2483-97. doi: 10.1046/j.1432-1327.2000.01280.x.
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.
我们分析了一种定位于质膜胆固醇含量低与胆固醇含量高的微结构域中的G蛋白偶联受体的特性。为此，对膜胆固醇水平变化非常敏感的人催产素受体在HEK293细胞中稳定表达。为了在不依赖配体结合研究的情况下计算受体总数，催产素受体用增强型绿色荧光蛋白（EGFP）标记，该蛋白不会改变受体的功能特性。仅1%的催产素受体存在于富含胆固醇的去污剂不溶性结构域中。相比之下，采用一种保留受体功能活性的无去污剂分级分离方案，我们在富含胆固醇的低密度膜中检测到10%-15%的受体，其中高亲和力状态的受体富集了两倍。在胆固醇含量低与胆固醇含量高的结构域中，高亲和力催产素受体在激动剂结合动力学和GTP敏感性方面表现相似。然而，与定位于胆固醇含量低的高密度膜中的催产素受体相比，定位于富含胆固醇的低密度膜中的高亲和力催产素受体在37℃时稳定性显著增强（t(1/2)约为三倍）。向高密度膜中添加胆固醇可完全保护催产素受体免于功能丧失。用溶解的受体进行的实验支持了胆固醇对稳定催产素受体的重要性。胆固醇显著延迟了用Chapso溶解的催产素受体的失活。总之，本报告的数据表明，七螺旋受体蛋白的功能特性可能因其在不同膜微结构域中的定位而异。
