Membrane cholesterol affects stimulus-activity coupling in type 1, but not type 2, CCK receptors: use of cell lines with elevated cholesterol.
Membrane cholesterol affects stimulus-activity coupling in type 1, but not type 2, CCK receptors: use of cell lines with elevated cholesterol.
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DOI：
10.1007/s11745-012-3744-4
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发表时间：
2013-03
期刊：
影响因子：
1.9
通讯作者：
Miller LJ
中科院分区：
文献类型：
--
作者：
Harikumar KG;Potter RM;Patil A;Echeveste V;Miller LJ
膜蛋白的脂质微环境可以影响其结构、功能和调节。我们最近描述了急性膜胆固醇修饰对1型和2型CCK受体功能的不同影响。我们现在使用膜胆固醇升高的新型受体承载细胞系来探索慢性胆固醇调节对这些受体的影响。CCK1R和CCK2R在SCAP功能增强的25RA细胞和影响HMG CoA还原酶和SREBP的Insig-1和Insig-2酶缺陷的SRD15细胞中得到稳定表达。这些细胞质膜中胆固醇的增加被直接证明，受体结合和信号特征反映了对受体功能的预期效果。在这两种环境中，两种类型的CCK受体都被内化并正常循环，以响应激动剂的占用。在这些CHO来源的细胞系中，在光镜水平上没有发现膜内受体分布的差异。研究了荧光激动剂和拮抗剂所占据的受体的荧光各向异性以及用YFP标记时的荧光各向异性。这些研究表明，在胆固醇丰富的环境中，激动剂配体占据CCK1R的活性状态的各向异性增加，模拟该受体的非偶联、非活性状态的荧光，而增加胆固醇对CCK2R的荧光没有影响。这些细胞系对于检测可能在异常脂质环境中使用的潜在药物的功能特性应该是非常有用的。
The lipid microenvironment of membrane proteins can affect their structure, function, and regulation. We recently described differential effects of acute modification of membrane cholesterol on the function of type 1 and 2 cholecystokinin (CCK) receptors. We now explore the regulatory impact of chronic cholesterol modification on these receptors using novel receptor-bearing cell lines with elevated membrane cholesterol. Stable CCK1R and CCK2R expression was established in clonal lines of 25RA cells having gain-of-function in SCAP [sterol regulatory element binding protein (SREBP) cleavage-activating protein] and SRD15 cells having deficiencies in Insig-1 and Insig-2 enzymes affecting HMG CoA reductase and SREBP. Increased cholesterol in the plasma membrane of these cells was directly demonstrated, and receptor binding and signaling characteristics were shown to reflect predicted effects on receptor function. In both environments, both types of CCK receptors were internalized and recycled normally in response to agonist occupation. No differences in receptor distribution within the membrane were appreciated at the light microscopic level in these CHO-derived cell lines. Fluorescence anisotropy was studied for these receptors occupied by fluorescent agonist and antagonist, as well as when tagged with YFP. These studies demonstrated increased anisotropy of the agonist ligand occupying the active state of the CCK1R in a cholesterol-enriched environment, mimicking fluorescence of the uncoupled, inactive state of this receptor, while there was no effect of increasing cholesterol on fluorescence at the CCK2R. These cell lines should be quite useful for examining the functional characteristics of potential drugs that might be used in an abnormal lipid environment.
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影响因子：
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