Differential effects of modification of membrane cholesterol and sphingolipids on the conformation, function, and trafficking of the G protein-coupled cholecystokinin receptor
Differential effects of modification of membrane cholesterol and sphingolipids on the conformation, function, and trafficking of the G protein-coupled cholecystokinin receptor
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DOI：
10.1074/jbc.m410385200
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发表时间：
2005-01-21
影响因子：
4.8
通讯作者：
Miller, LJ
中科院分区：
文献类型：
--
作者：
Harikumar, KG;Puri, V;Miller, LJ
受体的脂质微环境可以影响它们的构象、功能和调节。胆囊收缩素（CCK）刺激的信号在某些形式的高脂血症中是异常的，这表明可能对其脂质环境具有独特的敏感性。在这里，我们研究了胆固醇和鞘脂对CCK受体的影响，在模型中国仓鼠卵巢细胞系统具有脂质水平的修改。胆固醇的调制化学或代谢，和鞘脂调制使用温度敏感的细胞系（SPB-1）。用荧光完全激动剂配体Alexa 488-缀合的Gly-[Nle（28，31）]CCK-（26-33）探测受体构象，先前显示当占据活性构象的受体时，各向异性和寿命降低（Harikumar，K. G.，Pinon，D. L.，Wessel，W.美国，Prendergast，F. G.，和米勒，L. J.（2002）J.Biol.Chem.277，18552-18560）。该探针的各向异性和寿命随着胆固醇的增加而增加和延长，随着胆固醇或鞘脂的消耗而减少和缩短。随着胆固醇富集，这些参数的增加可能反映了CCK受体构象向其非活性、解偶联状态的变化。事实上，胆固醇富集导致非生产性激动剂配体结合，结合亲和力高于正常，钙信号传导响应于此降低。在胆固醇和鞘脂耗尽的状态下，受体移动到低于最佳的构象。随着胆固醇的消耗，配体结合和信号传导都降低，但内化和运输未受干扰。随着鞘脂消耗，配体结合和信号传导是正常的，但内化和贩运显着抑制。值得注意的是，在这些条件下，通过相同的网格蛋白依赖性途径的正常转铁蛋白受体运输得以维持。因此，CCK受体的脂质微环境特别重要，不同的脂质具有不同的作用。
The lipid microenvironment of receptors can influence their conformation, function, and regulation. Cholecystokinin (CCK)-stimulated signaling is abnormal in some forms of hyperlipidemia, suggesting the possibility of unique sensitivity to its lipid environment. Here we examined the influence of cholesterol and sphingolipids on CCK receptors in model Chinese hamster ovary cell systems having lipid levels modified. Cholesterol was modulated chemically or metabolically, and sphingolipids were modulated using a temperature-sensitive cell line (SPB-1). Receptor conformation was probed with a fluorescent full agonist ligand, Alexa 488-conjugated Gly-[Nle(28,31)]CCK-(26-33), shown previously to decrease in anisotropy and lifetime when occupying a receptor in the active conformation (Harikumar, K. G., Pinon, D. L., Wessels, W. S., Prendergast, F. G., and Miller, L. J. (2002) J. Biol. Chem. 277, 18552-18560). Anisotropy and lifetime of this probe were increased and prolonged with cholesterol enrichment, and decreased and shortened with depletion of cholesterol or sphingolipids. The increase in these parameters with cholesterol enrichment may reflect change in CCK receptor conformation toward its inactive, uncoupled state. Indeed, cholesterol enrichment resulted in nonproductive agonist ligand binding, with affinity of binding higher than normal and calcium signaling in response to this reduced. In cholesterol- and sphingolipid-depleted states, the receptor moved into conformations that were less than optimal. With cholesterol depletion, both ligand binding and signaling were decreased, yet internalization and trafficking were unperturbed. With sphingolipid depletion, ligand binding and signaling were normal, but internalization and trafficking were markedly inhibited. Of note, normal transferrin receptor trafficking through the same clathrin-dependent pathway was maintained under these conditions. Thus, lipid microenvironment of the CCK receptor is particularly important, with different lipids having distinct effects.