Harikumar Kaleeckal G, Puri Vishwajeet, Singh Raman Deep, Hanada Kentaro, Pagano Richard E, Miller Laurence J
Mayo Clinic Scottsdale, Cancer Center and Department of Molecular Pharmacology and Experimental Therapeutics, Scottsdale, Arizona 85259, USA.
J Biol Chem. 2005 Jan 21;280(3):2176-85. doi: 10.1074/jbc.M410385200. Epub 2004 Nov 10.
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.
受体的脂质微环境可影响其构象、功能及调节。在某些类型的高脂血症中，胆囊收缩素（CCK）刺激的信号传导异常，提示其对脂质环境可能具有独特的敏感性。在此，我们在脂质水平经修饰的中国仓鼠卵巢细胞模型系统中，研究了胆固醇和鞘脂对CCK受体的影响。通过化学或代谢方式调节胆固醇，利用温度敏感细胞系（SPB - 1）调节鞘脂。用荧光完全激动剂配体Alexa 488标记的甘氨酸 - [Nle(28,31)]CCK - (26 - 33)探测受体构象，先前研究表明，当该配体占据活性构象的受体时，其各向异性和寿命会降低（哈里库马尔，K.G.，皮农，D.L.，韦塞尔，W.S., 普伦德加斯特，F.G.，以及米勒，L.J.（2002年）《生物化学杂志》277卷，18552 - 18560页）。随着胆固醇富集，该探针的各向异性和寿命增加且延长；随着胆固醇或鞘脂耗竭，其各向异性和寿命降低且缩短。随着胆固醇富集，这些参数的增加可能反映CCK受体构象向其无活性、未偶联状态的转变。事实上，胆固醇富集导致无效的激动剂配体结合，结合亲和力高于正常水平，且对此的钙信号传导减弱。在胆固醇和鞘脂耗竭状态下，受体转变为次优构象。随着胆固醇耗竭，配体结合和信号传导均降低，但内化和转运未受干扰。随着鞘脂耗竭，配体结合和信号传导正常，但内化和转运受到显著抑制。值得注意的是，在这些条件下，通过相同网格蛋白依赖性途径的正常转铁蛋白受体转运得以维持。因此，CCK受体的脂质微环境尤为重要，不同脂质具有不同作用。
