Bolivar Juan H, Muñoz-García Juan C, Castro-Dopico Tomas, Dijkman Patricia M, Stansfeld Phillip J, Watts Anthony
Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Biochim Biophys Acta. 2016 Jun;1858(6):1278-87. doi: 10.1016/j.bbamem.2016.02.032. Epub 2016 Mar 21.
Information about lipid-protein interactions for G protein-coupled receptors (GPCRs) is scarce. Here, we use electron spin resonance (ESR) and spin-labelled lipids to study lipid interactions with the rat neurotensin receptor 1 (NTS1). A fusion protein containing rat NTS1 fully able to bind its ligand neurotensin was reconstituted into phosphatidylcholine (PC) bilayers at specific lipid:protein molar ratios. The fraction of motionally restricted lipids in the range of 40:1 to 80:1 lipids per receptor suggested an oligomeric state of the protein, and the result was unaffected by increasing the hydrophobic thickness of the lipid bilayer from C-18 to C-20 or C-22 chain length PC membranes. Comparison of the ESR spectra of different spin-labelled lipids allowed direct measurement of lipid binding constants relative to PC (Kr), with spin-labelled phosphatidylethanolamine (PESL), phosphatidylserine (PSSL), stearic acid (SASL), and a spin labelled cholesterol analogue (CSL) Kr values of 1.05±0.05, 1.92±0.08, 5.20±0.51 and 0.91±0.19, respectively. The results contrast with those from rhodopsin, the only other GPCR studied this way, which has no selectivity for the lipids analysed here. Molecular dynamics simulations of NTS1 in bilayers are in agreement with the ESR data, and point to sites in the receptor where PS could interact with higher affinity. Lipid selectivity could be necessary for regulation of ligand binding, oligomerisation and/or G protein activation processes. Our results provide insight into the potential modulatory mechanisms that lipids can exert on GPCRs.
关于G蛋白偶联受体（GPCRs）的脂-蛋白相互作用的信息很少。在这里，我们使用电子自旋共振（ESR）和自旋标记脂质来研究脂质与大鼠神经降压素受体1（NTS1）的相互作用。一种含有大鼠NTS1且完全能够结合其配体神经降压素的融合蛋白，以特定的脂质:蛋白摩尔比重组到磷脂酰胆碱（PC）双层膜中。每个受体中运动受限脂质的比例在40:1至80:1脂质范围内，这表明该蛋白处于寡聚状态，并且增加脂质双层从C-18到C-20或C-22链长的PC膜的疏水厚度，结果不受影响。比较不同自旋标记脂质的ESR光谱，可以直接测量相对于PC的脂质结合常数（Kr），自旋标记的磷脂酰乙醇胺（PESL）、磷脂酰丝氨酸（PSSL）、硬脂酸（SASL）和自旋标记胆固醇类似物（CSL）的Kr值分别为1.05±0.05、1.92±0.08、5.20±0.51和0.91±0.19。这些结果与视紫红质（唯一以这种方式研究的其他GPCR）的结果形成对比，视紫红质对这里分析的脂质没有选择性。双层膜中NTS1分子动力学模拟与ESR数据一致，并指出受体中PS可能以更高亲和力相互作用的位点。脂质选择性对于调节配体结合、寡聚化和/或G蛋白激活过程可能是必要的。我们的结果为脂质对GPCRs可能发挥的潜在调节机制提供了见解。
