Molecular Mechanism of Allosteric Modulation of the Oxytocin Receptor by Sterols

描述(申请人提供)：这项建议集中于催产素受体(OXTR)和胆固醇之间的复合体的X射线结构测定，催产素受体(OXTR)是G蛋白偶联受体(GPCR)家族的成员，胆固醇是哺乳动物细胞中普遍存在的一种脂肪，已知调节各种膜蛋白的功能。将致力于通过利用脂类的稳定作用来促进膜蛋白结构测定的技术的开发。长期以来，催产素系统被认为与支持哺乳动物的两个关键生殖功能有关：分娩期间的子宫收缩和哺乳期的乳汁排出。最近，人们对OXTR在社会和情绪行为中的参与产生了浓厚的兴趣。OXTR是由一种循环的非肽激素催产素激活的。OXTR已被证明对细胞膜中的胆固醇水平具有显着的敏感性。胆固醇的耗尽或添加可逆地将受体从低亲和力状态(KD&GT；100 nM)转变为高亲和力状态(KD~1 nM)。这一建议的主要假设是，胆固醇通过与一个不同的变构位点(S)结合来调节OXTR的活性，从而导致受体构象的变化。建议的结构和功能研究将利用脂基立方相(LCP)作为稳定和结晶困难膜蛋白的有希望的基质。具体目的是：1)将与GPCRs结合的天然脂质的特性和数量与LCP中晶体的成核和生长成功相关联；2)建立胆固醇对LCP中OXTR配体结合亲和力的调节作用；3)获得与胆固醇形成络合物的OXTR晶体。对于结构研究，我们将遵循非常成功的策略，用T4溶菌酶结合LCP中的结晶来取代受体的第三个细胞内环，这是最近产生了人b2肾上腺素能和腺苷A2AGPCRs的高分辨率结构。 与公众健康相关：该研究项目将开发新技术，以加快膜蛋白结构测定的成功率，并将尝试解决人类催产素G蛋白偶联受体的结构。G蛋白偶联受体家族的成员参与关键的细胞和生理过程，因此是重要的药物靶点。高分辨率的晶体结构可以深入了解信号转导的机制，并为合理设计更有效、更安全的新一代药物提供必要的模板。

DESCRIPTION (provided by applicant): This proposal is focused on the X-ray structure determination of a complex between the oxytocin receptor (OXTR), a member of the G Protein-Coupled Receptor (GPCR) family, and cholesterol, a ubiquitous lipid in mammalian cells known to modulate functions of a variety of membrane proteins. Significant efforts will be devoted to developments of technologies facilitating structure determination of membrane proteins through the use of the stabilizing effect of lipids. For a long time, the oxytocin system was linked with supporting two critical reproductive functions in mammals: uterine contraction during labor and milk ejection during lactation. Recently, a significant interest has emerged concerning the involvement of the OXTR in social and emotional behavior. The OXTR is activated by a cyclic nonapeptide hormone oxytocin. The OXTR has been demonstrated to have a remarkable sensitivity to the levels of cholesterol in the membrane. Depletion or addition of cholesterol reversibly transform the receptor between low (Kd>100 nM) and high (Kd~1 nM) affinity states. The main hypothesis of this proposal is that cholesterol modulates the activity of the OXTR by binding to a distinct allosteric site(s) thus inducing a change in the receptor conformation. The proposed structural and functional studies will take advantage of the Lipidic Cubic Phase (LCP) as a promising matrix for stabilizing and crystallizing difficult membrane proteins. The specific aims are: 1) Correlate the identity and amounts of natural lipids bound to GPCRs with successes of crystal nucleation and growth in LCP; 2) Establish the effect of cholesterol on modulating ligand binding affinities of the OXTR in LCP; 3) Obtain crystals of the OXTR in complex with cholesterol. For structural studies, we will follow the highly successful strategy of replacing the third intracellular loop of the receptor with T4 lysozyme combined with crystallization in LCP, which has recently yielded high resolution structures of human b2 adrenergic and adenosine A2A GPCRs. PUBLIC HEALTH RELEVANCE: This research project will develop new technologies that will accelerate the success rate of membrane protein structure determination and will attempt to solve a structure of the human oxytocin G protein-coupled receptor. Members of the G protein-coupled receptor family are involved in critical cellular and physiological processes and therefore are important drug targets. High resolution crystal structures give insights into mechanism of signal transduction and provide necessary templates for the rational design of a new generation of more efficient and safe drugs.