Molecular Mechanism of Allosteric Modulation of the Oxytocin Receptor by Sterols

描述（由申请人提供）：该提案专注于催产素受体（OXTR） （G蛋白偶联受体（GPCR）家族成员）和胆固醇之间复合物的x射线结构测定，胆固醇是哺乳动物细胞中普遍存在的脂质，已知可调节多种膜蛋白的功能。重大的努力将致力于通过使用脂质稳定作用来促进膜蛋白结构测定的技术的发展。长期以来，催产素系统与哺乳动物的两个关键生殖功能有关：分娩时的子宫收缩和哺乳时的泌乳。最近，人们对OXTR在社会和情感行为中的作用产生了极大的兴趣。OXTR被一种循环非肽激素催产素激活。OXTR已被证明对膜中的胆固醇水平具有显著的敏感性。消耗或添加胆固醇可使受体在低（Kd~ 100 nM）和高（Kd~1 nM）亲和状态之间可逆地转变。该提议的主要假设是，胆固醇通过结合一个不同的变构位点来调节OXTR的活性，从而诱导受体构象的变化。提出的结构和功能研究将利用脂质立方相（LCP）作为稳定和结晶困难膜蛋白的有前途的基质。具体目的是：1)将gpcr结合的天然脂质的身份和数量与LCP晶体成核和生长的成功联系起来；2)建立胆固醇对LCP中OXTR配体结合亲和力的调节作用；3)得到OXTR与胆固醇复合物的结晶。对于结构研究，我们将遵循非常成功的策略，用LCP中的T4溶菌酶结合结晶取代受体的第三胞内环，该策略最近获得了人类b2肾上腺素能和腺苷A2A gpcr的高分辨率结构。

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.