Molecular Mechanism of Allosteric Modulation of the Oxytocin Receptor by Sterols

描述（由申请人提供）：该提案的重点是对催产素受体（OXTR）（G蛋白偶联受体（GPCR）家族的成员）和胆固醇（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）之间的复合物进行X射线结构测定。将投入大量精力开发利用脂质的稳定作用促进膜蛋白结构测定的技术。长期以来，催产素系统与支持哺乳动物的两种关键生殖功能有关：分娩期间的子宫收缩和哺乳期间的排乳。最近，人们对 OXTR 参与社会和情感行为产生了浓厚的兴趣。 OXTR 由环状九肽激素催产素激活。 OXTR 已被证明对膜中的胆固醇水平具有显着的敏感性。胆固醇的消耗或添加可逆地将受体在低 (Kd>100 nM) 和高 (Kd~1 nM) 亲和力状态之间转变。该提议的主要假设是胆固醇通过与不同的变构位点结合来调节 OXTR 的活性，从而诱导受体构象的变化。拟议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。具体目标是： 1) 将与 GPCR 结合的天然脂质的特性和数量与 LCP 中晶体成核和生长的成功相关联； 2) 确定胆固醇对调节LCP中OXTR配体结合亲和力的影响； 3)获得OXTR与胆固醇复合物的晶体。对于结构研究，我们将遵循非常成功的策略，用 T4 溶菌酶取代受体的第三个细胞内环，并结合 LCP 中的结晶，最近已经产生了人 b2 肾上腺素和腺苷 A2A GPCR 的高分辨率结构。 公共健康相关性：该研究项目将开发新技术，加快膜蛋白结构测定的成功率，并尝试解析人类催产素 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。高分辨率晶体结构可以深入了解信号转导机制，并为合理设计新一代更高效、更安全的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.