Structure of Sphingosine 1-phosphate Receptors

Alan卡伯特森总结NIH F32 鞘氨醇1-磷酸（S1 P）是一种溶血磷脂信号分子，其存在是 正常血管发育和内皮完整性所需，尽管急性增加， S1 P浓度导致心源性死亡。S1 P结合五种G蛋白偶联受体（GPCR; S1 PR 1 -5），每一种都与各种G蛋白（Gi、Gq和G12/13）形成复合物。的阵列 小鼠遗传模型提供了对S1 P信号通路的更好理解。 这一知识使得S1 P受体成为各种药物的靶标，包括芬戈莫德， 复发性多发性硬化症的一线治疗此外，S1 P产量增加 导致S1 PR 1的刺激，其导致转录因子NF-κB和 STAT 3，导致许多类型的癌症，包括乳腺癌和淋巴瘤。尽管 这种潜在的生物学和医学重要性，很少有体外分析这些 受体和结构信息仅可用于与拮抗剂结合的S1 PR 1， 非活性构象我的目标是确定S1 PR 1的高分辨率结构 在其功能性信号传导复合物中与Gi蛋白结合， 显微镜（cryo-EM）。然后，我将使用诱变来表征这些相互作用， 生物物理技术，如微尺度热泳和表面等离子体共振。在 此外，我努力获得S1 PR 1 -5在其载脂蛋白或配体结合状态下的结构信息， 筛选各种构建体以使S1 PR的质量更大，从而允许在制备过程中进行颗粒对齐。 冷冻电镜数据处理这些研究将促进对GPCR-G蛋白的理解 相互作用，并使靶向S1 PR的新型药物的工程化能够治疗许多 疾病或癌症。

Alan Culbertson Summary NIH F32 Sphingosine 1-phosphate (S1P) is a lysophospholipid signaling molecule whose presence is required for normal vascular development and endothelial integrity, though acute increases in S1P concentrations result in cardiac death. S1P binds five G protein coupled receptors (GPCRs; S1PR1-5), each of which form complexes with various G proteins (Gi, Gq, and G12/13). An array of genetic models in mice have provided a greater understanding of the S1P signaling pathways. This knowledge has led the S1P receptors to be the target of various drugs including fingolimod, the first-line treatment for relapsing multiple sclerosis. In addition, increased production of S1P leads to stimulation of S1PR1, which leads to activation of the transcription factors NF-κB and STAT3, leading to numerous types of cancer including breast cancer and lymphomas. Despite this underlying biological and medical importance, there is little in vitro analysis of these receptors and structural information is only available for S1PR1 bound to an antagonist in the inactive conformation. My objective is to determine the high-resolution structure of S1PR1 bound to Gi protein in its functional signaling complex using single-particle cryo-electron microscopy (cryo-EM). I will then characterize these interactions using mutagenesis and biophysical techniques such as microscale thermophoresis and surface plasmon resonance. In addition, I strive to obtain structural information for S1PR1-5 in its apo or ligand bound state by screening various constructs to make S1PRs larger in mass, allowing for particle alignment during cryo-EM data processing. These studies will advance the understanding of GPCR-G protein interactions and enable the engineering of novel drugs that target the S1PRs to treat numerous diseases or cancer.