Regulation of KRAS Trafficking and Signaling by GPR31

GPR31 对 KRAS 贩运和信号传输的监管

• 批准号: 10047185
• 负责人: MARK Reid PHILIPS
• 金额: $ 16.95万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10047185
• 关键词: Affinity; Antineoplastic Agents; Biological Assay; Biology; Cell Proliferation; Cell membrane; Cells; Cellular Membrane; Cellular biology; Clinic; Co-Immunoprecipitations; Data; Differentiation and Growth; Drug Targeting; Eicosanoids; Endoplasmic Reticulum; Enzymes; Family; Farnesyl Transferase Inhibitor; Fluorescence Resonance Energy Transfer; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gs; Genes; Genome; Goals; Growth; Guanosine Triphosphate; Human; Hydroxyeicosatetraenoic Acids; Journals; KRAS2 gene; Ligation; Luciferases; MAP Kinase Gene; Malignant Neoplasms; Measures; Membrane; Membrane Proteins; Molecular; Molecular Chaperones; Molecular Conformation; Monomeric GTP-Binding Proteins; Mutate; Mutation; Oncogenes; Oncogenic; Pathway interactions; Peripheral; Pharmaceutical Preparations; Post-Translational Protein Processing; Proteins; Publishing; RNA Interference; Regulation; Reporting; Rhodopsin; Role; Screening Result; Series; Signal Pathway; Signal Transduction; Small Interfering RNA; Subgroup; Testing; base; cancer therapy; confocal imaging; design; drug discovery; experience; farnesylation; genome-wide; interest; live cell imaging; neoplastic cell; novel; prenylation; protein protein interaction; receptor; response; trafficking

PROJECT SUMMARY KRAS is the oncogene most frequently mutated in human cancer. KRAS functions as a molecular switch that regulates signaling pathways only when associated with cellular membranes. KRAS associates with membranes as a consequence of farnesylation that operates in conjunction with a polybasic C-terminus. Efforts to defeat KRAS by blocking farnesylation failed because of alternative enzymes capable of prenylating KRAS. We therefore took an unbiased approach to identify previously unrecognized genes that participate in the membrane association of KRAS. We devised a dual luciferase assay that reports loss of KRAS affinity for membranes and used this assay in a genome-wide siRNA screen. Among the 13 genes identified we were surprised to find a G protein coupled receptor (GPCR) designated GPR31, which is a high affinity receptor for 12-(S)- HETE and has been shown to stimulate MAPK signaling. GPR31 is an understudied GPCR that is included in the Illuminating the Druggable Genome project. We were also surprised to find by co-immunoprecipitation a physical interaction between GPR31 and KRAS, suggesting that GPR31 might act as a secretory pathway chaperone for KRAS as it traffics from endomembrane to the plasma membrane (PM). In preliminary studies we have found that GRP31 and KRAS colocalized on endomembrane and PM and that silencing of GPR31 with siRNA inhibits KRAS dependent cell proliferation and macropinocytosis. We now propose to determine if 12-(S)-HETE signaling through GPR31 regulates KRAS trafficking and signaling with three Specific Aims. Aim 1. Ligation of GPR31 with 12-(S)-HETE and Interaction of GPR31 with KRAS. We will measure the interaction between GPR31 and KRAS by co-immunoprecipitation and FRET ± 12-(S)-HETE or control eicosinoids. Aim 2. Ligation of GPR31 with 12-(S)-HETE and Trafficking of KRAS. We will study KRAS trafficking from endomembrane to PM using live cell imaging ± 12-(S)-HETE. Aim 3. Ligation of GPR31 with 12-(S)-HETE and KRAS activation. We will study KRAS signaling ± 12-(S)-HETE. The prosecution of these aims will determine if GPR31 signaling regulates KRAS and thereby prioritize GPR31 for anti- cancer drug discovery. !

项目总结 KRAS是人类癌症中突变最频繁的癌基因。KRAS的职能是 仅当与细胞相关时才调节信号通路的分子开关 膜。KRAS作为法尼化的结果与膜结合 与多碱的C-末端一起工作。通过屏蔽击败KRAS的努力 法尼化失败是因为替代的酶能够使KRAS提前甲基化。我们 因此采取了一种不偏不倚的方法来识别以前未被识别的基因 参加KRAS膜协会。我们设计了一种双荧光素酶测定法 报告了KRAS膜亲和力的丧失，并在全基因组范围内使用了这一分析方法 SiRNA筛选。在确认的13个基因中，我们惊讶地发现了一种G蛋白 偶联受体命名为GPR31，是12-(S)-的高亲和力受体。 HETE和已被证明刺激MAPK信号转导。GPR31是一个未被研究的 这是包括在照亮可药物基因组项目中的GPCR。我们是在 我还惊讶地发现，通过免疫共沉淀，GPR31之间存在物理相互作用 和KRAS，提示GPR31可能作为KRAS的分泌途径伴侣 因为它从内膜运输到质膜(PM)。在初步研究中 我们发现GRP31和KRAS共定位于内膜和PM上，并且 SiRNA沉默GPR31抑制KRAS依赖的细胞增殖和 巨噬细胞增多症。我们现在建议确定12-(S)-HETE信号是否通过 GPR31管理KRAS贩运和信号，有三个具体目标。目标1。 GPR31与12-(S)-HETE的连接及与KRAS的相互作用。我们会 免疫共沉淀和FRET检测GPR31与KRAS的相互作用 ±12-(S)-HETE或对照二十烷基类化合物。目的2.用12-(S)-HETE连接GPR31和 贩卖KRAS。我们将研究KRAS从子宫内膜到PM的运输 活细胞成像±12-(S)-HETE。目的3.GPR31与12-(S)-HETE和KRAS的连接 激活。我们将研究KRAS信号转导±12-(S)-HETE。对这些目标的起诉 将确定GPR31信号是否调节KRAS，从而确定GPR31对抗 抗癌药物的发现。 好了！