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. !

项目摘要 KRAS是人类癌症中最常突变的癌基因。KRAS作为一个 一种分子开关，仅当与细胞内的 膜。KRAS作为法尼基化的结果与膜结合， 与多元C-末端结合起作用。通过封锁来击败KRAS的努力 法尼基化失败是因为能够使KRAS异戊烯化的替代酶。我们 因此采取了一种公正的方法来鉴定以前未被识别的基因， 参与KRAS的膜结合。我们设计了一个双荧光素酶检测 该研究报告了KRAS对膜的亲和力丧失，并在全基因组范围内使用该测定法。 siRNA筛选。在这13个基因中，我们惊讶地发现了一个G蛋白 偶联受体（GPCR），称为GPR 31，其是12-（S）- HETE并已显示刺激MAPK信号传导。GPR 31是一个待研究项目 GPCR被纳入Illuminating the Druggable Genome项目。我们 还惊讶地发现，通过免疫共沉淀，GPR 31 提示GPR 31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GRP 31和KRAS共定位于内膜和PM， 用siRNA沉默GPR 31抑制KRAS依赖性细胞增殖， 巨胞饮作用我们现在提出确定12-（S）-HETE信号传导是否通过 GPR 31通过三个特定目的调节KRAS运输和信号传导。目标1. GPR 31与12-（S）-HETE的连接和GPR 31与KRAS的相互作用。我们将 通过免疫共沉淀和FRET检测GPR 31和KRAS之间的相互作用 ± 12-（S）-HETE或对照类花生酸。目标二。GPR 31与12-（S）-HETE的连接和 贩运KRAS。我们将研究KRAS从内膜到PM的运输， 活细胞成像± 12-（S）-HETE。目标3。GPR 31与12-（S）-HETE和KRAS的连接 activation.我们将研究KRAS信号± 12-（S）-HETE。实现这些目标 将确定GPR 31信号传导是否调节KRAS，从而优先考虑GPR 31用于抗- 癌症药物发现 !