Regulation of KRAS Trafficking and Signaling by GPR31
GPR31 对 KRAS 贩运和信号传输的监管
基本信息
- 批准号:10047185
- 负责人:
- 金额:$ 16.95万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AffinityAntineoplastic AgentsBiological AssayBiologyCell ProliferationCell membraneCellsCellular MembraneCellular biologyClinicCo-ImmunoprecipitationsDataDifferentiation and GrowthDrug TargetingEicosanoidsEndoplasmic ReticulumEnzymesFamilyFarnesyl Transferase InhibitorFluorescence Resonance Energy TransferG-Protein-Coupled ReceptorsGTP-Binding Protein alpha SubunitsGTP-Binding Protein alpha Subunits, GsGenesGenomeGoalsGrowthGuanosine TriphosphateHumanHydroxyeicosatetraenoic AcidsJournalsKRAS2 geneLigationLuciferasesMAP Kinase GeneMalignant NeoplasmsMeasuresMembraneMembrane ProteinsMolecularMolecular ChaperonesMolecular ConformationMonomeric GTP-Binding ProteinsMutateMutationOncogenesOncogenicPathway interactionsPeripheralPharmaceutical PreparationsPost-Translational Protein ProcessingProteinsPublishingRNA InterferenceRegulationReportingRhodopsinRoleScreening ResultSeriesSignal PathwaySignal TransductionSmall Interfering RNASubgroupTestingbasecancer therapyconfocal imagingdesigndrug discoveryexperiencefarnesylationgenome-wideinterestlive cell imagingneoplastic cellnovelprenylationprotein protein interactionreceptorresponsetrafficking
项目摘要
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!
项目成果
期刊论文数量(0)
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会议论文数量(0)
专利数量(0)
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{{ truncateString('MARK Reid PHILIPS', 18)}}的其他基金
FASEB SRC: Structure and Function of Small GTPases
FASEB SRC:小 GTP 酶的结构和功能
- 批准号:
10463260 - 财政年份:2022
- 资助金额:
$ 16.95万 - 项目类别:
Differential function and tumor vulnerabilities revealed by RAS membrane trafficking
RAS 膜运输揭示的差异功能和肿瘤脆弱性
- 批准号:
10468873 - 财政年份:2020
- 资助金额:
$ 16.95万 - 项目类别:
Differential function and tumor vulnerabilities revealed by RAS membrane trafficking
RAS 膜运输揭示的差异功能和肿瘤脆弱性
- 批准号:
10688011 - 财政年份:2020
- 资助金额:
$ 16.95万 - 项目类别:
Differential function and tumor vulnerabilities revealed by RAS membrane trafficking
RAS 膜运输揭示的差异功能和肿瘤脆弱性
- 批准号:
10237382 - 财政年份:2020
- 资助金额:
$ 16.95万 - 项目类别:
Differential function and tumor vulnerabilities revealed by RAS membrane trafficking
RAS 膜运输揭示的差异功能和肿瘤脆弱性
- 批准号:
10053541 - 财政年份:2020
- 资助金额:
$ 16.95万 - 项目类别:
Role of nonsense mediated RNA decay in pancreatic cancer
无义介导的RNA衰变在胰腺癌中的作用
- 批准号:
10229380 - 财政年份:2018
- 资助金额:
$ 16.95万 - 项目类别:
Role of nonsense mediated RNA decay in pancreatic cancer
无义介导的RNA衰变在胰腺癌中的作用
- 批准号:
9447641 - 财政年份:2018
- 资助金额:
$ 16.95万 - 项目类别:
Role of nonsense mediated RNA decay in pancreatic cancer
无义介导的RNA衰变在胰腺癌中的作用
- 批准号:
10410447 - 财政年份:2018
- 资助金额:
$ 16.95万 - 项目类别:
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