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是一个未被研究的
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2. 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
!
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
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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