摘要： MP29-06

关键词： M3 mAChR   IQGAP1   Rac1   VE-cadherin   beta-catenin  

摘要： The objective was to investigate whether M-3 muscarinic acetylcholine receptor (mAChR) dysfunction disrupts the linkage between the vascular endothelial (VE)-cadherin in the adherens junctional complex and the actin-based cytoskeleton, increasing vascular permeability in atherosclerosis. Western blotting revealed that a selective M-3 receptor antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), and M-3 receptor siRNA decrease VE-cadherin and beta-catenin in Triton X-100-insoluble fractions, indicating that M-3 receptor inhibition weakens the linkage between the VE-cadherin/beta-catenin complex and the actin cytoskeleton. Co-immunoprecipitation assays showed that M-3 receptor inhibition reduces Rac1 activity and the association of IQ motif-containing GTPase-activating protein 1 (IQGAP1) with Ras-related C3 botulinum toxin substrate 1 (Rac1), while increasing the interaction between IQGAP1 and beta-catenin. Using IQGAP1 siRNA, we found that IQGAP1 is required for stable interaction between VE-cadherin/beta-catenin and the actin cytoskeleton in quiescent endothelial cells;IQGAP1 siRNA augments the M-3 receptor inhibitioninduced dissociation between them. Moreover, S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide (NO) donor, attenuates this disassociation and Rac1 activity inhibition. The M-3 receptor facilitates interaction of the VE-cadherin-based adherens junctional complex and the actin-based cytoskeleton by maintaining Rac1 activity, which regulates the interaction between IQGAP1/Rac1 and IQGAP1/beta-catenin, and may contribute to endothelial barrier function under physiological conditions.

关键词： Cell Signaling   Estrogen   Estrogen Receptor   Nuclear Receptors   Protein-Protein Interactions   Scaffold Proteins   Transcription   IQGAP1  

摘要： Background: IQGAP1 is a scaffold protein that modulates diverse signaling pathways. Results: IQGAP1 binds to estrogen receptor- (ER) and ER, and knockdown of IQGAP1 impairs 17-estradiol (E-2)-stimulated transcriptional activation in human breast epithelial cells. Conclusion: IQGAP1 modulates ER function and is required for maximal E-2 function. Significance: IQGAP1 might be a therapeutic target for patients with breast carcinoma. The estrogen receptor (ER) is a steroid hormone receptor that acts as a transcription factor, modulating genes that regulate a vast range of cellular functions. IQGAP1 interacts with several signaling proteins, cytoskeletal components, and transmembrane receptors, thereby serving as a scaffold to integrate signaling pathways. Both ER and IQGAP1 contribute to breast cancer. In this study, we report that IQGAP1 binds ER and ER. In vitro analysis with pure proteins revealed a direct interaction between IQGAP1 and ER. Investigation with multiple short fragments of each protein showed that ER binds to the IQ domain of IQGAP1, whereas the hinge region of ER is responsible for binding IQGAP1. In addition, IQGAP1 and ER co-immunoprecipitated from cells, and the association was modulated by estradiol. The interaction has functional effects. Knockdown of endogenous IQGAP1 attenuated the ability of estradiol to induce transcription of the estrogen-responsive genes pS2, progesterone receptor, and cyclin D1. These data reveal that IQGAP1 binds to ER and modulates its transcriptional function, suggesting that IQGAP1 might be a target for therapy in patients with breast carcinoma.

关键词： 足细胞   细胞凋亡   血管紧张素Ⅱ   ras   GTP酶激活蛋白质类   细胞外   调节蛋白激酶1   2  

摘要： 目的研究IQ结构域GTP酶活化蛋白1（IQdomainGTPase-activatingprotein1，IQGAPl）在血管紧张素Ⅱ（Angll）诱导足细胞凋亡中的作用以及可能的分子机制。方法体外培养条件永生性小鼠足细胞（MPC），以不同浓度（0、10-12、10-10、10-8、10-6mol／L）AngⅡ处理MPC或10-8mol／LAngII刺激不同时间（0、1、3、6、12、24h），采用流式细胞术检测细胞凋亡率，免疫荧光、Western印迹法检测IQGAPl的表达及分布。IQGAPlsiRNA转染以及丝裂原活化蛋白激酶（MAPK）通路（包含p38、JNK和ERK1／23条主要通路）抑制剂SB202190（10μmol／L）、SP600125（25μmol／L）或U0126（10μmol／L）预处理MPC后，分别检测MAPK信号蛋白磷酸化水平及细胞凋亡率，并采用免疫共沉淀法研究IQGAPl与ERKl／2结合水平的变化。结果（1）AngⅡ可以诱导足细胞凋亡，且凋亡率随着刺激时间和刺激浓度的增加而进一步增加。（2）正常足细胞IQGAPl主要分布于细胞膜和胞质，随AngⅡ刺激浓度和刺激时间的增加，胞膜和胞质IQGAPl表达逐渐增高，且随剂量和时间增高。（3）SB202190、SP600125或U0126分别预处理足细胞，可显著降低Ang11诱导的足细胞凋亡（P〈0．05），但不影响IQGAPl蛋白表达。（4）IQGAPlsiRNA转染足细胞显著下调ERKl／2磷酸化水平（P〈0．05），降低IQGAPl与ERKl／2结合量（P〈0．05），并抑制AnglI诱导的细胞凋亡（P〈0．05），但对于p38以及JNK通路无显著影响。结论IQGAPl通过ERKl／2MAPK信号通路介导AngⅡ诱导的足细胞凋亡。

关键词： IQ motif containing GTPase activating   protein 1   K-Ras   Pancreatic cancer  

摘要： K-Ras is frequently mutated and activated especially in pancreatic cancers. To analyze K-Ras function, we have searched for K-Ras interacting proteins and found IQ motif containing GTPase activating protein 1 (IQGAP1) as a novel K-Ras binding protein. IQGAP1 has been known as a scaffold protein for B-Raf, MEK1/2 and ERK1/2. Here we showed that IQGAP1 selectively formed a complex with K-Ras but not with H-Ras, and recruited B-Raf to K-Ras. We found that IQ motif region of IQGAP1 interacted with K-Ras. Both active and inactive K-Ras interacted with IQGAPI, and effector domain mutants of K-Ras also associated with IQGAP1, indicating that IQGAP1 interacts with K-Ras irrespective of Ras-effectors like B-Raf. We also found that overexpression or knock-down of IQGAPI affected the interaction between K-Ras and B-Raf, and IQGAP1 overexpression increased ERK1/2 phosphorylation in K-Ras dependent manner in PANC1 cells. Our data suggest that IQGAP1 has a novel mechanism to modulate K-Ras pathway. (C) 2014 Elsevier Inc. All rights reserved.

关键词： IQGAP1   小干扰RNA   转移   非小细胞肺癌  

摘要： 目的:探究IQGAP1对非小细胞肺癌细胞株PC14/B生物学行为的影响。方法:构建干扰IQGAP1的质粒,转染至PC14/B细胞株,体外增殖、迁移、侵袭实验观察下调IQGAP1表达后对非小细胞肺癌细胞株PC14/B生物学行为影响。结果:成功构建IQGAP1表达下调的细胞株RNAi-PC14/B。与转染空载体的细胞株相比,下调IQGAP1的表达能够抑制PC14/B细胞增殖、迁移和侵袭能力。结论:IQGAP1表达可能与肺癌转移相关。

关键词： IQGAP   Repeats   ezrin   FERM domain   ITC  

摘要： Mammalian IQGAP proteins all feature multiple similar to 50 amino acid sequence repeats near their N-termini, and little is known about the function of these "Repeats". We have expressed and purified the Repeats from human IQGAP1 to identify binding partners. We used mass spectrometry to identify 42 mouse kidney proteins that associate with the IQGAP1 Repeats including the ERM proteins ezrin, radixin, and moesin. ERM proteins have an N-terminal FERM domain (4.1, ezrin, radixin, moesin) through which they bind to protein targets and phosphatidylinositol 4,5-bisphosphate (PIP2) and a C-terminal actin-binding domain and function to link the actin cytoskeleton to distinct locations on the cell cortex. Isothermal titration calorimetry (ITC) reveals that the IQGAP1 Repeats directly bind to the ezrin FEW domain, while no binding is seen for full-length "autoinhibited" ezrin or a version of full-length ezrin intended to mimic the activated protein. ITC also indicates that the ezrin FERM domain binds to the Repeats from IQGAP2 but not the Repeats from IQGAP3. We conclude that IQGAP1 and IQGAP2 are positioned at the cell cortex by ERM proteins. We propose that the IQGAP3 Repeats may likewise bind to FERM domains for signaling purposes.

关键词： hoof disorder   sole hemorrhage   association study   IQ motif-containing GTPase-activating protein 1 (IQGAP1)  

摘要： Feet and leg problems have a major effect on the well-being and lifespan of the dairy cow and thus are economically important to the dairy farmer. Apart from approaches using genetic selection for classical traits from conformation scoring, attempts for genetic improvement can be based either on records of individual disease cases or on records of disorder status at time of hoof trimming. In this study, 1,962 first-lactation cows were subjected to hoof trimming with an assessment of disorder status for sole hemorrhage as a binary trait. Cows were from 7 large commercial herds in Mecklenburg-Western Pomerania (northeastern Germany) that had similar housing with cubicles, slatted flooring, little use of straw for bedding, and total mixed ration feeding. Cows were trimmed and assessed once, focusing on cows in the first half of the lactation. Herds were visited at intervals to enable recording of cohorts at a similar stage of lactation. Each cohort or herd-visit included between 31 and 165 cows. Additional measurements included body weight, back fat thickness, and body condition at time of trimming. Further data on dairy production, conformation scores, and reproductive performance were merged after collection of records had finished. The DNA extracted from blood of 1,183 cows was used for analysis with a custom-made array of 384 single nucleotide polymorphisms (SNP). The SNP were selected according to results from the literature for effects in classical conformation traits, from biochemical pathway analysis, and from comparative analysis of putative candidate genes in cattle, pigs, and sheep. Selection of cohorts of cows for SNP chip analysis was such that cohorts with extreme frequencies of disorders and cohorts with slightly deviating housing systems were excluded in this first step. The results from a mixed threshold model analysis with genotype included as a fixed effect and accounting for relationships among animals revealed that the intronic SNP rs29017173 (A/G)

关键词： IQGAP1   卵巢癌   肿瘤干细胞   分化   侵袭  

摘要： 目的： 探索支架蛋白IQGAP1在卵巢癌肿瘤干细胞的表达,并观察IQGAP1对卵巢癌肿瘤干细胞分化过程中的侵袭能力的影响。 方法： 以浆液性卵巢癌3AO细胞株为亲本细胞,无血清悬浮培养法获得肿瘤干细胞。采用流式细胞术验证肿瘤干细胞的标记分子CD24表达,利用细胞免疫荧光实验观察干性分子OCT4和SOX2的定位。利用含10%胎牛血清培养基诱导其分化,期间光镜下观察细胞形态变化。 qRT-PCR法和Western-blot法分别检测IQGAP1mRNA和蛋白的表达水平变化。通过划痕实验和Transwell小室观察细胞迁移侵袭能力;采用wst-1法检测细胞增殖能力。 结果： 1.利用3AO细胞株通过无血清培养法获得表型CD24(-)表型的肿瘤干细胞。 2.相比3AO细胞株中,IQGAP1在肿瘤干细胞中呈低表达。 3.在3AO细胞株中,沉默IQGAP1后减弱了肿瘤迁移、侵袭能力,而不会改变细胞的增殖活性。 4.在卵巢肿瘤干细胞分化过程中,OCT4和SOX2表达下降,而IQGAP1表达增加;同时肿瘤细胞的侵袭能力增强。 5.在卵巢肿瘤干细胞分化过程中,IQGAP1沉默后细胞侵袭能力减弱。 结论： 1.无血清培养法是获得肿瘤干细胞比较成熟简便的实验技术。 2.在肿瘤干细胞分化过程中其侵袭能力逐步增强,且IQGAP1可能参与了该变化。

摘要： IQGAP1 interacts with numerous binding partners through a calponin homology domain (CHD), a WW motif, IQ repeats, a Ras GAP related domain (GRD), and a conserved C-terminal (CT) domain. Among various biological and cellular functions, IQGAP1 plays a role in cell-matrix interactions and actin cytoskeleton dynamics in membrane ruffling and lamellipodium protrusion. Phosphorylation in the CT domain regulates intramolecular interaction and IQGAP1 cellular activity. In a recent study, we discovered that IQGAP1 surprisingly localizes to actively retracting edges, instead of protruding areas, in B16F10 mouse melanoma cells and some other cells types. In these current studies we examined localization of IQGAP1 mutants to retracting versus protruding areas in phorbol ester-stimulated B16F10 cells. Cells were co-transfected with GFP-IQGAP1 full length (GFP-IQGAP1-FL), as an internal control, and one of five Myc-tagged IQGAP1 constructs (FL, CA, DeltaCHD, DeltaGRD or DeltaCT). The cotransfected cells were plated onto laminin for 30 minutes, stained with anti-Myc and anti-WAVE2 antibodies, and normalized fluorescence measurements were made in retracting and protruding areas. Retracting cell areas were defined as GFP-IQGAP1-FL positive and WAVE2 negative, while protruding cell areas were defined as GFP-IQGAP1-FL negative and WAVE2 positive. In retracting areas there were large decreases in both DeltaGRD and DeltaCT localization, a slight decrease in DeltaCHD localization, and normal localization of the CA mutant. In areas of cell protrusion there were large increases in both DeltaGRD and DeltaCT localization, and normal localization of DeltaCHD and CA mutants. These results indicate that two domains, GRD and CT, are essential for normal localization of IQGAP1 to retracting cell areas. Furthermore, our results suggest a model in which IQGAP1 in the areas of cell retraction is in the open, phosphorylated, conformation. Additionally we investigated the knockdown of IQGAP1 in B16F10