共查询到20条相似文献,搜索用时 31 毫秒
1.
2.
Nicole E. Brown Devrishi Goswami Mary Rose Branch Suneela Ramineni Eric A. Ortlund Patrick R. Griffin John R. Hepler 《The Journal of biological chemistry》2015,290(14):9037-9049
RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gαi/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gαi1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gαi1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gαo-AlF4−. Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gαi1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gαo-AlF4− and an AlF4−-insensitive mutant (G42R) of Gαi1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gαi1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gαo-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity. 相似文献
3.
Berlin S Tsemakhovich VA Castel R Ivanina T Dessauer CW Keren-Raifman T Dascal N 《The Journal of biological chemistry》2011,286(38):33223-33235
G protein-activated K(+) channels (Kir3 or GIRK) are activated by direct interaction with Gβγ. Gα is essential for specific signaling and regulates basal activity of GIRK (I(basal)) and kinetics of the response elicited by activation by G protein-coupled receptors (I(evoked)). These regulations are believed to occur within a GIRK-Gα-Gβγ signaling complex. Fluorescent energy resonance transfer (FRET) studies showed strong GIRK-Gβγ interactions but yielded controversial results regarding the GIRK-Gα(i/o) interaction. We investigated the mechanisms of regulation of GIRK by Gα(i/o) using wild-type Gα(i3) (Gα(i3)WT) and Gα(i3) labeled at three different positions with fluorescent proteins, CFP or YFP (xFP). Gα(i3)xFP proteins bound the cytosolic domain of GIRK1 and interacted with Gβγ in a guanine nucleotide-dependent manner. However, only an N-terminally labeled, myristoylated Gα(i3)xFP (Gα(i3)NT) closely mimicked all aspects of Gα(i3)WT regulation except for a weaker regulation of I(basal). Gα(i3) labeled with YFP within the Gα helical domain preserved regulation of I(basal) but failed to restore fast I(evoked). Titrated expression of Gα(i3)NT and Gα(i3)WT confirmed that regulation of I(basal) and of the kinetics of I(evoked) of GIRK1/2 are independent functions of Gα(i). FRET and direct biochemical measurements indicated much stronger interaction between GIRK1 and Gβγ than between GIRK1 and Gα(i3). Thus, Gα(i/o)βγ heterotrimer may be attached to GIRK primarily via Gβγ within the signaling complex. Our findings support the notion that Gα(i/o) actively regulates GIRK. Although regulation of I(basal) is a function of Gα(i)(GDP), our new findings indicate that regulation of kinetics of I(evoked) is mediated by Gα(i)(GTP). 相似文献
4.
5.
《武汉生物工程学院学报》2017,(3)
正按照最新标准,专著是指以单行本或多卷册(在限定的期限内出齐)形式出版的印刷型或非印刷型出版物,包括普通图书、古籍、学位论文、会议文集、汇编、报告、多卷书、丛书等。汇编类的参考文献应该按照专著的著录格式书写:主要责任者.题名:其他题名[G].其他责任者.版本项.出版地:出版者,出版年:引文页码[引用日期].获取和访问路径.数字对象唯一标识符. 相似文献
6.
<正>IgG亚类之间在免疫化学和功能上是互不相同的,某种特异性亚类单一缺失不一定与某种疾病有关,通常用敏感的放射免疫分析法测定出血清IgG_4的缺损即使不是全部也常与严重的绿脓杆菌感染复发有关,一般指呼吸道疾病。IgG_2与IgG_4缺陷趋向于同时出现,通常与免疫球蛋白A(lgA)和免疫球蚕白E(lgE)缺陷有关。在某些方面上IsG_1的特性和IsG_3相似,而IgG_2似乎与IgG_4相似,某种IgG亚类缺陷病人,似乎 相似文献
7.
8.
The mammalian fauna from Gülpinar (Canakkale, Turkey), dated Turolian, comprises threeHipparion taxa:H. cf.matthewi, H. sp. medium-sized, andH. sp. large. Two metatarsals may represent a fourth taxon or may derive from a different locality. The hipparion material is described here. 相似文献
9.
Martens-Lobenhoffer J Rodionov RN Drust A Bode-Böger SM 《Analytical biochemistry》2011,419(2):234-240
Nitric oxide is an ubiquitary cell signaling substance. Its enzymatic production rate by nitric oxide synthase is regulated by the concentrations of the substrate L-arginine and the competitive inhibitor asymmetric dimethylarginine (ADMA). A newly recognized elimination pathway for ADMA is the transamination to α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid (DMGV) by the enzyme alanine-glyoxylate aminotransferase 2 (AGXT2). This pathway has been proven to be relevant for nitric oxide regulation, but up to now no method exists for the determination of DMGV in biological fluids. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of DMGV. D(6)-DMGV was used as internal standard. Samples were purified online by column switching, and separation was achieved on a porous graphitic carbon column. The calibration was linear over ranges of 10 to 200 nmol/L for plasma and 0.1 to 20 μmol/L for urine. The intra- and interday accuracies and precisions in plasma and urine were better than 10%. In plasma samples, DMGV was present in concentrations between 19.1 and 77.5 nmol/L. In urine samples, concentrations between 0.0114 and 1.03 μmol/mmol creatinine were found. This method can be used as a tool for the scientific investigation of the ADMA conversion to DMGV via the enzyme AGXT2. 相似文献
10.
Styrax japonica Siebold et al Zuccarini (SJSZ) has been used to heal inflammation and bronchitis as folk medicine in Korea. Firstly, glycoprotein isolated from SJSZ (SJSZ glycoprotein) has a molecular weight with 38 kDa and consists of carbohydrate (57.64%) and protein (42.35%). In the composition of SJSZ glycoprotein, carbohydrate mostly consists of glucose (28.17%), galactose (21.85%), and mannose (2.62%) out of 52.64%, respectively. The protein consists of Trp (W, 7.01%), Pro (P, 6.72%), and Ile (I, 5.42%) out of 42.35% as three major amino acids, while total amount of other amino acids is 23.20%. The purpose of this study is to know whether the SJSZ glycoprotein (38 kDa) induces the cell cycle arrest and apoptosis in HepG2 cells. Cytotoxicity was evaluated using MTT and lactate dehydrogenase assay and amount of intracellular reactive oxygen species (iROS) and nitric oxide (NO) was measured using fluorescence microplate reader. Activities of cell cycle-related proteins [p53, p21, p27, Cyclin D1, and cyclin-dependent kinase (CDK)4] and apoptosis-related factors [iNOS, Bid, Bcl-2/bax, cytochrome c, caspase-9, caspase-3, and poly-(ADP-ribose) polymerase (PARP)] were assessed by Western blot and fluorescence-activated cell sorter (FACS) analysis. In the cell cycle-related proteins, SJSZ glycoprotein (50 μg/ml) significantly enhances the expression of p53, p21, and p27, whereas it suppressed the activity of cyclin D1/CDK4. In the apoptosis-related factors, SJSZ glycoprotein (50 μg/ml) stimulates to increase iROS, and NO, to activate iNOS, Bid, Bcl-2/bax, cytochrome c, caspase-9, caspase-3, and PARP. SJSZ glycoprotein (50 μg/ml) has potent effect to arrest cell cycle from G(0) /G(1) to S and to induce apoptosis in HepG2 cells. 相似文献
11.
Rebois RV Maki K Meeks JA Fishman PH Hébert TE Northup JK 《Cellular signalling》2012,24(11):2051-2060
β-Adrenergic receptors (βAR) and D(2)-like dopamine receptors (which include D(2)-, D(3)- and D(4)-dopamine receptors) activate G(s) and G(i), the stimulatory and inhibitory heterotrimeric G proteins, respectively, which in turn regulate the activity of adenylyl cyclase (AC). β(2)-Adrenergic receptors (β(2)AR) and D(4)-dopamine receptors (D(4)DR) co-immunoprecipitated when co-expressed in HEK 293 cells, suggesting the existence of a signaling complex containing both receptors. In order to determine if these receptors are closely associated with each other, and with other components involved in G protein-mediated signal transduction, β(2)AR, D(4)DR, G protein subunits (Gα(i1) and the Gβ(1)γ(2) heterodimer) and AC were tagged so that bioluminescence resonance energy transfer (BRET) could be used to monitor their interactions. All of the tagged proteins retained biological function. For the first time, FlAsH-labeled proteins were used in BRET experiments as fluorescent acceptors for the energy transferred from Renilla luciferase-tagged donor proteins. Our experiments revealed that β(2)AR, D(4)DR, G proteins and AC were closely associated in a functional signaling complex in cellulo. Furthermore, BRET experiments indicated that although activation of G(i) caused a conformational change within the heterotrimeric protein, it did not cause the Gβγ heterodimer to dissociate from the Gα(i1) subunit. Evidence for the presence of a signaling complex in vivo was obtained by purifying βAR from detergent extracts of mouse brain with alprenolol-Sepharose and showing that the precipitate also contained both D(2)-like dopamine receptors and AC. 相似文献
12.
13.
14.
应用SDS-聚丙烯酰胺电泳可以从小麦丛矮病毒中分离出5种结构蛋白。经过碘酸—Schiff′s试剂染色证明,其中分子量为66K的是糖蛋白,是组成病毒外膜突起的G蛋白。应用不同的植物凝集素对完整病毒进行凝集反应试验证实,只有ConA凝集素对病毒有凝集作用,葡萄糖有抑止凝集的作用。G蛋白氨基酸组成分析证明,酸性氨基酸的含量较高。用同位素~(125)I标记的G蛋白和N蛋白的双向图谱表明,植物弹状病毒的结构蛋白之间无共同的肽段。说明植物弹状病毒和动物弹状病毒一样,其蛋白也是由同一病毒基因组的不同片段转录和翻译产生的。 相似文献
15.
Ripley's指数的一个新变形——G(d) 总被引:2,自引:1,他引:1
在对Ripley's指数的物理背景进行分析的基础上提出了一个新的变形--G(d)指数.原始Ripley's指数K(d)是一个半径为d(d为尺度)的圆的面积估计量,是有量纲的,且K(d)随着d的增大迅速增大,应用起来不太方便.Ripley's指数的变形L(d)将K(d)换算成d的估计量,再减去d,使得L(d)在随机分布的假定下有数学期望0,这使得L(d)的应用比K(d)要方便.但L(d)还是一个具有长度单位的量,其上下包迹线呈明显的喇叭形状,对于应用还不是十分方便.提出的G(d)指数是一个比值,无量纲,数学期望也是0.文章给出了4个例子,这些例子说明,它保持了L(d)指数区分分布类型的能力,同时具有稳定这个良好特性,到了一定尺度以后,上下包迹线趋向常数.进一步分析得知,这些常数与单位面积的个体密度相关,呈对数关系,其相关系数r2达到0.9左右.这样在实际应用中,包迹线只要模拟到稳定点即可,余下部分可通过回归公式计算,从而节省计算工作量. 相似文献
16.
目的:探讨葡萄糖转运体9(GLUT9)基因启动子区的rs13124007(C/G)及rs6850166(A/G)位点的单核苷酸多态性(SNP)与中国汉族女性人群痛风易感性之间的相关性.方法:选取185例痛风患者和300例正常对照者,提取基因组DNA,采用聚合酶链式反应(PCR技术),特异性扩增GLUT9基因所需要的目的片段,对扩增的目的片段进行测序后,比较痛风组和正常对照组的基因型频率及等位基因频率分布情况.结果:女性痛风组中GLUT9基因的启动子区rs13124007和rs6805116两个位点的基因型频率分布与正常对照组相比,统计学上无明显的差异(X2=0.906,P=0.636;X2=3.335,P=0.189),rs13124007 SNP位点的C等位基因频率和rs6850166SNP位点的A的等位基因频率与正常对照组相比也无明显的统计学差异(X2=0.506,P=0.477;X=3.268,P=0.071).结论:葡萄糖转运体9(GLUT9)基因启动子区的rs 13124007(C/G)及rs6850166(A/G)位点的单核苷酸多态性(SNP)与中国汉族女性人群痛风易感性无明显的相关性. 相似文献
17.
Shai Berlin Tal Keren-Raifman Ruth Castel Moran Rubinstein Carmen W. Dessauer Tatiana Ivanina Nathan Dascal 《The Journal of biological chemistry》2010,285(9):6179-6185
Stable complexes among G proteins and effectors are an emerging concept in cell signaling. The prototypical Gβγ effector G protein-activated K+ channel (GIRK; Kir3) physically interacts with Gβγ but also with Gαi/o. Whether and how Gαi/o subunits regulate GIRK in vivo is unclear. We studied triple interactions among GIRK subunits 1 and 2, Gαi3 and Gβγ. We used in vitro protein interaction assays and in vivo intramolecular Förster resonance energy transfer (i-FRET) between fluorophores attached to N and C termini of either GIRK1 or GIRK2 subunit. We demonstrate, for the first time, that Gβγ and Gαi3 distinctly and interdependently alter the conformational states of the heterotetrameric GIRK1/2 channel. Biochemical experiments show that Gβγ greatly enhances the binding of GIRK1 subunit to Gαi3GDP and, unexpectedly, to Gαi3GTP. i-FRET showed that both Gαi3 and Gβγ induced distinct conformational changes in GIRK1 and GIRK2. Moreover, GIRK1 and GIRK2 subunits assumed unique, distinct conformations when coexpressed with a “constitutively active” Gαi3 mutant and Gβγ together. These conformations differ from those assumed by GIRK1 or GIRK2 after separate coexpression of either Gαi3 or Gβγ. Both biochemical and i-FRET data suggest that GIRK acts as the nucleator of the GIRK-Gα-Gβγ signaling complex and mediates allosteric interactions between GαiGTP and Gβγ. Our findings imply that Gαi/o and the Gαiβγ heterotrimer can regulate a Gβγ effector both before and after activation by neurotransmitters. 相似文献
18.
Vošahlíková M Svoboda P 《Physiological research / Academia Scientiarum Bohemoslovaca》2011,60(3):541-547
The effect of monovalent cations on trimeric G protein G(i)1α was measured at equimolar concentration of chloride anion in pertussis-toxin (PTX)-treated HEK293 cells stably expressing PTX-insensitive DOR- G(i)1α (Cys(351)-Ile(351)) fusion protein by high-affinity [(35)S]GTPgammaS binding assay. The high basal level of binding was detected in absence of DOR agonist and monovalent ions and this high level was inhibited with the order of: Na(+) > K(+) > Li(+). The first significant inhibition was detected at 1 mM NaCl. The inhibition by monovalent ions was reversed by increasing concentrations of DOR agonist DADLE. The maximum DADLE response was also highest for sodium and decreased in the order of: Na(+) > K(+) ~ Li(+). Our data indicate i) an inherently high activity of trimeric G protein G(i)1α when expressed within DOR- G(i)1α fusion protein and determined in the absence of monovalent cations, ii) preferential sensitivity of DOR- G(i)1alpha to sodium as far as maximum of agonist response is involved. 相似文献
19.
G protein-gated inwardly rectifying potassium channel (GIRK) plays a crucial role in regulating heart rate and neuronal excitability. The gating of GIRK is regulated by the association and dissociation of G protein βγ subunits (Gβγ), which are released from pertussis toxin-sensitive G protein α subunit (Gα(i/o)) upon GPCR activation in vivo. Several lines of evidence indicate that Gα(i/o) also interacts directly with GIRK, playing functional roles in the signaling efficiency and the modulation of the channel activity. However, the underlying mechanism for GIRK regulation by Gα(i/o) remains to be elucidated. Here, we performed NMR analyses of the interaction between the cytoplasmic region of GIRK1 and Gα(i3) in the GTP-bound state. The NMR spectral changes of Gα upon the addition of GIRK as well as the transferred cross-saturation (TCS) results indicated their direct binding mode, where the K(d) value was estimated as ~1 mm. The TCS experiments identified the direct binding sites on Gα and GIRK as the α2/α3 helices on the GTPase domain of Gα and the αA helix of GIRK. In addition, the TCS and paramagnetic relaxation enhancement results suggested that the helical domain of Gα transiently interacts with the αA helix of GIRK. Based on these results, we built a docking model of Gα and GIRK, suggesting the molecular basis for efficient GIRK deactivation by Gα(i/o). 相似文献