Name: rhoc gtpase activation assay
Uploaded: 2010-08-22T17:00:00.000+00:00
Duration: 9 min 58 s
Description: Watch this Scientific Journal Video about RhoC GTPase Activation Assay at JoVE.com

防衛W81XWH - 05 - 1 - 0005、W81XWH - 06 - 1から0495まで、W81XWH - 08 - 1から0029とW81XWH - 08 - 1から0356までの部

1。 GST -融合タンパク質を準備<ol><li> BamH1/EcoR1 pGEX3xベクターにサブクローニングrhotekinのRho結合ドメイン（RBD）の最初のN末端90アミノ酸を含むJM109コンピテント細菌のグリセロールストックが行われます。 高効率を確保するために、次の手順は、各実験のために実行しなければなりません。我々の経験では、新しく調製したGST - RBDは、堅牢で正確な分子量GTPase活性化アッセイの鍵となります。 </li><li> LBアンプ50mlにグリセロールストックのμL50（おおよそ、解凍しないで）追加し、振盪しながら37℃で一晩を成長させる。 </li><li> 500mLのLBアンプで1:10に希釈し、1時間拡大</li><li> 2時間のためにIPTGを0.1mMのでGST -タンパク質の産生を誘導する。 </li><li> 5000に遠心ボトルや遠心分離機、Sorval GSA3ローターに均等に配布するRPM 4℃で20分間。 </li><li>遠心ボトルにペレットを再懸濁します、溶解緩衝液10ml * *細菌溶解バッファー：20％ショ糖、10％グリセロール、50mMのトリスpH8.0、0.2mMののNa 2 S 2 O 5、2mMのMgCl 2、2mMのDTTは、新鮮なPMSF、ベンズアミド、アプロチニンおよびロイペプチンを追加。 </li><li> 2〜3分間（これは超音波処理器の種類によって異なります。私たちは、マーク6で設定されたフィッシャーソニックDismembranatorモデル500、50％のサイクルを使用する）超音波洗浄します。 </li><li>遠心分離機、ソーバルSS34、4℃で20分10,000回転℃、この時点で小アリコートを取り出すことができるとSDS - PAGEによって実行されます。ゲルはクマシーで染​​色することができますし、バンドは46 kDaの明らかなはずである。 </li><li>上清を除去し、50％glutatione -セファロース4Bスラリーの1 mLを加える。 </li><li> 4℃で30分間インキュベート° Cを回転した。溶解緩衝液で3回洗浄する。 </li><li> GST -魚のバッファを使用して50％スラリー（約1ml）にGST-RBD/glutatione-Sepharoseビーズを再懸濁します** ** GST -魚のバッファ：10％グリセロール、50mMのトリスpH7.4の、100mMのNaCl、1％NP - 4、2mMのMgCl 2、新鮮なPMSF、ベンズアミド、アプロチニンおよびロイペプチンを追加する</li></ol> 2。 GST -融合は、アッセイをプルダウン<ol><li> 5〜10 × 10 6細胞/アッセイを使用して、 </li><li>一度、氷冷PBSで細胞を洗浄、氷上にプレートを保つ（我々が氷で満たされた平らなガラス皿を使用）と0.5mLのGST -魚のバッファーで溶解する。 </li><li>氷上で5分間インキュベートする、ラバーポリスマンやフラットスクラッパーを使用して、収穫の細胞は、チューブを遠心し、4℃で20,000 rpmの5分を遠心分離して譲渡℃に</li><li>新鮮なマイクロ遠心チューブに上清を移す。合計ローを（すなわちGDP + GTP結合）を決定するために50μLを取る。 </li><li> 0.5mLのGST-RBD/glutatione-Sepharoseビーズを追加し、4℃で回転で一晩℃で、インキュベートする。 </li><li> GST -魚のバッファ付き6X洗います。 </li><li> 40μL2X - Laemelliサンプルバッファーで再懸濁し、ビーズ この時点で2.4にかかった合計Rhoタンパク質のアリコートの濃度を測定し、30μgのは、対応するアクティベーションサンプルでゲル上で実行されるようにしておく必要があります。 </li><li>沸騰サンプルは90℃で5分間、軽く遠心し、上清（すなわち、ビーズを避けるために）負荷がかかると12ウェル4-20％グラジエントSDS - PAGEゲル上で実行されます。 </li><li>ないより高い150よりボルト、Towbinバッファーを用いてメンブレンにトランスファーし、100 Wの定電流で泳動する</li><li> RhoC - specfic抗体と5％ミルク/ TBST液とプローブをメンブレンにブロックする。 </li></ol> 3。代表的な結果良い結果は約22 kDaの単一バンドを生成する必要があります。悪い結果では、複数のバンドや高いバックグラウンドを生成する。これは、タンパク質分解、サンプルの不完全な洗浄または古いGST - RBDの使用を示している。 <img src="/files/ftp_upload/2083/2083fig1.jpg" alt="図1" /> 図1。RhoC GTP加水分解酵素の異なるレベルを表現する3つの別々の細胞株は、アクティブおよび総RhoCなしRhoCのアクティブと合計RhoC、高レベルの低レベルを実証するために示されている。ブロットは、取り除かれ、合計RhoCタンパク質のためのローディングコントロールとしてハウスキーピング遺伝子のアクチンに対する抗体で再プローブした。 <img src="/files/ftp_upload/2083/2083fig2.jpg" alt="図2" /> 図2。タンパク質分解、サンプルの不完全な洗浄または古いGST - RBDの使用から複数のバンドや高いバックグラウンドの結果は。

<ol>
	<li>van Golen, K. L., Wu, Z. F., Qiao, X. T., Bao, L. W., Merajver, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoC+GTPase,+a+novel+transforming+oncogene+for+human+mammary+epithelial+cells+that+partially+recapitulates+the+inflammatory+breast+cancer+phenotype.">RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype.</a> Cancer Res. 60 (20), 5832-5838 (2000).</li><li>van Golen, K. L., Wu, Z. F., Qiao, X. T., Bao, L., Merajver, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoC+GTPase+overexpression+modulates+induction+of+angiogenic+factors+in+breast+cells.">RhoC GTPase overexpression modulates induction of angiogenic factors in breast cells.</a> Neoplasia. 2 (5), 418-425 (2000).</li><li>Golen, K. L. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversion+of+RhoC+GTPase-induced+inflammatory+breast+cancer+phenotype+by+treatment+with+a+farnesyl+transferase+inhibitor.">Reversion of RhoC GTPase-induced inflammatory breast cancer phenotype by treatment with a farnesyl transferase inhibitor.</a> Mol Cancer Ther. 1 (8), 575-583 (2002).</li><li>Golen, K. L. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogen+activated+protein+kinase+pathway+is+involved+in+RhoC+GTPase+induced+motility,+invasion+and+angiogenesis+in+inflammatory+breast+cancer.">Mitogen activated protein kinase pathway is involved in RhoC GTPase induced motility, invasion and angiogenesis in inflammatory breast cancer.</a> Clin Exp Metastasis. 19 (4), 301-311 (2002).</li><li>Yao, H., Dashner, E. J., van Golen, C. M., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoC+GTPase+is+required+for+PC-3+prostate+cancer+cell+invasion+but+not+motility.">RhoC GTPase is required for PC-3 prostate cancer cell invasion but not motility.</a> Oncogene. 25 (16), 2285-2296 (2006).</li><li>Hall, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+I+Collagen+receptor+(alpha2beta1)+signaling+promotes+prostate+cancer+cell+invasion+through+RhoC+GTPase.">Type I Collagen receptor (alpha2beta1) signaling promotes prostate cancer cell invasion through RhoC GTPase.</a> Neoplasia. 10 (8), 797-803 (2008).</li><li>Sequeira, L., Dubyk, C. W., Riesenberger, T. A., Cooper, C. R., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+GTPases+in+PC-3+prostate+cancer+cell+morphology,+invasion+and+tumor+cell+diapadesis.">Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell diapadesis.</a> Clin Exp Metastasis. 25, 569-579 (2008).</li><li>van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL2+Induces+Prostate+Cancer+Transendothelial+Cell+Migration+via+Activation+of+the+Small+GTPase+Rac.">CCL2 Induces Prostate Cancer Transendothelial Cell Migration via Activation of the Small GTPase Rac.</a> J Cell Biochem. 104, 1587-1597 (2008).</li><li>Chatterjee, M., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Int J Cancer. , (2010).</li><li>Sander, E. E., ten Klooster, J. P., van Delft, S., van der Kammen, R. A., Collard, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac+downregulates+Rho+activity:+reciprocal+balance+between+both+GTPases+determines+cellular+morphology+and+migratory+behavior.">Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior.</a> J Cell Biol. 147 (5), 1009-1022 (1999).</li></ol>

利害の衝突は宣言されません。

手順の最も重要なステップは、新鮮なGST - RBDの世代です。このステップに細心の注意を払うために失敗は失敗の主な理由です。すべてのステップは、細胞溶解液（2.5）で一晩インキュベーションにGST - RBD（1.2）の世代から、一日で行う必要があります。もう一つの重要なステップは、細胞ライセートを生成するために十分な細胞があることを一定にすることです。セル内の低いレベルで存在?...

<table><tbody><tr><td>Gluthione-sepharose 4B beads</td><td>GE Healthcare</td><td>17-0756-01</td><td></td></tr><tr><td>Criterion 4-20% Tris-HCl gels</td><td>Bio-Rad</td><td>345-0032</td><td></td></tr></tbody></table>

rhoc gtpase activation assay

RhoC GTPアーゼは、RhoAのGTPアーゼ〜91％の相同性を持っています。ために細胞内での有病率から、RhoAのGTPアーゼのための多くの試薬および技術が開発されている。しかし、RhoC GTPアーゼは、比較的低レベルでの転移性癌細胞に発現している。したがって、少数のRhoC固有の試薬が開発されている。私たちはRhoC低分子量GTPaseを検出するためのRho活性化アッセイを適応している。この手法は、アクティブなRhoC GTPアーゼを引き出すためにGST - Rhoの結合ドメイン融合タンパク質を利用しています。さらに、我々は全体のレベル（GTPおよびGDP結合）RhoCのGTPアーゼを決定するアッセイの開始時に総タンパク質を収穫することができます。これは、セル内のアクティブな対総RhoC GTP加水分解酵素の測定が可能になります。この手順のいくつかの商用バージョンがしかし、市販のキットはRhoAのGTP加水分解のために最適化されており、通常はRhoC GTPアーゼのためにうまく動作しない開発されています。アッセイの部分はRhoC特異的抗体の開発だけでなく、変更されています。

Watch this Scientific Journal Video about RhoC GTPase Activation Assay at JoVE.com

RhoC GTPase Activation Assay

このプロトコルは、細胞内のアクティブなRhoC低分子量GTPaseのレベルを決定するためにプルダウンアッセイを利用しています。

RhoC GTPアーゼ活性化アッセイ

RhoC GTPase has 91% homology to RhoA GTPase.  Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed.  ...

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

12.4K ビュー.  University of Delaware. このプロトコルは、細胞内のアクティブなRhoC低分子量GTPaseのレベルを決定するためにプルダウンアッセイを利用しています。

ビデオ: RhoC GTPase Activation Assay

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a major pharmacological target for multiple indications, including analgesia, blood pressure regulation, and the treatment of psychiatric disease. Upon ligand binding, GPCRs catalyze the activation of intracellular G-proteins by stimulating the incorporation of guanosine triphosphate (GTP). Activated G-proteins then stimulate signaling pathways that elicit cellular responses. GPCR signaling can be monitored by measuring the incorporation of a radiolabeled and non-hydrolyzable form of GTP, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), into G-proteins. Unlike other methods that assess more downstream signaling processes, [35S]GTP&#947;S binding measures a proximal event in GPCR signaling and, importantly, can distinguish agonists, antagonists, and inverse agonists. The present protocol outlines a sensitive and specific method for studying GPCR signaling using crude membrane preparations of an archetypal GPCR, the &#181;-opioid receptor (MOR1). Although alternative approaches to fractionate cells and tissues exist, many are cost-prohibitive, tedious, and/or require non-standard laboratory equipment. The present method provides a simple procedure that enriches functional crude membranes. After isolating MOR1, various pharmacological properties of its agonist, [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO), and antagonist, naloxone, were determined.

Guanosine triphosphate (GTP) binding is one of the earliest events in G-Protein-Coupled Receptor (GPCR) activation. This protocol describes how to pharmacologically characterize specific GPCR-ligand interactions by monitoring the binding of the radio-labeled GTP analog, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), in response to a ligand of interest.

ラジオ標識GTP結合を介したGタンパク質共役型受容体シグナルの測定

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a ...

生化学

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

小さい gtp アーゼ プレニル化および分子量 GTP 結合を使用して膜分画と gtp アーゼ抗体法の検出

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

組換えGタンパク質共役型受容体のハイスループットスクリーニングのための「二重付加」カルシウム蛍光アッセイ

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

<meta charset="utf8"></head><body>ハイスループットスクリーニングのためのER-ミトコンドリア接触を測定するためのシンプルなアッセイ

Split-Luciferase Reassembly Assay to Measure Endoplasmic Reticulum-Mitochondria Contacts in Live Cells

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid homeostasis, mitochondrial dynamics, autophagosome and mitophagosome biogenesis, and apoptosis. Failure to maintain normal ER-mitochondrial coupling is implicated in many neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegia. It is of considerable significance to explore how the dysregulation of ER-mitochondrial contacts could lead to cell death and whether repairing these contacts to the normal level could ameliorate neurodegenerative conditions. Thus, improved assays that measure the level of these contacts could help to illuminate the pathogenic mechanisms of these diseases. Ultimately, establishing simple and reliable assays will facilitate the development of new therapeutic strategies. Here we describe&#160;a split-luciferase assay to quantitatively measure the level of ER-mitochondria contacts in live cells. This assay can be used to study the pathophysiological role of these contacts as well as to identify their modulators in high-throughput screening.

<meta charset="utf8"></head><body>著者スポットライト:ER-ミトコンドリア接触の調節と調節不全—神経変性疾患の病因への影響

We have established a split-luciferase reassembly assay to monitor the endoplasmic reticulum-mitochondria contacts in live cells. Using this assay, we describe a protocol to quantitatively measure the level of these inter-organelle couplings in HEK293T cells, under the condition of chemical treatment.

生細胞における小胞体-ミトコンドリア接触を測定するためのスプリットルシフェラーゼ再構成アッセイ

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

イメージングG -タンパク質共役受容体（GPCR）を介するシグナル伝達事象その制御走キイロタマホコリカビ</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

生物学

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion 1. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities 2. Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab 3,4 and we have used it in kidney tubular cell lines 5,6,7. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay8. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli.

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates

The method presented here describes an assay to follow activation of RhoA specific GDP/GTP Exchange Factors (GEFs) in cultured cells by making use of a mutant RhoA GST fusion protein that has high affinity for activated GEFs. GEFs are precipitated from cell lysates, detected by Western blotting and quantified by densitometry.

上皮細胞ライセートからのGSTタグ付き突然変異体のRhoタンパク質（GST-RhoAは（G17A））を使用してアクティブロー-GEFの親和性降水

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

蛍光ベースのカルシウム動員アッセイによるGタンパク質共役受容体のキャラクタリゼーション

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.

This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

競合アッセイを使用してGTPアーゼ結合タンパク質の親和性を比較すると、

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the ...

RhoC GTPアーゼ活性化アッセイ

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ラジオ標識GTP結合を介したGタンパク質共役型受容体シグナルの測定

小さい gtp アーゼプレニル化および分子量 GTP 結合を使用して膜分画と gtp アーゼ抗体法の検出

組換えGタンパク質共役型受容体のハイスループットスクリーニングのための「二重付加」カルシウム蛍光アッセイ

生細胞における小胞体-ミトコンドリア接触を測定するためのスプリットルシフェラーゼ再構成アッセイ

生細胞における小胞体-ミトコンドリア接触を測定するためのスプリットルシフェラーゼ再構成アッセイ

生細胞における小胞体-ミトコンドリア接触を測定するためのスプリットルシフェラーゼ再構成アッセイ

イメージングG -タンパク質共役受容体（GPCR）を介するシグナル伝達事象その制御走キイロタマホコリカビ

上皮細胞ライセートからのGSTタグ付き突然変異体のRhoタンパク質（GST-RhoAは（G17A））を使用してアクティブロー-GEFの親和性降水

蛍光ベースのカルシウム動員アッセイによるGタンパク質共役受容体のキャラクタリゼーション

競合アッセイを使用してGTPアーゼ結合タンパク質の親和性を比較すると、

RhoC GTPアーゼ活性化アッセイ

要約

さらに動画を探す

この動画の章

ラジオ標識GTP結合を介したGタンパク質共役型受容体シグナルの測定

小さい gtp アーゼ プレニル化および分子量 GTP 結合を使用して膜分画と gtp アーゼ抗体法の検出

組換えGタンパク質共役型受容体のハイスループットスクリーニングのための「二重付加」カルシウム蛍光アッセイ

生細胞における小胞体-ミトコンドリア接触を測定するためのスプリットルシフェラーゼ再構成アッセイ

生細胞における小胞体-ミトコンドリア接触を測定するためのスプリットルシフェラーゼ再構成アッセイ

生細胞における小胞体-ミトコンドリア接触を測定するためのスプリットルシフェラーゼ再構成アッセイ

イメージングG -タンパク質共役受容体（GPCR）を介するシグナル伝達事象その制御走キイロタマホコリカビ

上皮細胞ライセートからのGSTタグ付き突然変異体のRhoタンパク質（GST-RhoAは（G17A））を使用してアクティブロー-GEFの親和性降水

蛍光ベースのカルシウム動員アッセイによるGタンパク質共役受容体のキャラクタリゼーション

競合アッセイを使用してGTPアーゼ結合タンパク質の親和性を比較すると、

小さい gtp アーゼプレニル化および分子量 GTP 結合を使用して膜分画と gtp アーゼ抗体法の検出