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

국방학과 W81XWH - 05 - 1-0005, W81XWH - 06 - 1-0495, W81XWH - 08 - 1-0029 및 W81XWH - 08 - 1-0356

1. 준비 GST - 융합 단백질 <ol><li> BamH1/EcoR1 pGEX3x 벡터로 subcloned rhotekin Rho 구속력을 도메인 (RBD)의 첫 번째 N - 터미널 90 아미노산을 포함하는 JM109 관할 세균의 글리세롤 주식이 만들어집니다. 고효율을 보장하기 위해 다음 단계는 각 실험에 대해 수행되어야합니다. 우리의 경험에서 갓 GST - RBD는 강력하고 정확한 GTPase 활성화 분석의 열쇠입니다 준비. </li><li> LB의 A 50 ML에 글리세롤 주식의 (대략, 해동하지 않음) 50 μL 추가 잡고 37 ° C 하룻밤 성장. </li><li> 500 ML LB - A에 1시 10분을 희석 1 H.위한 성장 </li><li> 2 시간에 대한 IPTG 0.1 밀리미터와 GST 단백질 생산을 유도. </li><li> 5000에서 원심 분리기 병 및 원심 분리기, Sorval GSA3 로터에 균일하게 배포 RPM 4 ° C 20 분. </li><li> 원심 분리기 병에 Resuspend 펠렛, 용해 완충액 10 ML *. * 박테리아 용해 완충액 : 20 % 자당, 10 % 글리세롤, 50 MM 트리스 산도 8.0, 0.2 MM 2 S 2 O 5, 2 MM MgCl 2, 2 MM의 DTT 나는 신선한 PMSF, benzamide, aprotinin과 류펩틴을 추가합니다. </li><li> 2-3 분 (이 sonicator의 종류에 따라 다릅니다. 우리는 마르크 6 세트 피셔 소닉 Dismembranator 모델 500, 50 %의 사이클을 사용) Sonicate. </li><li> 4 원심 분리기, Sorvall SS34, 20 분 10,000 RPM ° C. 이 시점에서 작은 나누어지는는 제거 할 수 있습니다 SDS - PAGE에 의해 실행합니다. 겔은 쿠매시 자국이있을 수 있으며, 밴드 46 KDA에서 명확해야합니다. </li><li> 뜨는 제거 50 % glutatione - 세파 로스 4B 슬러리 1 ML를 추가합니다. </li><li> 4 30 분 품어 ° C를 회전. 용해 완충액 3 번 씻으십시오. </li><li> GST - 생선 버퍼와 50 %의 슬러리 (약 1 ML)로 GST-RBD/glutatione-Sepharose 비즈를 Resuspend **. ** GST - 피시 버퍼 : 10 % 글리세롤, 50 MM 트리스 산도 7.4, 100 MM NaCl는, 1 % NP - 4, 2 MM MgCl 2, 신선한 PMSF, benzamide, aprotinin과 류펩틴 추가 </li></ol> 2. GST - 융합은 분석을 내리다 <ol><li> 50-10 X 10 6 세포 / 분석을 사용 </li><li> 일단 얼음 차가운 PBS에서 얼음에 접시를 유지 (우리가 얼음 가득한 평면 유리 접시를 사용)와 0.5 ML GST - 물고기 버퍼 lyse 세포를 씻으십시오. </li><li> 얼음에 5 분 품어, 고무 경찰이나 평면 scrapper를 사용하여 수확 세포는 튜브를 microfuge 4에서 20,000 RPM 5 분 원심 분리기로 전송 ° C. </li><li> 신선한 microfuge 튜브에 뜨는 전송합니다. 총 Rho (예 : GDP + GTP 바운드) 결정 50 μL를 가져가라. </li><li> 0.5 ML의 GST-RBD/glutatione-Sepharose 비즈를 추가하고 4 회전 하룻밤 ° C, 알을 품다. </li><li> GST - 생선 버퍼와 배의 씻으십시오. </li><li> 40 μL 2X - Laemelli 샘플 버퍼에 Resuspend 비즈 이 시점에서 2.4 촬영한 총 Rho 단백질 나누어지는의 농도는 결정 30 μg는 해당 인증 샘플로 젤에서 실행할 수 있도록 준비한다. </li><li> 종기 샘플은 90 ° C에서 5 분, 짧게 원심 분리기 및 뜨는 (예 : 구슬을 피하) 부하를 가지고 12 잘 40~20% 기울기 SDS - PAGE 젤에서 실행됩니다. </li><li> 100 W.의 Towbin 버퍼와 상수가 현재 사용하는 멤브레인로 전송, 아니 높은 150 볼트에서 젤을 실행 </li><li> RhoC - specfic 항체와 함께 5 % 우유 / TBST 솔루션과 프로브와 멤브레인를 차단합니다. </li></ol> 3. 대표 결과 좋은 결과는 약 22 KDA에서 단일 밴드를 생산한다. 나쁜 결과는 여러 개의 밴드 혹은 높은 배경을 생산하고 있습니다. 이것은 단백질 저하, 샘플 불완전 세척 또는 오래된 GST - RBD의 사용을 나타내는 것입니다. <img src="/files/ftp_upload/2083/2083fig1.jpg" alt="그림 1" /> 그림 1. RhoC GTPase의 다른 레벨을 표현하는 세 가지 세포 라인은 활성 및 총 RhoC 활성 및 총 RhoC 높은 수준의 여부 RhoC의 낮은 수준을 보여주는 표시됩니다. 얼룩 그런 다음 옷을 벗긴 채 총 RhoC 단백질에 대한로드 컨트롤 역할을하는 집 지키는 유전자 굴지으로 항체와 reprobed했다. <img src="/files/ftp_upload/2083/2083fig2.jpg" alt="그림 2" /> 그림 2. 여러 밴드 또는 단백질 저하, 샘플의 불완전한 세척 또는 오래된 GST - RBD의 사용으로 높은 배경 결과입니다.

<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>

관심 없음 충돌 선언하지 않습니다.

절차의 가장 중요한 단계는 신선한 GST - RBD의 생성이다. 이 단계에주의하지 않으면 실패에 대한 주요 이유입니다. GST - RBD (1.2)의 세대에서 세포 lysate (2.5)와 함께 밤새 배양하기위한 모든 단계는, 하루에 수행되어야합니다. 또 다른 중요한 단계는 세포 lysates을 생산하기에 충분한 세포가있다는 것을 확실하게하는 것입니다. 세포의 낮은 수준에 존재하는 경향이 RhoC GTPase에서 볼 때 이것은 특히 중요하다.

<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 GTPase RhoA GTPase가에 91%의 상동 있습니다. 때문에 세포에 그 유행의 RhoA GTPase 많은 시약 및 기술이 개발되었습니다. 그러나, RhoC GTPase는 상대적으로 낮은 수준에서 전이성 암세포에 표시됩니다. 따라서, 몇 가지 RhoC 특정 시약이 개발되었습니다. 우리는 RhoC GTPase를 감지 Rho 활성화 분석을 적응있다. 이 기술은 활성 RhoC의 GTPase를 빼내 GST - Rho 바인딩 도메인 융합 단백질을 활용합니다. 또한, 우리는 전체의 수준 (GTP와 GDP 바운드) RhoC의 GTPase을 결정하기 위해 분석의 시작 부분에서 총 단백질을 수확 수 있습니다. 이것은 세포에 적극적으로 대 총 RhoC GTPase의 결정을 허용합니다. 이 절차의 여러 상업적인 버전은 그러나 개발되었습니다, 상업 키트 RhoA GTPase에 최적화되어 있으며, 일반적으로 RhoC GTPase를 위해 잘 작동하지 않습니다. 분석의 부품은 RhoC 특정 항체의 개발뿐만 아니라 수정되었습니다.

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

RhoC GTPase Activation Assay

이 프로토콜은 세포 내에서 활성 RhoC GTPase의 수준을 결정하기 위해 분석 아래로 끌어을 활용합니다.

RhoC GTPase 활성화 분석

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

Nanyang Technological University

Research

JoVE Journal

Biology

12.4K 조회수.  University of Delaware. 이 프로토콜은 세포 내에서 활성 RhoC GTPase의 수준을 결정하기 위해 분석 아래로 끌어을 활용합니다.

비디오: 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.

무선 표지 된 GTP 결합을 통한 G- 단백질 결합 수용체 신호 측정

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

연구

생화학

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.

작은 GTPase Prenylation와 GTP 바인딩 사용 하 여 막 분류 및 GTPase 연결 된 Immunosorbent 분석 결과의 검색

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

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.

이미징 G - 단백질 결합 수용체 (GPCR) - 중재 신호 행사 그 제어 Chemotaxis Dictyostelium Discoideum</em

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

생물학

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.

상피 세포 Lysates에서 GST-태그가 돌연변 Rho 단백질을 (GST-RhoA (G17A))를 사용하여 활성 Rho-GEFs의 친화력 강수량

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

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include well-validated targets for therapeutic intervention. Discovery of lead compounds, including small synthetic molecules, that specifically inhibit the receptor&#39;s function, is an important initial step in drug development and relies on sensitive, specific, and robust cell-based assays. Here, we describe a kinetic cellular assay with a fluorescent readout primarily designed to identify receptor-specific antagonists that inhibit the intracellular Ca2+ release evoked upon the activation of the CXC chemokine receptor 4 (CXCR4) by its endogenous ligand, the CXC chemokine ligand 12 (CXCL12). A key advantage of this method is that it also enables screening of compounds endowed with intrinsic agonistic properties (i.e., compounds eliciting an increase in intracellular Ca2+ concentration in the absence of CXCL12) or compounds modulating the receptor&#39;s function via interaction with allosteric binding sites (i.e., positive and negative allosteric modulators (PAMs and NAMs, respectively)). On the down side, autofluorescent compounds might interfere with the assay&#39;s readout, thereby hampering reliable data interpretation. Most likely this assay can be implemented, with minimal adaptations, as a generic drug discovery assay for many other GPCRs of which the activation leads to a release of intracellular Ca2+.

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

The described cellular assay is designed for the identification of CXC chemokine receptor 4 (CXCR4)-interacting agents that inhibit or stimulate, either competitively or allosterically, the intracellular Ca2+ release initiated by CXCR4 activation.

운동 형광 기반 Ca2 + 동원 분석 결과 G 단백질 결합 된 수용 체 주 작동 근, 길 항 근, 그리고 Allosteric 변조기를 식별 하

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include ...

의학

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).

형광 기반의 칼슘 동원 분석에 의해 G 단백질 결합 수용체의 특성

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.

경쟁 분석법을 이용는 GTPase - 결합 단백질의 친화 비교

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

Competition Binding Assay to Study Competing GTPase-Binding Protein Partners

Source: Williamson, R. C., et al.&#160; <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53254">Comparing the Affinity of GTPase-binding Proteins using Competition Assays.</a> J. Vis. Exp. (2015)
This video demonstrates a competition assay to study GTPase-binding protein partners. Utilizing nucleotide-bound GTPase protein immobilized on magnetic beads, the competitive binding between two interacting protein partners for the same binding site on the GTPase can be studied to assess the binding affinities of the protein partners.

경쟁 GTPase 결합 단백질 파트너를 연구하기 위한 경쟁 결합 분석

Source: Williamson, R. C., et al.&#160; Comparing the Affinity of GTPase-binding Proteins using Competition Assays. J. Vis. Exp. (2015)
This video ...

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-protein interactions

Small GTPases - Ras and Rho

Ras and Rho are small monomeric GTPases that act downstream of receptor tyrosine kinase (RTK) and regulate various cellular processes. These GTPases switch between active and inactive states by binding to guanine nucleotides.
Three regulatory proteins control their activity:
<ul>
	<li>Guanine nucleotide exchange factors or GEF,</li>
	<li>GTPase-activating proteins or GAPs, and</li>
	<li>Guanine nucleotide-dissociation inhibitors or GDIs.</li>
</ul>
The GEF activates the GTPase by exchanging the bound-GDP with GTP. The GTPases&#8217; activity is turned off by GAP. They hydrolyze the bound GTP to GDP. The GDI further arrests the GTPase in its GDP-bound inactive form.
Ras is a membrane-bound GTPase and attaches to the plasma membrane via prenyl chains. There are three types of Ras present in mammals: H-Ras, K-Ras, and N-Ras. The binding of growth factor ligand activates RTKs and triggers Ras-GEF to displace GDP with GTP. The active Ras-GTP can now recruit and activate the first kinase of the MAPK signaling cascade, such as MAPKKK/Raf, triggering MAPK signaling, thereby initiating cell proliferation. However, it quickly hydrolyzes the GTP and is turned off, preventing uncontrolled cell proliferation. Hyperactive mutant forms of Ras stay in the GTP-bound state and often lead to tumorigenesis.
Rho family proteins such as Cdc42, Rho, and Rac modulate cell shape, movement, and migration. Rho can be soluble or membrane-bound. Soluble RhoGDP binds GDI and stays inactive. Following stimulation, GDI dissociates, facilitating membrane localization of Rho in one of the following ways:
<ol>
	<li>Electrostatic attractive forces between positively charged C termini of Rho and the negatively charged plasma membrane lipids promote its localization.</li>
	<li>The truncated tropomyosin-related kinase B receptor or T1 binds RhoGDI and sequesters it on the plasma membrane, allowing Rho to escape to the membrane for activation. Alternatively, displacement factors such as the ezrin (E), radixin (R), and moesin (M) or ERM family proteins interact with the RhoGTPases-GDI complex, subsequently displacing the GDI from Rho.</li>
	<li>Alternatively, phosphorylation of RhoGDI by protein kinases destabilizes the RhoGTPases-GDI complex, releasing Rho for membrane localization.</li>
</ol>
Once localized to the membrane, Rho interacts with a nearby activated RhoGEF and undergoes GDP/GTP exchange. Activated RhoGTP now promotes downstream signaling and regulates various cellular processes.

소형 GTPase - Ras 및 Rho

Ras and Rho are small monomeric GTPases that act downstream of receptor tyrosine kinase (RTK) and regulate various cellular processes. These GTPases ...

교육

JoVE Core

Cell Biology

Unit 1

Signaling Networks of Kinase Receptors

주요 용어 및 개념

Activation and Inactivation of G Proteins

Heterotrimeric G proteins are guanine nucleotide-binding proteins. As the name suggests, heterotrimeric G proteins are composed of three subunits: alpha, beta, and gamma. They remain GDP-bound or GTP-bound inside the cells and switch between inactive/active states. The G&#945;&#160;subunit possesses the nucleotide-binding pocket that binds guanine nucleotides and switches between GDP or GTP-bound states. In contrast, the G&#42933; and G&#947;&#160;subunits are always bound together with high affinity and are together referred to as G&#42933;&#947;&#160;subunits.
Heterotrimeric G proteins regulate signaling downstream of G protein-coupled receptors&#160; (GPCRs) and remain anchored to the membrane by lipid modifications. Myristoylation or palmitoylation at the N-terminus of the G&#945;&#160;subunit, and prenylation at the C-terminus of the G&#947;&#160;subunit anchor these subunits to the membrane and stabilize the heterotrimeric complex.
Upon ligand binding, GPCRs bind the G&#945;&#160;subunit of heterotrimeric G proteins with high affinity. The receptor-G protein interaction leads to the opening of the nucleotide-binding site of the G&#945;&#160;subunit, releasing the GDP. GPCRs function as guanine nucleotide exchange factors&#160; (GEFs) that facilitate G&#945;-GTP binding.
The activated GTP-G&#945;&#160;subunit undergoes a conformational change and detaches from the receptor and G&#42933;&#947;&#160; subunit.&#160; GTP-G&#945;&#160;and&#160; G&#42933;&#947;&#160;subunits now individually participate in signal transduction pathways and activate effectors like adenylyl cyclase (AC), phospholipase C&#42933; (PLC&#42933;) and&#160; Na+, K+, and Ca2+-specific ion channels, thereby triggering the production of different second messengers.
The human genome encodes&#160; 21 different G&#945;&#160;subunits that can be classified into subfamilies such as G&#945;s, G&#945;i, G&#945;q, Gt&#945;, Golf, G12&#945;, and G13&#945;. Each of these G&#945;&#160;subunits performs a specific function upon binding their effector. For example, the binding of G&#945;s to adenylyl cyclase activates adenylyl cyclase&#160; to produce cAMP, an important second messenger. cAMP regulates muscle contraction and the metabolism of fats or sugars. In contrast, the binding of G&#945;i inhibits adenylyl cyclase activity and cAMP synthesis.
Other subfamilies of G&#945; subunits perform various other cellular processes. The G&#945;q family activates PLC&#42933;, which produces second messengers like inositol trisphosphate (IP3) and diacylglycerol (DAG). Both IP3 and DAG affect various cellular pathways, including growth and differentiation. The transducin or Gt&#945;&#160;family binds to rhodopsin to transmit information from&#160; visual stimuli by activating specific phosphodiesterases, which hydrolyze cGMP to GMP.&#160; Golf is the variant of the G&#945;s family and is used&#160; in odorant signaling pathways. The family of G12&#945; and G13&#945;&#160;&#160;proteins are involved in cytoskeleton regulation.
G&#945;&#160;subunits have intrinsic GTPase activity, and binding of GTP-G&#945;&#160;to the effector enhances their rate of GTP hydrolysis. The activated GTP-G&#945;&#160;form is thus short-lived and hydrolyzed to GDP-G&#945;&#160; within minutes, switching back to the inactive state. This feedback mechanism helps avoid the overstimulation of cells in response to a signal. G proteins are also regulated by another family of GAP, Regulators of G protein Signaling (RGS). The human genome encodes 25 RGS proteins, each interacting with a particular set of G&#945;&#160;subunits, and helps shut off G protein-mediated cellular responses.

G 단백질의 활성화 및 불활성화

Heterotrimeric G proteins are guanine nucleotide-binding proteins. As the name suggests, heterotrimeric G proteins are composed of three subunits: alpha, ...

RhoC GTPase 활성화 분석

요약

더 많은 비디오 탐색

이 비디오의 챕터