This work was supported by Wellcome Trust grant 088419 to MDB.

GST-태그는 GTPase 1. 정제 <ol><li> 문화 E. 예컨대 220 rpm으로 흔들면서 37 ℃에서 유전자 pGEX-RAC1 O / N로 형질 전환 된 BL21로서 대장균, autoinduction 배지 500 ㎖ (25 mM의 나트륨 2 HPO 4, 25 mM의 KH 2 PO 4, 50 mM의 NH 4 CL, 5 mM의 SO 4 나 2, 2 mM의 황산, 2 mM의 CaCl2를, 0.5 % 글리세롤, 0.05 % 글루코스, 0.2 % 락토오스, 5g 트립 톤, 2.5 g 효모 추출물, 100 μg의 / ㎖ 암피실린). </li><li> 10,000 XG, 4 ° C에서 10 분 동안 원심 분리하여 수확 박테리아. </li><li> 20 ㎖의 단백질 추출 시약, 1X 프로테아제 억제제 세균 펠렛을 재현 탁하고 반전하여 실온에서 20 분 동안 배양한다. </li><li> 30 분 동안 40,000 XG에서 원심 분리하여 해물을 명확히. </li><li> 인산염 완충 식염수로 세척 2 ㎖의 글루타티온 자석 구슬, 추가 (PBS : 10 밀리미터 나 2 HPO 4, 1.8 mM의 KH 2 PO 4, 137 mM의 NaCl을, 2.7 밀리미터의 KCl). </li><li> 4 ° C에서 반전하여 혼합, 2 시간 동안 품어. </li><li> 각 단계에서 비즈를 침전 자분 분류기를 사용하여, 10 ml의 PBS로 단백질 로딩 비드 네 번 씻는다. </li><li> 필요할 때까지 재현 탁의 100 μL 씩에 -80 ° C에서 2 ml의 PBS에서 구슬과 상점을 단백질로드. </li></ol> 는 GTPase 결합 단백질의 2 식 <ol><li> 다음 실험 하루 전에, 녹색 형광 단백질 (GFP)을 코딩하는 플라스미드를 형질 HEK293T 별도 75 cm-2는 GTPase 플라스크에 각각 결합 단백질의 버전 -tagged. 염기로드의 검증을 위해, 트랜의 HEK293T의 세 번째 75 cm 2 플라스크에 TrioD1을 GFP - 태그. <ol><li> 100 μL에 1 ㎎ / ㎖ 멸균 150 mM의 염화나트륨에 polyethylamine을 희석. </li><li> 223 μL 감소 혈청 미디어에 27 μl를 희석 polyethylamine를 추가합니다. </li><li> 250 μL 감소 혈청 배지에 12 μg의 플라스미드 DNA를 추가합니다. </li><li> 실온에서 2 분 동안 각 튜브를 품어. </리&gt; <li> polyethylamine을 결합하고 DNA는 2 분에 대해 하나의 튜브와 소용돌이에 혼합. </li><li> 실온에서 15 ~ 20 분 동안 품어. </li><li> 5 ml의 신선한 성장 배지에 90 % 합류에 HEK293T 성장 배지 (둘 베코 변형 이글 미디어, 10 % 소 태아 혈청, 2 mM L- 글루타민, 항생제)를 대체. </li><li> 플라스크에 결합 polyethylamine / DNA 믹스를 추가하고, 37 ℃, 5 % CO 2에서 O / N을 품어. </li></ol></li></ol> 는 GTPase 결합 단백질의 3 정화 <ol><li> PBS에서 형질 세포의 플라스크를 씻어 무료로 액체를 흡입, 5 분 동안 플라스크 드레인. </li><li> 500 ㎕의 용해 완충액에 세포를 긁어 (50mM 트리스 - 염산 (pH7.8), 1 % Nonidet P-40, 1X 프로테아제 억제제) 미세 원심 분리 튜브에. </li><li> 30 분 동안 4 ° C에서 반전하여 혼합하여 세포를 Lyse. </li><li> 용해시, 세척 사이에 2 분 동안 2,700 XG에 40 μL GFP - 트랩 구슬이 많은 신선한 용해 완충액으로 3 회, 침강 구슬을 씻어. </li><li> 10 분 동안 21,000 XG에서 원심 분리 용 해물을 명확히. </li><li> 전사는 4 ° C에서 반전하여 혼합, 세척 GFP 트랩 비드를 분리 및 GFP 융합 단백질은 2 시간 동안 결합​​하게 경쟁 단백질의 각각의 용 해물을 정화. 얼음에 GFP - TrioD1 세포에서 해물을 유지합니다. </li><li> 50 mM 트리스 -HCl (pH 7.8), 50 mM의 NaCl을 두번로드 GFP 트랩 구슬 씻어 0.7 % (W / V) Nonidet P-40 회 50 mM 트리스 -HCl (pH 7.6)에서, 20 밀리미터의 MgCl 2 , 세척 사이에 2 분 동안 2,700 XG에 구슬을 침강. </li><li> 40 ㎕의 0.2 M 글리신 (PH 2.5)을 추가하고 30 초 동안 아래로 피펫 팅에 의해 GFP - 융합 단백질을 용출. 4 μL 1 M 트리스 - 염산 (PH 10.4)를 포함하는 새로운 미세 원심 분리 튜브에 21,000 60 초 동안 XG 및 전송 액체에서 즉시 침전물 구슬. 정제 된 단백질의 손상을 제한하기 위해 신속하게이 작업을 수행합니다. </li><li> 정량적 blott를 사용 상대적인 수율을 확립 항 GFP 항체와 함께 웨스턴 블랏에 의한 각 프로브와 정제 된 단백질의 1 μL를 분석제조 업체의 프로토콜에 따라 시스템을 보내고. 대안 적으로, bicinchoninic 산 (BCA) 단백질 분석으로 농도를 결정하지만, 단백질과 동일한 방식으로 분석과 반응하지 않거나 오염 단백질이 존재하면 에러를 도입한다. </li><li> 50 mM 트리스 -HCl (pH 7.6), 20 mM의의 MgCl 2를 첨가하여 단백질의 몰 농도를 등화. </li></ol> 는 GTPase의 4 염기로드 <ol><li> 단계 1에서 제조 한 GST-RAC1 자성 비드, 하나의 분취 량을 해동. </li><li> 각 단계에서 비즈를 침전 자분 분류기를 사용하여, GST-RAC1 비드의 90 μl를 취하여 20 mM 트리스 - 염산 (PH 7.6), 25 mM의 염화나트륨, 0.1 mM의 DTT, 4 mM의 EDTA로 3 회 세척 하였다. </li><li> 대기음 구슬에서 버퍼와 100 μL 20 mM 트리스 - 염산 (PH 7.6), 25 mM의 NaCl을 0.1 mM의 DTT, 4 mM의 EDTA를 추가합니다. </li><li> 국내 총생산 (GDP), GTP 또는 전혀 염기 로딩 경쟁 실험에 필요한지 여부에 따라, 12 μl의 100 mM의 국내 총생산 (GDP), 12 ㎕의 10 mM의 번지를 추가anosine 5 &#39;- [γ-티오] 트리 포스페이트 (GTPγS) 또는 60 μL GST-RAC1 비즈없이 염기. </li><li> 염기 로딩 컨트롤, 세 10 μL 분취에 남아있는 구슬을 분할하여 2 μL 100 mM의 국내 총생산 (GDP), 2 μL 10 밀리미터 GTPγS 각 튜브없이 염기를 추가합니다. </li><li> 교반하면서 30 ℃에서 30 분 동안 비드 혼합물을 배양한다. </li><li> 실험 믹스 3 μL (단계 4.4), 제어 믹스 (단계 4.5)의 각각 0.5 μL 1 M의 MgCl 2를 첨가하여 핵산 - 결합 RAC1 안정화. </li></ol> 바인딩 5. 경쟁. <ol><li> 6의 microfuge 튜브를 설정, 각 포함 : 200 ㎕의 50 mM 트리스 -HCl (pH 7.6), 20 mM의의 MgCl 2 (단계 4.7에서) 10 μL 실험 염기로드 RAC1 비즈 5 μL RAC1 결합 단백질 A (단백질 결합 상수) </li><li> 각 튜브에, 0, 1, 2.5, 5, 10 또는 20 μL의 RAC1 - 결합 단백질 B (가변 결합 단백질)를 추가한다. 이 볼륨은 가정pproximately 동일한 주식 상수와 변수 결합 단백질의 농도를 조정해야 할 수도 있습니다. <ol><li> 이 두 단백질의 결합 친화도에 큰 차이가 있으며 이것이 반복 실험을 통해 경험적으로 결정되어야하는 경우 결합 단백질의 양 및 B를 조정한다. 50 mM 트리스 -HCl (pH 7.6), 20 mM의의 MgCl 2를 첨가하여 235 μL로 결합 혼합물의 전체 부피를 차지한다. </li></ol></li><li> 포함하는 미세 원심 분리 튜브를 설정합니다 : 200 ㎕의 50 mM 트리스 -HCl (pH 7.6), 20 mM의의 MgCl 2 (단계 4.7에서) 10 μL 실험 염기로드 RAC1 비즈 10 μL RAC1 결합 단백질 A (단백질 결합 상수) </li><li> 국내 총생산 (GDP), GTPγS없이 염기 제어 튜브를 설정합니다 : 200 ㎕의 50 mM 트리스 -HCl (pH 7.6), 20 mM의의 MgCl 2 10 μL 제어 RAC1 비즈는 국내 총생산 (GDP), GTPγS 또는 전혀 염기와 4.5 단계에서로드 단계 4.7에서 안정화. 180 μL H단계 3.6에서와 같이 제조 EK293T GFP - TrioD1 용 해물, 4 μL 1 M의 MgCl 2 </li><li> 4 ° C에서 반전하여 혼합, 2 시간 동안 상기 혼합물을 배양한다. </li><li> 50 mM 트리스 -HCl (pH 7.6), 20 mM의의 MgCl 2 비드 세 번 세척 하였다. </li><li> 용출 시료 완충액을 20 μL 환원 단백질을 결합 (50 mM 트리스 -HCl (pH 7), 5 % SDS, 20 % 글리세롤, 0.02 ㎎ / ㎖ 브로 모 페놀 블루, 5 % β 메르 캅토 에탄올). </li></ol> 경쟁 6. 분석 <ol><li> 소듐 도데 실 설페이트 폴리 아크릴 아미드 겔 전기 영동 (SDS-PAGE) 및 웨스턴 블롯에 의해 결합 된 단백질 (단계 5.6) 10 μL를 해결. </li><li> 39 °에서 CO 막을 인큐베이션 / N 안티 GFP 항체로 블로킹 완충액으로 1/1000 희석하여 PBS로 1 배 희석하고, 0.1 % 트윈 -20은이 태그는 GTPase - 결합 단백질의 양을 검출한다. </li><li> PBS로 10 분, 0.1 % 트윈 20 막 세 번 씻으십시오. </li><li> 800 - 공액 안티 - 토끼 초 DyLight에 RT에서 30 분 동안 인큐베이션 막을ondary 항체, 버퍼를 차단에서 1 / 10,000을 희석하면, 0.1 % 트윈 20 PBS에 1 배 희석. </li><li> PBS로 10 분, 0.1 % 트윈 20 막 세 번 씻으십시오. </li><li> 제조사의 프로토콜에 따라 밴드 강도를 측정하기 위해 소프트웨어를 사용하여, 적외선 이미지 시스템을 사용하여 멤브레인을 스캔. </li><li> 가변 경쟁자 (단백질 B)의 양에 대해 각 단백질 밴드의 강도를 플롯. </li><li> 라인이 평형이 달성되는 경쟁자 비율을 결정하는 상수 경쟁자 (단백질 A, 5 μL)의 체적 교차하는 점에서 가변 경쟁자의 볼륨을 나눈다. </li><li> 단계 6.1-6.6에 설명 된대로 P21 활성화 키나제 1 (PAK1) (이펙터)와 GFP - TrioD1 (GEF)에 대한 염기 로딩 상태, 프로브 막 확인하십시오. </li></ol>

<ol>
	<li>Burridge, K., Rho Wennerberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=and+Rac+take+center+stage.">and Rac take center stage.</a> Cell. 116 (2), 167-179 (2004).</li><li>Raftopoulou, M., Hall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration:+Rho+GTPases+lead+the+way.">Cell migration: Rho GTPases lead the way.</a> Dev Biol. 265 (1), 23-32 (2004).</li><li>Rossman, K. L., Der, C. J., Sondek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GEF+means+go:+turning+on+RHO+GTPases+with+guanine+nucleotide-exchange+factors.">GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors.</a> Nat Rev Mol Cell Biol. 6 (2), 167-180 (2005).</li><li>Worthylake, D. K., Rossman, K. L., Crystal Sondek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=structure+of+Rac1+in+complex+with+the+guanine+nucleotide+exchange+region+of+Tiam1.">structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1.</a> Nature. 408 (6813), 682-688 (2000).</li><li>Scheffzek, K., Ahmadian, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTPase+activating+proteins:+structural+and+functional+insights+18+years+after+discovery.">GTPase activating proteins: structural and functional insights 18 years after discovery.</a> Cell Mol Life Sci. 62 (24), 3014-3038 (2005).</li><li>Del Pozo, ., A, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+regulate+GTP-Rac+localized+effector+interactions+through+dissociation+of+Rho-GDI.">Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI.</a> Nat Cell Biol. 4 (3), 232-239 (2002).</li><li>Williamson, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronin-1C+and+RCC2+guide+mesenchymal+migration+by+trafficking+Rac1+and+controlling+GEF+exposure.">Coronin-1C and RCC2 guide mesenchymal migration by trafficking Rac1 and controlling GEF exposure.</a> J Cell Sci. 127 (Pt 19), 4292-4307 (2014).</li><li>Del Pozo, M. A., Price, L. S., Alderson, N. B., Ren, X. D., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+to+the+extracellular+matrix+regulates+the+coupling+of+the+small+GTPase+Rac+to+its+effector+PAK.">Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK.</a> Embo J. 19 (9), 2008-2014 (2000).</li><li>Van Rijssel, J., Hoogenboezem, M., Wester, L., Hordijk, P. L., Van Buul, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+N-terminal+DH-PH+domain+of+Trio+induces+cell+spreading+and+migration+by+regulating+lamellipodia+dynamics+in+a+Rac1-dependent+fashion.">The N-terminal DH-PH domain of Trio induces cell spreading and migration by regulating lamellipodia dynamics in a Rac1-dependent fashion.</a> PLoS. 7 (1), e29912 (2012).</li><li>Self, A. J., Hall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+intrinsic+nucleotide+exchange+and+GTP+hydrolysis+rates.">Measurement of intrinsic nucleotide exchange and GTP hydrolysis rates.</a> Methods Enzymol. 256, 67-76 (1995).</li></ol>

저자가 공개하는 게 없다.

This protocol describes a method for comparing the relative affinities of pairs of small GTPase-binding proteins. The key steps are the preparation of purified GTPase-binding proteins and the nucleotide loading of the GTPase. The use of GTPase-binding proteins with the same GFP tag, allows the concentrations at which similar amounts of each competitor binds to be accurately determined. The use of recombinant nucleotide-loaded GTPase allows interrogation of the binding properties of the GTPase under specific activity conditions. This step is also the most sensitive as nucleotides will both hydrolyze and detach from the GTPase if the magnesium conditions are not maintained precisely.
In the cell, the large number of GTPase-binding proteins combined with the rapid nucleotide turnover makes such pathways difficult to interpret. The simplicity of this method in comparing only pairs of binding proteins and using carefully controlled nucleotide-loading conditions allows signaling pathways to be elucidated. However, the greatest strength of the protocol is also the greatest weakness as it is a simplification of the in vivo situation. Competition assays can be used to build a robust hypothesis, but this should then be tested in cells by knockdown experiments.
There are three features that must be considered when selecting the GFP-tagged GTPase-binding proteins to be used in the experiment. First, the fusion proteins must express well in mammalian cells, such as HEK293T, as competition assays require a reasonable amount of protein. Second, it must be possible to purify the recombinant protein without significant degradation, and where this is not possible, cloning of a GTPase-binding fragment should be considered. Third, the two GTPase-binding proteins must resolve from one another on SDS-PAGE to allow analysis in section 6.
There are a number of potential caveats to the experiment that need to be considered, and possibly addressed:
Possible denaturation of purified GTPase-binding proteins during the acid elution step or steric hindrance by the GFP tag. In our hands, these have not been a problem, but must be tested. The purified proteins can be tested in functional assays 10. Commercial kits now exist for testing the activity of GEFs or GAPs without the need for isotope-labeled nucleotides. Sequestering proteins, by their nature protect GTPases from GEF or GAP activity, so can be used as competitive inhibitors in the commercial GEF or GAP assays, as we did in our recent publication 7. The relevant feature of proteins that traffic GTPase are the capacity to bind the GTPase, and this can be tested easily in a pull down assay. An alternative approach to testing protein integrity that is applicable to all binding proteins is to titrate protein eluted from GFP-trap beads with glycine with the same protein removed from GFP-trap beads by enzymatic cleavage. The experiment would be analyzed by probing both the GFP-tagged and cleaved protein with an antibody against the protein itself. If the protein is undamaged by elution, equilibrium should be achieved at a 1:1 ratio. This approach would also indicate whether the presence of the GFP tag itself compromises the binding properties of the candidate protein, though this does require the production of a construct with an enzymatic cleavage site between the tag and the binding protein. Whether the protein is compromised by the tag or the elution step, the problem could be addressed by modifying the protocol to use an alternative purification method. Rather than GFP, binding proteins could be His-tagged, purified using Ni-NTA and analyzed using an antibody against the His-tag. The important feature is that both binding proteins must share a common tag although, if necessary, two tags could be added to a protein, one for purification and the other for detection.
The protocol is designed to investigate competition between interactions with the switch I/II domains. Although the majority of GTPase interactions are mediated by this motif, there are some exceptions, most notably the interactions of GDIs that bind to the prenyl tail, as well as obscuring the switch domains. In principle, the protocol could be adapted to use GTPase purified from mammalian cells, so that the GTPase is prenylated, however, the presence of multiple binding sites or allosteric effects complicate the interpretation of competition-binding data. Further problems associated with such a modification are that GDIs co-purify with GTPase from mammalian cells, compromising the purity of the isolated proteins and the hydrophobic nature of the prenyl groups means that prenylated GTPases are associated with either GDI or lipid membrane and such factors would need to be considered in the experiment.
The amount of GST-Rac1 being used in the assay. The constant GTPase binding protein must be at a greater concentration than the Rac1, or when the competitor is added, it will simply bind to free Rac1. It will be immediately obvious if this has happened as binding of the competitor, without a loss of the constant protein, will be detected in much the same way as when the two competing proteins bind to one another as shown in Figure 3B. As an additional control (Step 5.3), a binding reaction containing double the amount of constant binding protein and no variable binding protein should be included (Step 5.3). If the Rac1 in the titration experiment is saturated, doubling the amount of constant binding protein will have no effect on the output. The volumes suggested in the protocol should be appropriate, but the amount of Rac1 can be easily reduced. If binding of the competitor without loss of the constant binding partner is observed, reducing the amount of Rac1 should be attempted before trying to map binding sites to avoid ternary complex formation.
Non-specific interaction of GTPase-binding proteins with the GST or bead, as well as specifically with Rac1. This problem would be manifested by residual binding of the constant GTPase-binding protein, even when the variable GTPase-binding protein has reached a plateau at high concentration. Identification of this issue will be aided by conducting reciprocal experiments where the constant and variable GTPase-binding proteins are swapped. Reciprocal experiments will also greatly improve the accuracy of the estimate of equilibrium point, so should always be included. In cases of non-specific binding, the relative concentrations at which equilibrium is achieved can still be calculated by comparing band intensity between the maxima and minima for each protein, or by measuring the extent of non-specific binding by using GST beads as bait, rather than GST-Rac1.
Pull down assays using different nucleotide-loading conditions should be used to complement the competition assay described in this protocol. Determining the nucleotide preference of partners is important for both understanding the competition events and understanding the signaling pathway that the GTPase-binding protein is involved in. In Figure 2B we analyze binding of proteins with established preference for GTP-loaded or nucleotide-free GTPase as a means to validate nucleotide loading. However, it is sensible to investigate the effect of nucleotide loading on each of the competitors as well. If the hypothetical competitors show different preferences, competition will make less of a contribution to the signaling pathway, and indeed nucleotide turnover is likely to be the mechanism that directs exchange of the binding proteins.

The actin cytoskeleton that determines the shape, polarity and migratory properties of mammalian cells is regulated by the Rho-family of small GTPases. The Rho-family GTPases include RhoA that stimulates cytoskeletal contraction, Rac1 that stimulates actin branching and membrane protrusion, and Cdc42 that has similar effects on actin polymerization to Rac1 and causes the formation of filopodia 1,2. GTPase signaling activity is determined by binding of a nucleotide, which controls the contraction and relaxation of the switch I and switch II loops that mediate the protein-protein interactions with both regulators and effectors. Guanosine 5&#8217;-triphosphate (GTP)-bound GTPases activate downstream effectors, whereas the Guanosine 5&#8217;-diphosphate (GDP)-bound form is inactive. In the cell, cycles of GTP hydrolysis and nucleotide exchange allow rapid turnover of GTPase signals that are necessary for cytoskeletal dynamics. Nucleotide turnover is regulated by three mechanisms. Guanine nucleotide exchange factors (GEFs) stabilize the nucleotide-free GTPase, catalyzing exchange of GDP for GTP, and thereby stimulating GTPase signaling activity 3,4. GTPase-activating proteins (GAPs) catalyze hydrolysis of GTP to GDP, thereby inhibiting GTPase signaling activity 5. Sequestering molecules such as regulator of chromatin condensation 2 (RCC2) and guanine nucleotide dissociation inhibitors (GDIs) obscure the switch loops and in the case of GDIs remove the GTPase from the membrane by interaction with the prenyl tail 6,7. Each of the three classes of regulatory molecule interact with the switch loops, as do the downstream effectors and some trafficking regulators such as coronin-1C 7. The purpose of this protocol is to measure competition for the switch I/II binding site between putative regulators and downstream signaling molecules. It should be noted that competition assays test binding to a shared binding site, so that this protocol is not suitable for testing interactions with other sites, such as binding of GDIs to the prenyl tail.
The subtlety of the conformation differences between active and inactive forms, combined with the labile nature of the bound nucleotide, has made study of GTPase-binding events difficult. The role of the bound nucleotide means that conventional binding assays such as immunoprecipitation or surface plasmon resonance are not well suited to investigation, as the nucleotide cannot be controlled. This obstacle is compounded by the overlap in the binding sites of GEFs, GAPs, effectors, sequestering molecules and trafficking molecules, which make binding data for a single interaction difficult to interpret in the context of the competition that will occur in the cell. Immunoprecipitation, in particular, is compromised by competition between binding partners, as under certain cellular conditions, one binding partner might be identified at the expense of all others, while under other conditions, another partner might dominate. The dynamic nature of GTPase signaling is essential to GTPase function and must be considered when analyzing the relationships between the binding interactions of different regulators. Indeed, we recently described a pathway that relied heavily on competitive binding. We identified coronin-1C as a trafficking molecule that bound to the switch loops of GDP-Rac1 7. In areas of low GEF activity, trafficking would dominate, removing Rac1 from those regions. However, when Rac1 is delivered to regions of the cell where GEF activity is high, the GEF would outcompete coronin-1C, thereby both activating Rac1 and preventing coronin-1C-mediated removal of Rac1 from that area. The model goes further, because the action of the GEF exchanges bound GDP for GTP, shifting the equilibrium still further from coronin-1C. Consequently, Rac1 activity could be explained entirely in terms of competition and relative affinity.
In this protocol, we describe a method for comparing the relative affinities of different binding partners for small GTPases, using Rac1 as an example. By using a purified protein approach, it is possible to piece together a chain of signaling events by pair wise comparison, in an experiment where the bound nucleotide can be closely controlled.

<table><tbody><tr><td>Bugbuster</td><td>Novagen</td><td>70584-3</td><td></td></tr><tr><td>COMPLETE protease inhibitor</td><td>Roche</td><td>05 056 489 001</td><td></td></tr><tr><td>Glutathione magnetic beads</td><td>Pierce</td><td>88821</td><td></td></tr><tr><td>Polyethylenimine, branched, average M&lt;sub&gt;w&lt;/sub&gt; ~25,000</td><td>Sigma Aldrich</td><td>408727-100ML</td><td></td></tr><tr><td>OPIMEM</td><td>Life Technologies</td><td>31985-047</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Media</td><td>Sigma Aldrich</td><td>D5796</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>10270-1-6</td><td></td></tr><tr><td>L-Glutamine</td><td>Life Technologies</td><td>25030-024</td><td></td></tr><tr><td>GFP-Trap_A</td><td>Chromotec</td><td>gta-20</td><td></td></tr><tr><td>GDP</td><td>Sigma Aldrich</td><td>G7127</td><td>Highly unstable. Aliquot and store at -80 immediately upon reconstritution</td></tr><tr><td>GTP&amp;gamma;S</td><td>Sigma Aldrich</td><td>G8634</td><td>Highly unstable. Aliquot and store at -80 immediately upon reconstritution</td></tr><tr><td>Blocking Buffer</td><td>Sigma Aldrich</td><td>B6429</td><td></td></tr><tr><td>Tween-20</td><td>Sigma Aldrich</td><td>P9416</td><td></td></tr><tr><td>Anti-GFP antibody</td><td>Living Colors</td><td>632592</td><td>Use at 1/1000 dilution</td></tr><tr><td>DyLight 800 conjugated goat anti-rabbit secondary antibody</td><td>Fisher Scientific</td><td>10733944</td><td></td></tr><tr><td>Anti-PAK1 antibody</td><td>Cell Signaling</td><td>2602S</td><td>Use at 1/1000 dilution</td></tr><tr><td>Odyssey SA Infrared Imaging System</td><td>Li-cor</td><td>9260-11PC</td><td></td></tr></tbody></table>

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.

이 프로토콜은 경쟁사의 정확한 농도를 알 필요 (그림 1)하지 않고, RAC1 바인딩 파트너의 상대적 친화도를 계산하도록 설계되었습니다. 단백질 농도의 측정 오류를 소개하고 신호 경로의 분자 사이의 경쟁을 고려할 때 필요하지 않습니다. 그러나, 두 경쟁 분석법으로 상이한 양을 추가 할 때 단순 비율이 계산 될 수 있도록하는 스톡 용액에 동일 몰 농도가 있음을 아는 것이 중요하다. GFP 트랩 비드 40 ㎕를 300 pmol의 그래서 합류 75cm 높은 두 개의 다른 결합 단백질의 제제는 조정 전의 유사 할 것이라는 결과, 비드 포화 것이다 발현 세포의 2 플라스크 ~의 결합능을 가지고 (도 2A). 단백질 중 하나가 잘못 표현하면,이 문제는 셀의 하나 이상의 플라스크에서 그 단백질을 정제에 의해 극복 될 수있다. <p claSS = &quot;jove_content&quot;&gt; 대부분의는 GTPase 이펙터와 규제의 결합은 미끼는 GTPase의 염기 로딩에 따라 달라집니다, 그래서 로딩이 성공 여부를 테스트하는 것이 중요합니다. 로드 중 세포 용 해물로부터 공지 결합 단백질을 침전에 의해 검증 될 수있다. 이러한 GTP-RAC1과에 PAK1 바인드로 이펙터 단백질은 쉽게 해물에서 침전 및 웨스턴 블로 팅 (8) (그림 2B)에 의해 검출 될 수있다. 전이 상태를 안정화시키기는 GTPase없는 뉴클레오티드 우선적 결합 GEFs. GEFs는 일반적으로 비활성 낮은 풍부,의이고 자주 가난시킨다, 그것은 시험 염기 프리는 GTPase에 GEF 또는 GEF 단편을 과발현하는 것이 좋습니다. 우리는 자주 트리오의 첫 번째 DBL 동성을 사용하여, GFP 융합 (GFP - TrioD1 9) (그림 2B)로 표현하지만 어떤 GEF는 작동합니다. 국내 총생산 (GDP)로드는 GTPase에 결합하는 단백질은 희소하다. BINDI로 간단하게 검증 할 수있는 하나의 단백질 (7), 또는 국내 총생산 (GDP) 로딩으로 우리는 최근 RCC2보고도 지구 환경 기금이나 이펙터에 겨. 실험의 출력에 결합는 GTPase 두 GFP 표지 된 결합 파트너를 나타내는 웨스턴 블롯 것이다. 두 단백질을 검출하기 위해 하나의 항체를 사용하여, 두 경쟁 유사한 양 결합되는 농도를 결정할 수 있으므로 상대적 친화 유추. RAC1 인신 매매 단백질의 프로펠러 도메인 사이에이 예 대회에서, coronin-1C (단백질을 RAC1가 결합) 및 RAC1 - 금속 이온 봉쇄 단백질, RCC2은 (단백질 B를 RAC1가 결합), (그림 3A)를 시연한다. coronin-1C 프로펠러 (5 μL)의 일정한 볼륨을 사용하고, RCC2의 증가 볼륨을 추가함으로써, 우리는 RCC2가 강하게 가지고 있음을 보여주는 GFP에서 그 평형이 RCC2 (별표)의 1.25-2.5 μL에 도달시킨다 볼 수 있습니다 coronin-1C보다 RAC1에 대한 친화력. 각 competito 평균치를 정량적 웨스턴 블 롯팅을 사용하여 밴드의 강도를 측정하고, 플롯에 의해R, 평형 점은 볼륨되는 곡선 (도 3b)를 교차를 식별하여 정확하게 계산 될 수있다. 결합 파트너가 서로뿐만 아니라 RAC1 결합에 결합하는 경우 성공적인 경쟁 분석에 사용할 수있는 하나의 장애물이다. 그림 3A의 + B의 우리는 오히려 전체 길이 coronin-1C보다 RCC2과 coronin-1C의 프로펠러 도메인 간의 경쟁을 보여줍니다. 절두 coronin를 사용하는 이유는 coronin-1C는 테일 도메인 통해 RCC2 결합한다는 것이다. 전장 coronin-1C는 RCC2 대해 적정하면, 두 단백질의 결합으로 인해 삼원 복합체 형성보다는 경쟁 (도 3c)로 검출된다. 경쟁이 발생하는 경우, 하나의 단백질의 결합은 다른 감소하면서 증가하고 총 결합 GFP 융합은 일정하게 유지 될 것이다. 경우 삼원 복합체는는 GTPase 결합 단백질 중 하나자를 형성 할 필요가 있기 때문에 여기서 compet적인 억제제는 더 이상 상호 작용하지 않습니다. <img alt="그림 1" src="/files/ftp_upload/53254/53254fig1.jpg" /> 그림 1. 워크 플로우. 경쟁 분석을 사용하여는 GTPase 결합 단백질의 친 화성을 ​​결정하기위한 워크 플로우의 도식 표현. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53254/53254fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/53254/53254fig2.jpg" /> 정제 된 단백질의 그림 2. 검증. (A)으로 정제하여 GFP - 태그 두 단백질의 상대 수익률을 결정하는 안티 GFP와 프로빙, 웨스턴 블롯 분석 결합 단백질 RAC1. 실험 기간 동안 보상이 유형들이 결합 실험에 일치하도록 두 단백질의 농도가 조절 될 수있다. (B) GDP, GTPγS없이 염기로드 GST-RAC1이 내생 PAK1 또는 과발현 GFP - TrioD1에 대한 플롯에 의해 감지 단백질 GFP - TrioD1을 표현 HEK293T에서 해물 배양과 결합되었다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53254/53254fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/53254/53254fig3.jpg" /> 상대 단백질의 그림 3. 웨스턴 블롯 분석을 결합. 예 출력을 경쟁 결합 분석에서. (A) RAC1 5 μL GFP - coronin-1C 프로펠러 도메인과 혼합에 적정 된 GFP - RCC2의 볼륨을 증가하고 국내 총생산 (GDP)로드.으로 GFP에 대한 웨스턴 블롯 결합 단백질, 두 단백질의 검출 시차의 문제가 회피되고, GFP 신호는 두 개의 융합 단백질 사이의 몰비를보고한다. 별표는 eithe의 경쟁 비율을 나타냅니다평형 점의 R 측. (B) 3 개의 독립적 인 실험으로부터 바운드 GFP 융합 단백질의 밴드 강도는 평형에 도달하는 데 필요한 형광 접합 된 이차 항체 및 평균 RCC2의 양을 계산하는 플롯하여 정량적 웨스턴 블롯으로 측정 하였다. (C RAC1 결합 단백질 오히려 경쟁보다는 서로에 결합 원 복합체를 형성 실험에서) 예 출력. 국내 총생산 (GDP)이 장착 된 RAC1 5 μL의 GFP - RCC2와 혼합 GFP - coronin-1C 전체 길이의 볼륨을 증가했다가에 적정 하였다. 바운드 GFP - coronin-1C의 증가를 결합 GFP - RCC2의 손실없이이 원 복합체 형성을 나타냅니다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53254/53254fig3large.jpg" target="_blank">하십시오 이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Comparing the Affinity of GTPase-binding Proteins using Competition Assays at JoVE.com

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

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 ...

comparing-affinity-gtpase-binding-proteins-using-competition

Nanyang Technological University

Research

JoVE Journal

Biology

9.1K 조회수.  University of Bristol. 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.

비디오: Comparing the Affinity of GTPase-binding Proteins using Competition Assays

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 ...

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생화학

Measuring Protein Binding to F-actin by Co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique &#8211; the actin co-sedimentation, or pelleting, assay &#8211; to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin.

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction.

공동 침강에 의한 F-actin에 대한 단백질 결합 측정

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

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 ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

소분자 억제제 스크리닝을 위한 NADH 결합 ATPase 분석의 반 높은 처리량 적응

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent &#946;-arrestin2 recruitment assay, the universality of &#946;-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango&#39;s sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient &#946;-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

PRESTO-탱고 분석기를 사용하여 GPCR 전체 규모의 리간드 스크리닝을 위한 β-Arrestin2 모집의 병렬 심문

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

RhoC GTPase Activation Assay

RhoC GTPase has 91% homology to RhoA GTPase. Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed. However, RhoC GTPase is expressed in metastatic cancer cells at relatively low levels. Therefore, few RhoC-specific reagents have been developed. We have adapted a Rho activation assay to detect RhoC GTPase. This technique utilizes a GST-Rho binding domain fusion protein to pull out active RhoC GTPase. In addition, we can harvest total protein at the beginning of the assay to determine levels of total (GTP and GDP bound) RhoC GTPase. This allows for the determination of active versus total RhoC GTPase in the cell. Several commercial versions of this procedure have been developed however, the commercial kits are optimized for RhoA GTPase and typically do not work well for RhoC GTPase. Parts of the assay have been modified as well as development of a RhoC-specific antibody.

This protocol utilizes a pull down assay to determine the levels of active RhoC GTPase within cells.

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.  ...

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 ...

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 ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

시차 주사 Fluorimetry를 사용하여 단백질 - 리간드 상호 작용의 결정

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).