这项工作得到了国家卫生研究院授予的DA-000266和医学科学家培训计划T32授权（CV，NWZ和PCS）的支持。作者还要感谢Somersault 18:24（somersault1824.com）的科学与医学插图图书馆。

1.重组HA-MOR1在培养细胞中的表达注意:按照无菌层流罩中的所有细胞培养方案。 <ol><li>用70％乙醇灭菌细胞培养层流罩，并在整个细胞培养物中保持无菌技术。 </li><li>制备人胚胎肾细胞293（HEK293）细胞培养基，补充有2mM L-谷氨酰胺，1％青霉素/链霉素和10％胎牛血清（FBS）的完全Dulbecco&#39;s修饰的Eagle培养基（DMEM）:DMEM，pH7.4 ）。 </li><li>将2.5×10 6 HEK293细胞在10毫升完全DMEM中的10厘米组织培养板上孵育，并在37℃和5％CO 2下孵育直至达到70-80％汇合（O / N）。 注意:为了收集足够的蛋白质进行完整的GTP结合实验，将至少3×10厘米的细胞平板。 </li><li>使用选择的转染试剂，用HA-MOR1转染细胞，的指导方针。孵育36-48小时。 注意:人MOR-1 cDNA（NCBI参考序列:NM_000914.4）是Gavril Pasternak的大量礼物，并被克隆到pCMV-HA中。 </li></ol>细胞分馏和膜收集<ol><li> 准备以下裂解缓冲液: <ol><li>缓冲液1:10mM 4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES），pH 7.4; 1mM乙二醇 - 双（β-氨基乙基醚）-N，N，N&#39;，N&#39;-四乙酸（EGTA）; 10％蔗糖;蛋白酶抑制剂鸡尾酒;和1mM二硫苏糖醇（DTT）。在隔膜当天新增DTT。 </li><li>缓冲液2:10mM HEPES，pH 7.4; 1mM EGTA; 1mM MgCl 2 ;和1mM DTT。在隔膜当天新增DTT。 </li></ol></li><li>从培养箱中取出转染的细胞（步骤1.4），吸出培养基，并用5 mL冰冷的磷酸盐缓冲盐水（PBS）冲洗细胞。吸出PBS和多余的液体碟子。 </li><li>向细胞中加入600μLBuffer 1。使用细胞刮刀，将细胞从板表面移开，并将细胞悬液移液至1.6 mL微量离心管中。 </li><li>立即在液氮中快速冷冻微量离心管。样品冷冻后，将溶解产物在冰上解冻，或将其置于-80°C下长时间储存​​。 </li><li>对所有细胞板重复步骤2.3和2.4。 </li><li> 一旦裂解物解冻，使用单脉冲微管匀浆器打开细胞。将均化器脉冲3-5次，持续10秒，将管放回冰上30秒钟之间。 <ol><li>收集20μL作为全细胞级分。 </li></ol></li><li>在1,000×g和4℃下将剩余样品离心10分钟。收集上清液并置于新的1.6 mL微量离心管中。 </li><li> 将沉淀物重悬于100μL缓冲液1中，并用研杵再造。脉冲同性恋生成器3-5次5秒钟，将管放回冰上30秒钟之间。 <ol><li>在1,000 xg和4°C下将样品再次离心10分钟。将上清液与步骤2.7的上清液合并。 注意:剩余的颗粒含有核和膜蛋白。 </li></ol></li><li>在11,000 xg和4℃下将合并的上清液离心20分钟。将上清液（细胞溶质级分）分离成新的1.6 mL微量离心管。 </li><li>将沉淀物重悬于200μL的缓冲液2中。将沉淀物均质化3-5次。在21,100 xg和4℃下将样品离心20分钟。吸出上清液。将含有粗膜级分的沉淀重悬于50μL缓冲液2中。 </li><li>立即进行GTPγS结合实验（第3部分）或在液氮中快速冷冻并储存在-80℃。 </li></ol> [ 35 S]GTPγS结合</p&gt; 注意:处理[ 35 S]GTPγS时和使用[ 35 S]GTPγS结合实验时，请使用标准放射化学安全性协议。随时戴防护手套和实验服。检查包装材料是否有泄漏或裂缝。根据制度方案处理废物和多余的试剂。 <ol><li>通过混合50mM HEPES，pH 7.4，制备不完全结合缓冲液（IBB）; 5mM MgCl 2; 100mM NaCl;和1mM乙二胺四乙酸（EDTA）。 </li><li>在10mM Tris-HCl，pH7.6和10mM DTT中稀释储备物[35S]GTPγS至50nM。制备500μL等分试样并将其储存在-80°C。在开始实验之前立即使用新鲜解冻的等分试样。在冰上解冻等分试样。 </li><li>通过将1mgGTPγS粉末溶解在177.62μL纯水中以制备10mM的原料浓度来制备非放射性标记的GTPγS。制作10-μL等分试样并存放-20°C。 </li><li>通过补充IBB来制备完整的结合缓冲液（CBB），包括终浓度为1mM DTT，0.1％（wt / vol）牛血清白蛋白（BSA），10μMGDP和0.1nM [35S]GTPγS。 注意:CBB现在包含放射性标记的GTP。在使用放射性溶液时采取适当的安全预防措施。 </li><li>在冰上从步骤2.11中解冻膜级分。 </li><li> 通过使用分光光度计在595nm的Bradford蛋白质测定来定量膜级分的蛋白质浓度。 <ol><li>用缓冲液2制备稀释系列的BSA蛋白标准品，终浓度为0,25,500,750,1,500,2,500和5,000μg/ mL。 </li><li>在比色杯中加入2μL每个标准品或膜1 mL的Bradford试剂。通过涡旋充分混合。 </li><li>使用0μg/ mL蛋白质的比色杯将分光光度计调整到595 nm波长，空白。 </li><li>等待5分钟，r每个标准和每个样品在595 nm波长。 </li><li>绘制标准品的吸光度与浓度。使用Beer-Lambert定律（A =εcl，其中ε=消光系数，l =比色皿长度，c =浓度），计算膜样品的浓度。 </li></ol></li><li>在IBB中将膜级分稀释至浓度为100μg蛋白/ mL。 注意:一个10厘米的HEK293细胞皿通常产生1和3毫克蛋白质/毫升之间。 </li><li> 在单个1.6 mL微量离心管中制备以下实验条件:配体稀释系列，测量基础GTP结合的基础活性条件和非特异性结合条件以测量非特异性GTP结合。 <ol><li>对于配体稀释系列，在最终体积为100μL的CBB中制备目标配体的稀释系列。将配体系列制备成2倍所需的最终浓度附件。空间配体浓度以充分覆盖一系列响应。 <ol><li>为了测定DAMGO对通过MOR1的GTP结合的影响，制备CBB中2mM，200μM，20μM，2μM，200nM，20nM，2nM，200pM，20pM和2pM的DAMGO稀释液体积:100μL）。 注意:例如，如果感兴趣的配体具有10μM的预期EC 50值，则制备200nM，2μM，20μM，200μM和2mM的配体稀释液。这五个条件涵盖了广泛的浓度，是测定所需浓度的2倍。 </li></ol></li><li>对于基底结合，只需准备100μLCBB的管。 </li><li>对于非特异性结合，制备配有99μLCBB的管，补充有1μL的2mM非放射性标记的GTPγS。 </li></ol></li><li>向每个实验条件中加入100μL稀释的膜溶液（步骤3.7）。 </li><li>孵化膜与各种实验在温热混合器或轨道振荡器中，在25℃下在1.6mL微量离心管中进行30分钟的条件。 </li></ol>膜过滤<ol><li>通过组合50mM Tris-HCl，pH 7.4制备洗涤缓冲液; 5mM MgCl 2 ;和50mM NaCl的蒸馏水中。 </li><li>将玻璃纤维过滤器预先放在水中10分钟。 注意:过滤器的孔径为1μm，直径为2.1cm，厚度为675μm。 </li><li>从温热混合器或轨道振动筛中取出样品（步骤3.10）。将样品短暂脉冲旋转5秒以收集管底部的每个样品。 </li><li>取下真空过滤装置的盖子。将预浸过滤器放置在设备的真空端口上。重新固定设备盖以形成真空密封。打开真空</li><li>将每个200μL实验条件的195μL吸管到过滤器上，以尽量减少吸附到管壁和/或移液管的误差婷。 </li><li>用1 mL冰冷的洗涤缓冲液清洗过滤器三次。 </li></ol>液体闪烁计数<ol><li>将5 mL闪烁计数小瓶放入计数架。向每个小瓶中加入5 mL闪烁液。 </li><li>关闭真空（步骤4.4）。取下真空过滤装置的盖子。使用镊子从过滤装置的真空端口中取出过滤器，并将每个过滤器放入单独的5 mL闪烁瓶中。 </li><li>用195μLCBB准备一个样品瓶，以确定最大信号。 </li><li>安全地盖住每个小瓶。在25°C的轨道振荡器上孵育小瓶10分钟。 </li><li>打开闪烁计数器。使用标准相关的闪烁程序对闪烁计数器进行编程，以测量每个样品35分钟同位素发射5分钟。 </li><li>按"开始"进行计数。 </li><li>计数完成后，处理计数的小瓶作为危险废物。 </li></ol>数据分析<ol><li> 将数据输入统计和分析软件。 <ol><li>从每个其他测量中减去非特异性结合以确定具体的结合。 注意:通过用非放射性标记的GTPγS孵育的样品测定非特异性结合程度（步骤3.8.3）。 </li><li>打开统计和分析软件并选择一个XY数据集条目。 </li><li>在[ 35 S]GTPγS结合实验中输入特定的结合数据到统计和分析软件中的数据表中。在"X"列中输入摩尔浓度的激动剂浓度。输入关联的35 S计数为"Y"值。 </li></ol></li><li> 转换数据。 <ol><li>通过单击"分析"将X值转换为各自的对数。从内置选择"变换"n分析。 </li><li>在"变换"对话框中，选择"使用变换X值"，然后选择"X = log（X）"功能。选择创建一个新的结果图。 </li><li>点击"确定"。 </li></ol></li><li> 归一化Y值。 <ol><li>显示变换结果后，单击"分析"。从内置分析中选择"规范化"。 </li><li>在"规范化"对话框中，选择单选按钮将0％定义为"每个数据集中的最小值"，100％作为"每个数据集中的最大值"。选择框以显示结果百分比。选择框以创建结果的新图形。 </li><li>点击"确定"。 </li></ol></li><li> 将非线性回归曲线拟合到绘制的数据。进行非线性回归分析。 <ol><li>显示标准化结果后，单击"分析"。从内置分析，选择"非线性回归（曲线拟合）"。 </li><li>如果调查激动剂，请从对话框中选择"log（激动剂）vs.归一化反应 - 可变斜率"。 </li><li>如果调查拮抗剂，则从对话框中选择对数（拮抗剂）与归一化反应 - 变量斜率。 </li><li>点击"确定"。 </li></ol></li><li>查看图表和结果，以确保合适和数据合理协议。确保回归与绘制数据的一般模式相匹配，并且残差不会大到使得剂量 - 反应曲线平坦。 注意:软件将总结非线性回归的结果，详细介绍引起半最大响应（EC 50 ），希尔系数（ H H ）和半最大抑制浓度（IC 50 ）的浓度。 </li><li> 衍生激动剂/拮抗剂参数效力。 <ol><li>推导出激动剂平衡解离常数（K b ）来自以下实验16,17,18 <ol><li>评估激动剂与固定浓度拮抗剂竞争的剂量反应的变化。这里EC 50 &#39;= EC 50 （1 + [拮抗剂] /拮抗剂Kb ），其中EC 50 &#39;是右移EC 50 。 </li><li>评估[ 35 S]GTPγS结合不同浓度的拮抗剂与固定浓度的激动剂竞争。这里，K b = IC 50 /（2 +（[激动剂] /激动剂EC 50 ） n ） 1 / n -1，其中n是激动剂的Hill系数。 </li></ol></li></ol></li><li>根据数据的变异性，对每个配体重复实验（步骤3.8-5.6）约3-5次，并使用统计和分析软件对结果进行平均是。 </li></ol>

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R., 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=Pharmacological+Assessment+of+M1+Muscarinic+Acetylcholine+Receptor-Gq/11+Protein+Coupling+in+Membranes+Prepared+from+Postmortem+Human+Brain+Tissue.">Pharmacological Assessment of M1 Muscarinic Acetylcholine Receptor-Gq/11 Protein Coupling in Membranes Prepared from Postmortem Human Brain Tissue.</a> J. Pharm. Exp. Ther. 325 (3), 869-874 (2008).</li><li>Milligan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles:+extending+the+utility+of+[35S]GTP+gamma+S+binding+assays.">Principles: extending the utility of [35S]GTP gamma S binding assays.</a> Trends. Pharmacol. Sci. 24 (2), 87-90 (2003).</li><li>Harrison, C., Traynor, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+[35S]GTPgammaS+binding+assay:+approaches+and+applications+in+pharmacology.">The [35S]GTPgammaS binding assay: approaches and applications in pharmacology.</a> Life Sci. 74 (4), 489-508 (2003).</li><li>Traynor, J. R., Nahorski, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+by+mu-opioid+agonists+of+guanosine-5'-O-(3-[35S]thio)triphosphate+binding+to+membranes+from+human+neuroblastoma+SH-SY5Y+cells.">Modulation by mu-opioid agonists of guanosine-5'-O-(3-[35S]thio)triphosphate binding to membranes from human neuroblastoma SH-SY5Y cells.</a> Mol. Pharmacol. 47 (4), 848-854 (1995).</li><li>Sittampalam, G. S., 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=GTP&#947;S+Binding+Assays.">GTP&#947;S Binding Assays.</a> Assay Guidance Manual. , (2004).</li><li>Strange, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+GTP&#947;S+([35S]GTP&#947;S+and+Eu-GTP&#947;S)+binding+assay+for+analysis+of+ligand+potency+and+efficacy+at+G+protein-coupled+receptors.">Use of the GTP&#947;S ([35S]GTP&#947;S and Eu-GTP&#947;S) binding assay for analysis of ligand potency and efficacy at G protein-coupled receptors.</a> Br. J. Pharmacol. 161 (6), 1238-1249 (2010).</li></ol>

作者宣称没有竞争的兴趣。

本方案描述了两种单独但互补的方法:将细胞和组织分成广泛但不同的隔室的简单方法，以及通过测量[ 35 S]GTPγS结合来研究GPCR信号的方法。 有效的细胞分馏具有广泛的应用范围，从蛋白质的提取和富集到蛋白质亚细胞定位的评估，到受体药理学的研究。虽然存在分选细胞和组织的替代方法，但是这里提出的方案比较便宜，更快，更简单。然而，与更成熟的方法不同，...

G蛋白偶联受体（GPCR）是一系列大量的细胞表面受体，负责一系列生理过程，包括镇痛，嗅觉和行为1 。 GPCR通过检测特定的外部信号并随后刺激细胞内信号传导而起作用。因此，它们标志着电池的外部和内部环境之间的关键连接。由于GPCR在生物学中的关键作用，它们已经成为基础研究和药物发现的主要目标2,3 。 与其他结合离散配体的受体家族不同，GPCR可以结合非常不同类型的分子。虽然一个GPCR可能与肽相互作用，但另一个可能会感测到光子，小分子或离子1,4 。虽然他们的配体是多样的，GPCR在他们的总体架构师是统一的ure和功能。单个GPCR由七个具有细胞外氨基末端和细胞内羧基末端的α螺旋跨膜蛋白5,6组成 。 GPCR与由α，β和γ亚基组成的细胞内G-蛋白 - 异源三聚蛋白复合物偶联 - 介导多种信号通路7 。 Gα亚基是鸟嘌呤核苷酸结合蛋白，当与鸟苷二磷酸（GDP）结合时是无活性的，当与鸟苷三磷酸（GTP）结合时，其活性为8,9 。当GPCR结合其配体时，它们经历构象变化，允许Gα从Gβγ解离，从而允许Gα与GTP 7交换GDP。受体本身在其羧基末端被各种丝氨酸/苏氨酸磷酸化内源性激酶10,11并且内化以减弱受体信号传导12,13,14 。同时，激活的Gα单体和Gβγ二聚体进行激活不同的信号通路7 。每个G蛋白亚基有几种同种型，每种同种型都针对特定的下游途径和二级信使系统。主要的Gα同种型包括G s ，G q ，G i / o和G 12-13 。通常，单个GPCR与特定的Gα同种型相关联，从而将外部刺激与特异性细胞反应1相联系 。 表征GPCR-配体相互作用对于了解受体的生物学至关重要。由于GDP / GTP交易是最早的一天遵循配体结合的nts，监测GTP结合可以测量GPCR活化或抑制。在GPCR信号传导中测定更多的下游事件通常不是定量的或化学计量的，可能不区分完全激动剂与部分激动剂，并且可能需要昂贵的试剂。此外，增加的GTP与Gα蛋白的结合是GPCR激活后几乎是普遍的事件，这意味着测量GTP结合是广泛适用于监测大多数GPCR的活性的测定。测量GTP结合是一种简单而快速的方法来监测过表达感兴趣受体或天然组织的细胞中的GPCR信号。本方案详细描述了使用原型GPCR（μ-阿片受体（MOR1））的功能性GTP结合测定来定量测定激动剂和拮抗剂对GPCR信号传导的活性。 该方案首先概述了如何从过表达MOR1的细胞中分离粗膜。注意到该方案不限于过表达系统，并且可以应用于许多来源的膜，包括表达多种受体和G蛋白的天然组织或制剂。然后该方案详细说明如何响应不同浓度的[D-Ala，N-MePhe，Gly-ol] - 脑啡肽（DAMGO）或纳洛酮（MOR1激动剂和拮抗剂）测量放射性GTP类似物与这些膜的结合，分别。 GTP类似物[ 35 S]鸟苷-5&#39;-O-（3-硫代）三磷酸（[ 35 S]GTPγS）是不可水解的。该性质是至关重要的，因为Gα亚基表现出内在的GTPase活性7 ，并且可消除可水解的GTP放射化学上的标记的γ磷酸盐。然后将膜捕获到玻璃纤维过滤器上并洗涤，之后通过液体闪烁计数定量放射性标记的GTP。可以推导出多个药理学参数包括受体 - 配体相互作用，包括激动剂的半最大反应（EC 50 ）和希尔系数（n H ）以及拮抗剂16,17的半最大抑制浓度（IC 50 ）和平衡解离常数（K b ） ， 18 。

<table><tbody><tr><td>DMEM, high glucose, pyruvate, no glutamine</td><td>Thermo Fisher Scientific</td><td>10313021</td><td>Warm in a 37&amp;deg;C water bath before use</td></tr><tr><td>L-glutamine</td><td>Thermo Fisher Scientific</td><td>25030081</td><td>Warm in a 37&amp;deg;C water bath before use</td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>Warm in a 37&amp;deg;C water bath before use</td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Thermo Fisher Scientific</td><td>31985070</td><td>Warm in a 37&amp;deg;C water bath before use</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Thermo Fisher Scientific</td><td>16000044</td><td>Warm in a 37&amp;deg;C water bath before use</td></tr><tr><td>Cell culture 10 cm plate</td><td>Sigma-Aldrich</td><td>CLS430167</td><td></td></tr><tr><td>Lipofectamine 3000 reagent</td><td>Thermo Fisher Scientific</td><td>L3000-008</td><td></td></tr><tr><td>1.6 mL microcentrifuge tubes</td><td>USA Scientific</td><td>1615-5500</td><td></td></tr><tr><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane (Trizma base)</td><td>Thermo Fisher Scientific</td><td>BP152-1</td><td></td></tr><tr><td>ethylene glycol-bis(&amp;beta;-aminoethyl ether)-N,N,N&#039;,N&#039;-tetraacetic acid (EGTA)</td><td>Sigma-Aldrich</td><td>E3889</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>E9884</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S5016</td><td></td></tr><tr><td>cOmplete ULTRA Tablets, Mini, EASYpack Protease Inhibitor Cocktail</td><td>Sigma-Aldrich</td><td>2900</td><td></td></tr><tr><td>DL-Dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>DO632</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Thermo Fisher Scientific</td><td>BP358-1</td><td></td></tr><tr><td>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>M1028-1</td><td></td></tr><tr><td>Pellet pestles motor</td><td>Sigma-Aldrich</td><td>Z359971</td><td></td></tr><tr><td>Pestles</td><td>Bel Art</td><td>F19923-0001</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Affymetrix</td><td>10857</td><td></td></tr><tr><td>[&lt;sup&gt;35&lt;/sup&gt;S]guanosine-5&amp;rsquo;-O-(3-thio)triphosphate ([&lt;sup&gt;35&lt;/sup&gt;S]GTP&amp;gamma;S)&amp;nbsp;</td><td>Perkin Elmer</td><td>NEG030H</td><td></td></tr><tr><td>nonradiolabeled guanosine-5&amp;rsquo;-O-(3-thio)triphosphate (GTP&amp;gamma;S)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>89378</td><td></td></tr><tr><td>guanosine diphosphate (GDP)</td><td>Sigma-Aldrich</td><td>51060</td><td></td></tr><tr><td>Bradford reagent</td><td>Bio-Rad</td><td>5000006</td><td></td></tr><tr><td>UV/VIS spectrophotometer</td><td>Beckman Coulter</td><td>DU640</td><td></td></tr><tr><td>spectrophotometer cuvettes</td><td>USA Scientific</td><td>9090-0460</td><td></td></tr><tr><td>orbital shaker</td><td>Thermo Fisher Scientific</td><td>2314</td><td></td></tr><tr><td>thermomixer</td><td>Eppendorf</td><td>535027903</td><td></td></tr><tr><td>glass fiber filters&amp;nbsp;</td><td>GE Healthcare Life Sciences</td><td>1821-021</td><td></td></tr><tr><td>vacuum filtration apparatus</td><td>Millipore Corporation</td><td>XX2702550</td><td></td></tr><tr><td>desktop microcentrifuge</td><td>Eppendorf</td><td>65717</td><td></td></tr><tr><td>Scintillation counter</td><td>Beckman Coulter</td><td>LS6500</td><td></td></tr><tr><td>scintillation fluid&amp;nbsp;</td><td>Ecoscint A</td><td>LS-273</td><td></td></tr><tr><td>scintillation counter vials</td><td>Beckman Coulter</td><td>592690</td><td></td></tr><tr><td>scintillation vial lids</td><td>Beckman Coulter</td><td>592928</td><td></td></tr><tr><td>Prism 6</td><td>GraphPad Software</td><td>PRISM 6</td><td></td></tr><tr><td>ATP1A1 antibody</td><td>Developmental Studies Hybridoma</td><td>a6F</td><td>1:1000 in 3% BSA</td></tr><tr><td>GAPDH antibody</td><td>EMD Millipore</td><td>CB1001</td><td>1:5000 in 3% BSA</td></tr><tr><td>H2B antibody</td><td>Cell Signaling</td><td>2934S</td><td>1:2500 in 3% BSA</td></tr><tr><td>PDI antibody</td><td>Cell Signaling</td><td>3501S</td><td>1:1000 in 3% BSA</td></tr><tr><td>HA antibody</td><td>Roche</td><td>11867423001</td><td>1:2000 in 3% BSA</td></tr></tbody></table>

measuring g-protein-coupled receptor signaling via radio-labeled gtp binding

G-蛋白偶联受体 （GPCR）是一系列跨膜受体，在正常细胞生理学中起关键作用，构成多种适应症的主要药理学靶点，包括镇痛，血压调节和精神病治疗。在配体结合后，GPCRs通过刺激三磷酸鸟苷（GTP）的掺入来催化胞内G蛋白的活化。活化的G蛋白刺激引发细胞反应的信号通路。可以通过测量GTP，[ 35 S]鸟苷-5&#39;-O-（3-硫代）三磷酸（[ 35 S]GTPγS）的放射性标记和不可水解形式掺入G蛋白来监测GPCR信号。与评估更多下游信号传导过程的其他方法不同，[ 35 S]GTPγS结合测量GPCR信号中的近端事件，并且重要的是可以区分激动剂ts，拮抗剂和反向激动剂。本方案概述了使用原型GPCR（μ-阿片样物质受体（MOR1））的粗膜制剂研究GPCR信号的敏感和具体方法。虽然存在分离细胞和组织的替代方法，但许多成本过高，繁琐和/或需要非标准的实验室设备。本方法提供了丰富功能性粗膜的简单方法。分离MOR1后，测定其激动剂[D-Ala，N-MePhe，Gly-ol] - 脑啡肽（DAMGO）和拮抗剂纳洛酮的各种药理学性质。

细胞分离可用于从细胞溶质和核蛋白中分离和富集膜相关蛋白。 图1是Western印迹，证实在亚细胞分馏过程中可以收集的三种主要级分的含量。具体来说， 图1显示了组蛋白H2B和甘油醛-3-磷酸脱氢酶（GAPDH），核蛋白和胞质蛋白质的分离干净地分离膜蛋白（ 即 Na + / K + ATP酶，蛋白质二硫键异构酶（PDI）和HA-MOR1）分别。另外?...

Watch this Scientific Journal Video about Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding at JoVE.com

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

三磷酸鸟苷（GTP）结合是G蛋白偶联受体（GPCR）激活中最早的事件之一。该方案描述了如何通过监测放射性标记的GTP类似物[ 35 S]鸟苷-5&#39;-O-（3-硫代）三磷酸（[ 35 S]GTPγS）的结合在药理学上表征特异性GPCR-配体相互作用对感兴趣配体的反应。

通过放射性标记的GTP结合测量G蛋白偶联的受体信号

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

measuring-g-protein-coupled-receptor-signaling-via-radio-labeled-gtp

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Unit 1

Pharmacodynamics

Signaling Networks of G Protein-coupled Receptors

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16.2K 次观看.  Johns Hopkins University School of Medicine. 三磷酸鸟苷（GTP）结合是G蛋白偶联受体（GPCR）激活中最早的事件之一。该方案描述了如何通过监测放射性标记的GTP类似物[ 35 S]鸟苷-5'-O-（3-硫代）三磷酸（[ 35 S]GTPγS）的结合在药理学上表征特异性GPCR-配体相互作用对感兴趣配体的反应。 

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

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.

膜分馏和 gtpase-关联免疫吸附法检测小 gtpase 预基化和 gtp 结合

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

科研

生物化学

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.

使用 PRESTO-Tango Assay 在 GPCR-宽范围范围内对 β-Arrestin2 进行配制筛查的平行审讯

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

A Pipeline to Investigate the Structures and Signaling Pathways of Sphingosine 1-Phosphate Receptors

Lysophospholipids (LPLs) are bioactive lipids that include sphingosine 1-phosphate (S1P), lysophosphatidic acid, etc. S1P, a metabolic product of sphingolipids in the cell membrane, is one of the best-characterized LPLs that regulates a variety of cellular physiological responses via signaling pathways mediated by sphingosine 1-phosphate receptors (S1PRs). This implicated that the S1P-S1PRs signaling system is a remarkable potential therapeutic target for disorders, including multiple sclerosis (MS), autoimmune disorders, cancer, inflammation, and even COVID-19. S1PRs, a small subset of the class A G-protein coupled receptor (GPCR) family, are composed of five subtypes: S1PR1, S1PR2, S1PR3, S1PR4, and S1PR5. The lack of detailed structural information, however, impedes the drug discovery targeting S1PRs. Here, we applied the cryo-electron microscopy method to solve the structure of the S1P-S1PRs complex, and elucidated the mechanism of activation, selective drug recognition, and G-protein coupling by using cell-based functional assays. Other lysophospholipid receptors (LPLRs) and GPCRs can also be studied using this strategy.

S1P exerts its diverse physiological effects through the S1P receptors (S1PRs) subfamily. Here, a pipeline is described to expound on the structures and function of S1PRs.

研究鞘氨醇1-磷酸受体结构和信号通路的管道

Lysophospholipids (LPLs) are bioactive lipids that include sphingosine 1-phosphate (S1P), lysophosphatidic acid, etc. S1P, a metabolic product 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.

用于重组G蛋白偶联受体高通量筛选的"双加法"钙荧光测定

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

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.

成像摹蛋白偶联受体（GPCR）介导的信号，控制趋化粘菌</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

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

量化在G蛋白偶联受体激动剂活性

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the 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.

一种基于动力学荧光的 Ca2 +动员法, 用于识别 G 蛋白偶联受体激动剂、拮抗器和构调制器

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

医学

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

基因编码的分子探针来研究G蛋白偶联受体

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

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).

G蛋白偶联受体的基于荧光的钙动员含量表征

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

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between G&#945; subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the &#946;2-adrenergic receptor and the G&#945;s C-terminus peptide upon treatment with 100 &#181;M isoproterenol hydrochloride as previously characterized1. Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a G&#945; C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.

Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, F&#1255;rster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

在活细胞悬浮液荧光测定G蛋白选择性GPCR构象测量使用FRET传感器

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group ...

通过放射性标记的GTP结合测量G蛋白偶联的受体信号

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