Name: measuring g-protein-coupled receptor signaling via radio-labeled gtp binding
Uploaded: 2017-06-09T12:30:00
Description: Watch this Scientific Journal Video about Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding at JoVE.com

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?...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="1034">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">DMEM, high glucose, pyruvate, no glutamine</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">10313021</td>
			<td style="width:264px;">Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">L-glutamine</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">25030081</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Penicillin-Streptomycin</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">15140122</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Opti-MEM I Reduced Serum Medium</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">31985070</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Fetal Bovine Serum</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">16000044</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Cell culture 10-cm plate</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">CLS430167</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Lipofectamine 3000 reagent</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">L3000-008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">1.6 mL microcentrifuge tubes</td>
			<td style="width:189px;">USA Scientific</td>
			<td style="width:119px;">1615-5500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Tris(hydroxymethyl)aminomethane (Trizma base)</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">BP152-1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:463px;">ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA)</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">E3889</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">E9884</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Sucrose</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5016</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:463px;">cOmplete ULTRA Tablets, Mini, EASYpack Protease Inhibitor Cocktail</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">2900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">DL-Dithiothreitol (DTT)</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">DO632</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Sodium chloride (NaCl)</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">BP358-1</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:463px;">Magnesium chloride (MgCl2)</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">M1028-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Pellet pestles motor</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z359971</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Pestles</td>
			<td style="width:189px;">Bel Art</td>
			<td style="width:119px;">F19923-0001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Bovine serum albumin (BSA)</td>
			<td style="width:189px;">Affymetrix</td>
			<td align="right" style="width:119px;">10857</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">[35S]guanosine-5&#8217;-O-(3-thio)triphosphate ([35S]GTP&#947;S)&#160;</td>
			<td style="width:189px;">Perkin Elmer</td>
			<td style="width:119px;">NEG030H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">non-radiolabeled guanosine-5&#8217;-O-(3-thio)triphosphate (GTP&#947;S)&#160;</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">89378</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">guanosine diphosphate (GDP)</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">51060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Bradford reagent</td>
			<td style="width:189px;">Bio-Rad</td>
			<td align="right" style="width:119px;">5000006</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">UV/VIS spectrophotometer</td>
			<td style="width:189px;">Beckman Coulter</td>
			<td style="width:119px;">DU640</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">spectrophotometer cuvettes</td>
			<td style="width:189px;">USA Scientific</td>
			<td style="width:119px;">9090-0460</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">orbital shaker</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">2314</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">thermomixer</td>
			<td style="width:189px;">Eppendorf</td>
			<td align="right" style="width:119px;">535027903</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">glass fiber filters&#160;</td>
			<td style="width:189px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">1821-021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">vacuum filtration apparatus</td>
			<td style="width:189px;">Millipore Corporation</td>
			<td style="width:119px;">XX2702550</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">desktop microcentrifuge</td>
			<td style="width:189px;">Eppendorf</td>
			<td align="right" style="width:119px;">65717</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Scintillation counter</td>
			<td style="width:189px;">Beckman Coulter</td>
			<td style="width:119px;">LS6500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">scintillation fluid&#160;</td>
			<td style="width:189px;">Ecoscint A</td>
			<td style="width:119px;">LS-273</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">scintillation counter vials</td>
			<td style="width:189px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">592690</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">scintillation vial lids</td>
			<td style="width:189px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">592928</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Prism 6</td>
			<td style="width:189px;">GraphPad Software</td>
			<td style="width:119px;">PRISM 6</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:463px;">ATP1A1 antibody</td>
			<td style="width:189px;">Developmental Studies Hybridoma</td>
			<td style="width:119px;">a6F</td>
			<td>1:1000 in 3% BSA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">GAPDH antibody</td>
			<td style="width:189px;">EMD Millipore</td>
			<td style="width:119px;">CB1001</td>
			<td>1:5000 in 3% BSA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">H2B antibody</td>
			<td style="width:189px;">Cell Signaling</td>
			<td style="width:119px;">2934S</td>
			<td>1:2500 in 3% BSA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">PDI antibody</td>
			<td style="width:189px;">Cell Signaling</td>
			<td style="width:119px;">3501S</td>
			<td>1:1000 in 3% BSA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">HA antibody</td>
			<td style="width:189px;">Roche</td>
			<td align="right" style="width:119px;">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 ...

Research

Education

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JoVE Core

Biochemistry

Cell Biology

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Unit 1

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Signaling Networks of G Protein-coupled Receptors

SCIENTISTS IN ACTION

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

使用硝酸纤维素结合分析细菌组氨酸激酶自身磷酸化的定量分析

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

科研

生物化学

Measuring Protein Binding to F-actin by Co-sedimentation

通过共沉淀测量蛋白与F-肌动蛋白的结合

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

Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

用耐热配体测量生物分子 DSC 剖面快速表征折叠和结合的相互作用

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

化学感受器配体结合域的高效复用与纯化方法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

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

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 ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

NEMO IKK绑定域的生产、结晶和结构确定

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

实时测量核苷酸与完整功能性膜蛋白的结合

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...