这项工作是由国家卫生部批准通用汽车69829研究院的支持。

1。测量激动剂浓度反应曲线：没有构活动<ol><li>相对激动剂K B值（RA I）估计，至少有两个激动剂浓度反应曲线是必需的。任何的G蛋白偶联受体的功能反应在体外检测可测激动剂浓度可以控制和单一类型的受体介导的响应。合适的细胞检测包括测量营地 4,7 和 8,9 inositolphosphate积累在细胞株表达了重组受体。整个组织检测的例子包括 10平滑肌或 M 2受体和β-肾上腺素受体 ​​介导的左心房 11日的现场刺激的大鼠收缩的变化收缩的测量。 </li><li>选择一系列激动剂进行分析，包括一个高度有效的激动剂（例如，内源性配体）。在一个给定的实验，测量完成每个激动剂浓度反应曲线。如果激动剂数量太大，在一次实验中完成检测，分为亚组，每个组成一个单一的实验管理的激动剂激动剂。对于每一个分组，测量与在该小组的其他激动剂的高度有效的激动剂浓度响应曲线（见图3）。重复每个小组的实验3 - 6次，约，取决于数据的可变性。 </li><li>对于每个浓度响应曲线，衡量在没有激动剂（基底回应）的响应，并在激动剂浓度的增加存在。空间激动剂浓度均匀日志规模大约每0.3 - 0.7日志10个单位，覆盖的响应范围和定义的最大响应（五最大 ）（见图3）。对于experim细胞株的经济需求，每个测量是一式三份。 </li><li>减去在每个浓度的激动剂存在测量基础响应。以这种方式绘制在图3中的反应进行了计算。 </li></ol> 2。初步分析激动剂浓度反应曲线：没有构活动<ol><li>数据输入到数据表中的棱镜浓度反应实验（例如，图5）。所有的日志激动剂浓度下标有X的列输入激动剂，似乎最大的电子最大值响应测量输入到A列，并为其他激动剂进入到相邻的字母列。进入一个给定的字母列分列复制一个浓度反应曲线的测量。 </li><li>选择图形表上的数据绘制和分析数据，非线性回归分析使用方程题为日志（激动剂）与响应 - 可变斜率（四个参数）。零约束“ 自下而上 ”的参数，并进行回归分析。复制“ 顶”（五最大值 ），并登录到Excel电子表格用于计算初始参数估计欧共体50个参数。 最大的电子最大估计激动剂被指定为“标准的激动剂”，而其他激动剂指定为“试验激动剂”。 </li><li>计算的日志τK初步估计价值标准激动剂（日志EC 50&#39;）的负对数EC 50 OBS（LOGR1）。 计算初步估算为日志日志RA 我的每个测试激动剂值（LOGRA）{（前* 50教统会“）/（顶&#39;* EC 50）}，其中标准激动剂的参数是一个撇号表示。 计算日志亲和力CON（LOGK2 - LOGK5）作为各自的EC 50值的负对数常数测试激动剂。 </li></ol> 3。激动剂RAI值使用非线性回归分析的估计：无构活动<ol><li>在棱镜输入到数据表的数据，描述在2.1以上。确保标准激动剂的数据在A列输入</li><li>输入用户定义的公式，“日志RAI”，几行到棱镜案件共涉及五个激动剂所示： <ul><li> &lt;A&gt; P = LOGK1 </li><li> &lt;A&gt; Q = LOGR </li><li> &lt;〜一个&gt; Q = LOGR + LOGRA </li><li> &lt;B&gt; P = LOGK2 </li><li> &lt;C&gt; P = LOGK3 </li><li> &lt;D&gt; P = LOGK4 </li><li> &lt;E&gt; P = LOGK5 </li><li>为Y = MSYS /（1 + {[（1 +10 ^（X + P））/（10 ^（X + Q））] ^ M}） </li></ul></li><li>输入初始参数估计如下： 输入一个任意低的价值标准激动剂（LOGK1）的日志亲和常数为0。 输入的初步估计LOGR和亲和常数（LOGK2 - LOGK5）和日志RAI值（LOGRA）测试激动剂。 分配传感器的斜坡因子（M）值为1.0的初步估计。 该系统的最大响应（MSYS）值相当于标准激动剂（前） 发送最大分配的初步估计。 </li><li>应用以下参数约束：LOGK1约束的常数，0; LOGR1，MSYS，共享所有数据集的价值M，LOGK2 - LOGK5和LOGRA，没有任何限制。 </li><li>启动非线性回归分析。结果产量日志K OBS和日志天雨我的测试激动剂值。如果标准激动剂是一个完整的激动剂的，那么日志K OB和日志RA I值是准确的。如果实际上是最有效的激动剂（标准激动剂）的部分激动剂，然后在 K OBS值更有效的AGonists可能被高估。尽管如此， 我的日志RA的估计仍然在这种情况下，准确。 </li><li>如果不衔接的回归，实验者不妨阴谋的理论曲线的初始参数估计定义。如果有数据点和理论曲线之间的偏差较大，检查计算初始参数估计。如果有一个或多个激动剂发送最大值相当于标准激动剂，它可能是必要的，以限制他们的日志 K OBS为0值回归衔接的解决方案。 </li></ol> 4。参展激动剂浓度反应曲线测量构受体细胞检测活动<ol><li>激动剂K B值估计的M - 1，细胞在体外实验中使用的展览构受体活性的绝对单位。三方tive受体活性被定义为在应对上述利益受体的表达引起的基础水平的增加。 </li><li>选择激动剂和设计上面1.2节中所描述的那样的实验。 </li><li>对于每一个浓度反应曲线，测量激动剂在非转染细胞的情况下（基底回应），并与利益受体（基底响应+构响应）转染细胞的反应。测量中存在不同浓度的上述1.3节中所述的不同激动剂的响应。 </li><li>计算构受体活性和每个激动剂浓度测量响应减去在未转染细胞与受体的基底响应的反应。图6显示了以这种方式计算，一个信号通路表现出非常低的构活动的各种激动剂的反应。 </li></ol> 5。初步的一个参展alysis激动剂浓度反应曲线构活动<ol><li>输入到一个数据表中的棱镜的数据（例如，图6）在3.1节所述，但还需要输入相应的字母列相应的日志激动剂浓度为-20构受体活性引起的反应。进入一个大的负对数近似零的激动剂浓度。 </li><li>分析如上所述，在2.2节，但按照“自下而上”参数的限制，以便它是“为所有的数据集共享的数据。”复制的“ 顶”（五最大 ），共享“底部”，登录到一个Excel电子表格用于计算初始参数估计的EC50参数。 </li><li>计算初始参数估计如下： 日志标准激动剂（LOGK1）或任何整，测试激动剂的亲和力常数必须确定在不同的实验， 描述以前3 </suP&gt; 计算日志的初步估计K OBS价值的每个部分的测试激动剂日志{（Top&#39; TOP）/ 50（Top&#39;底）（欧共体）}（即，只有在这种情况下LOGK2）， “底“表示构受体激活引起的反应。 计算的初步估计为每个激动剂作为日志的日志K B（LOGKb）{顶/（EC 50底）}。 计算初步估计作为日志的日志τSYS（LOGTsys）{/底部（顶部的“ -底）}。 </li></ol> 6。参展构受体的活性，使用非线性回归分析反应激动剂KB值的估计<ol><li>在棱镜输入到数据表的数据，描述在3.1以上</li><li>输入用户定义的方程，“登录KB”，几行到棱镜两个激动剂条件所示： <ul><li> &lt;A&gt; LOGKOBS = LOGK1 </li><li> &lt;B&gt; LOGKOBS = LOGK2 </li><li> =（10 ^（X + LOGKOBS））1 </li><li> B = 10 ^（X + LOGTSYS + LOGKB） </li><li> C =（10 ^（LOGTSYS）） </li><li>为Y = MSYS / {1 + [（A /（B + C））^ M]} </li></ul></li><li>输入初始参数估计如下： 输入每个激动剂的亲和常数（LOGK1 - 进入下步骤5.3中确定的值）。 输入的 K OBS估计每个测试激动剂。 输入激动剂KB估计。 输入日志τSYS（LOGTsys）估计。 分配的传感器斜率因子（M）和系统的最大响应（MSYS）值1.0和顶部的“（五标准激动剂最大 ），分别。 </li><li>应用以下参数限制：LOGTsys，MSYS，共享所有数据集的价值M，LOGK2和LOGKb，没有任何限制。 </li><li>启动非线性回归分析。结果产量K B的标准激动剂的日志，该日志K OBS </sub&gt;和日志K B部分激动剂，价值观和响应系统（MSYS），τ 系统自由受体复合物的价值（LOGTsys），和传感器斜率因子的运作模式（M）最大。 </li><li>如果不衔接的回归，按照上述点3.6下的步骤。 </li></ol> 7。代表性的成果图5显示了一些我们以前发布的数据毒蕈碱激动剂诱导中国仓鼠卵巢细胞中稳定表达的M 3受体12的磷酸水解。在这个实验中选定的毒蕈碱激动剂的浓度反应曲线测定。如上所述根据第3条估计每个激动剂K B值，表示相对oxotremorine - M的数据进行分析。这些日志RA 我估计，卡巴，-0.56 ± 0.063; areco线，-0.60 ± 0.074;毛果芸香碱，-1.20 ± 0.15; MCN - A - 343，-1.92 ± 0.31。理论曲线代表最小二乘回归方程的数据（3.2节）。该系统的最大响应（M系统）和传感器的斜率因子（M）的相应概算分别为53.9 ± 1.3和1.15 ± 0.09，分别。日志K OBS激动剂值oxotremorine - M的除外，卡巴，5.19 ± 0.14;槟榔碱，5.49 ± 0.12;毛果芸香碱，5.58 ± 0.16和MCN - A - 343，5.27 ± 0.33 。 图6显示了毒蕈碱激动剂刺激的HEK 293细胞稳定表达摹α153磷酸肌醇水解实验的结果。瞬时表达的M 3受体引起基础肌醇水解，这是由于构受体活性增加性。估计日志K B oxotremorine - M和MCN - A - 343的值，根据第6条所述的数据进行了分析。分析产生oxotremorine - M和MCN - A - 343的日志K b值8.30 ± 0.59和6.59 ± 0.77，分别。理论曲线代表数据的回归方程最小二乘（见6.2节）。日志τ 系统 ，男SYS和 M估计分别为-2.29 ± 0.59，95.8 ± 2.8和0.72 ± 0.08，分别。估计的 K OBS MCN - A - 343的价值为5.35 ± 0.46 。 <img alt="图1" src="/files/ftp_upload/3179/3179fig1.jpg" /> 图1单一受体的活性和激活受体人口的平均水平之间的关系。单一受体的活动，一个单一的受体发生transitio的理论行为。NS之间有效（ON）和无效（关）激动剂存在的状态显示。激动剂为有效和无效状态的亲和常数K B和K 一表示， 人口的行为 ，理论的几个受体激动剂存在有效和无效状态之间发生随机转换行为是显示人口显示， 平均人口在一个特定的激动剂浓度存在的受体激活受体的平均水平。激活受体的人口水平，被称为刺激。 <img alt="图2" src="/files/ftp_upload/3179/3179fig2.jpg" /> 图2受体激活功能和疗效（ε）和观察到的亲和常数（K OBS） 激动剂。人口平均之间的关系，平均水平。增加激动剂浓度显示。受体激活引起受体激活受体激活的平均水平是绘制激动剂的激动剂日志浓度。激动剂半最大受体激活所需的负对数浓度相当于日志观察激动剂（ 日志K OBS）的亲和常数。最大的受体激活功能疗效（ε）表示。 <img alt="图3" src="/files/ftp_upload/3179/3179fig3.jpg" /> 图3。[3]在中国仓鼠卵巢细胞表达人类的 M 3受体的各种毒蕈碱激动剂inositolphosphate积累的刺激浓度反应曲线的例子。共有九个激动剂浓度反应曲线进行了测量。实验可以分为两部分。对于每一个部分，标准激动剂（oxotremorinEM）总是额外激动剂一起测试。因此，两种类型的实验显示在 A 和 B都需要测量9激动剂浓度反应曲线，如果只有五激动剂可一次检测。数据Ehlert 等12。 <img alt="图4" src="/files/ftp_upload/3179/3179fig4.jpg" /> 图4。各种毒蕈碱激动剂刺激[3] inositolphosphate积累的HEK 293细胞中表达人类的M 2受体和Gα15浓度反应曲线的例子。对M 2受体表达造成了30 - 35％[3] inositolphosphate响应，这是由于构受体活性。数据Ehlert 等12。 <img alt="图5" src="/files/ftp_upload/3179/3179fig5.jpg" /> 图5。 </强&gt;毒蕈碱激动剂介导的中国仓鼠卵巢细胞的磷脂水解稳定表达人类的M 3受体。显示选定的毒蕈碱激动剂刺激磷酸肌醇水解的浓度反应曲线。从三个实验的平均值±扫描电镜显示。数据Ehlert 等12。 <img alt="图6" src="/files/ftp_upload/3179/3179fig6.jpg" /> 图6。Oxotremorine - M和MCN - A - 343 -介导的表达摹α15 的 M 3受体的HEK 293细胞磷酸肌醇水解。磷酸肌醇水解表示相对于发送 oxotremorine - M 的最大。激动剂的情况下，肌醇水解占约2％的响应的M 3受体的表达。三个实验± SEM手段。在DATA Ehlert 等3。

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没有利益冲突的声明。

由于我们估计RA ​​I（相对K B值）的方法只需要激动剂浓度反应曲线的测量，我们的分析可以做到随时测量这些曲线。 如果一天的到，每天的实验准备工作（例如，细胞或组织）的反应中的变化是大的，每个浓度-反应曲线的响应测量可以被归的“热门”的标准激动剂估计为每一天的实验。 顶部和日志欧共体50的估计，预计...

quantifying agonist activity at g protein-coupled receptors

当激动剂激活G蛋白偶联受体（GPCRs）的人口，就会引发一个信号通路在细胞或组织的反应达到高潮。这个过程可以在一个单一的受体，受体的人口，或下游的反应程度进行分析。在这里，我们描述如何分析下游的反应，以获得激动剂亲和力常数为单一受体的活跃状态的估计。 受体表现为量子开关，有效和无效状态（图1）之间的交替。激活状态的交互与特定的G蛋白或其他信号伙伴。在配位体的情况下，非国家占主导地位。激动剂约束力增加的可能性，受体将进入活跃状态的开关，因为其亲和常数为活跃状态（K B）比为无效状态（K一）要大得多。求和随机输出的人口中的所有受体产生一个恒定的水平在时间上的受体激活。引出半最大受体激活受体激动剂浓度的倒数相当于观测到的亲和常数（K OBS），并处于活动状态的激动剂-受体复合物的一小部分是定义为疗效（ε） （图2）。 已经开发了分析，对GPCRs下游反应的方法， 使估算的K OB和相对有效性激动剂1,2。在这份报告中，我们将展示如何修改此分析来估计激动剂K B值相对另一激动剂。我们的分析，展示构活动，显示如何估计 K B的M -1的绝对单位。 我们分析激动剂浓度反应曲线3,4的方法包括全球非线性回归使用的运作模式5。我们描述一个程序使用的应用软件，棱镜（GraphPad软件公司，加利福尼亚州圣迭戈）。分析得出K产品的OB和疗效（τ）成正比的参数估计。 τKOBS之一激动剂除以另一个，估计是一个相对尺度K B（RA I）6。任何参展构活动的受体，它是可能的估计参数免费受体复合物的疗效（τSYS）成正比。在这种情况下，K B激动剂的价值相当于τKOBS /τ 系统 3 。 我们的方法是有用的，确定为受体亚型激动剂的选择性和量化通过不同的G蛋白受体激动剂信令。

Watch this Scientific Journal Video about Quantifying Agonist Activity at G Protein-coupled Receptors at JoVE.com

Quantifying Agonist Activity at G Protein-coupled Receptors

一个活跃状态激动剂的亲和力常数估算方法（<em&gt; K<sub&gt; B</sub</emG蛋白偶联受体）的描述。分析提供了绝对或相对的措施<em&gt; K<sub&gt; B</sub</em&gt;根据构受体激活是否是可测量的。我们的方法适用于各种反应受体激活下游。

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 ...

quantifying-agonist-activity-at-g-protein-coupled-receptors

Research

JoVE Journal

Biology

18.2K 次观看.  University of California, Irvine. 一个活跃状态激动剂的亲和力常数估算方法（ K B

视频: Quantifying Agonist Activity at G Protein-coupled Receptors

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

Hui Zhang and Niko G. Gubernator contributed equally to this work.

A new means to measure neurotransmission optically using fluorescent dopamine analogs.

多巴胺的释放突触前货箱码头在个别的可视化与FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

科研

生物学

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

在体内 G蛋白偶联受体相互作用光谱分辨的双光子显微镜定量

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

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 ...

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 ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

通过全内反射荧光显微镜可视化G蛋白偶联受体的单事件分辨率网格蛋白介导的内吞作用

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

关联特定基因的DNA甲基化与表达的变化和星形胶质细胞的转录活性<em&gt; KCNJ10</em&gt;（Kir4.1）

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

人生长激素测定血清SPRI，纳米单层屋面和酶联免疫吸附测定比较分析

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

成像G蛋白偶联受体介导的趋化和信令活动在中性粒细胞样细胞HL60

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

配体激活的G蛋白偶联受体内在化的检测通过共聚焦显微镜

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...