Name: rhoc gtpase activation assay
Uploaded: 2010-08-22T17:00:00.000+00:00
Duration: 9 min 58 s
Description: Watch this Scientific Journal Video about RhoC GTPase Activation Assay at JoVE.com

Dept. of Defense W81XWH-05-1-0005, W81XWH-06-1-0495, W81XWH-08-1-0029 and W81XWH-08-1-0356

1. Prepare GST-fusion Protein
<ol>
 <li> Glycerol stocks of JM109 competent bacteria containing the first N-terminal 90 amino acids of the rhotekin Rho binding domain (RBD) subcloned into the BamH1/EcoR1 pGEX3x vector are made. 
 
 To ensure High efficiency the following steps MUST be performed for each experiment. In our experience, freshly prepared GST-RBD is key to a robust and accurate GTPase activation assay.</li>
 <li> Add 50 &mu;L (approximate, do not thaw) of glycerol stock to 50 mL of LB amp and grow 37&deg;C overnight with shaking.</li>
 <li> Dilute 1:10 in 500 mL LB-amp and grow for 1 h.</li>
 <li> Induce GST-protein production with 0.1 mM IPTG for 2 hrs.</li>
 <li> Distribute evenly into centrifuge bottles and centrifuge, Sorval GSA3 rotor at 5000 RPM 4&deg;C for 20 min.</li>
 <li> Resuspend pellet in centrifuge bottles, 10 mL of lysis buffer*. 
 *Bacterial Lysis Buffer: 20% Sucrose, 10% glycerol, 50 mM Tris pH 8.0, 0.2 mM Na2S2O5, 2 mM MgCl2, 2 mM DTT, Add fresh PMSF, benzamide, aprotinin and leupeptin.</li>
 <li> Sonicate 2-3 min (this depends on the type of sonicator. We use a Fischer Sonic Dismembranator Model 500 set at mark 6, 50% cycle).</li>
 <li> Centrifuge, Sorvall SS34, 20 min 10,000 rpm at 4&deg;C. At this point a small aliquot can be taken out and run by SDS-PAGE. The gel can be coomassie stained and a band should be apparent at 46 kDa.</li>
 <li> Remove supernatant and add 1 mL of 50% glutatione-Sepharose 4B slurry.</li>
 <li> Incubate for 30 min at 4&deg;C with rotation. Wash 3 times with lysis buffer.</li>
 <li> Resuspend GST-RBD/glutatione-Sepharose beads to a 50% slurry (approximately 1 mL) with GST-fish buffer**. **GST-Fish Buffer: 10% Glycerol, 50 mM Tris pH 7.4, 100 mM NaCl, 1% NP-4, 2 mM MgCl2, Add fresh PMSF, benzamide, aprotinin and leupeptin</li>
</ol>
2. GST-fusion pull down assay
<ol>
 <li> Use 5-10 x 106 cells/assay</li>
 <li> Wash cells once in ice cold PBS, keep plates on ice (we use a flat glass dish filled with ice) and lyse with 0.5 mL GST-fish buffer.</li>
 <li> Incubate 5 min on ice, harvest cells using a rubber policeman or flat scrapper, transfer to microfuge tube and centrifuge 20,000 r.p.m. 5 min at 4&deg;C.</li>
 <li> Transfer supernatant to a fresh microfuge tube. Take 50 &mu;L to determine total Rho (i.e. GDP + GTP bound).</li>
 <li> Add 0.5 mL GST-RBD/glutatione-Sepharose beads and incubate at 4&deg;C, overnight with rotation.</li>
 <li> Wash 6x with GST-fish buffer.</li>
 <li> Resuspend beads in 40 &mu;L 2x-Laemelli sample buffer 
 At this point the concentration of the total Rho protein aliquot taken in 2.4 should be determined and 30 &mu;g prepared to be run on the gel with the corresponding activation sample.</li>
 <li> Boil samples at 90&deg;C for 5 min, centrifuge briefly and take supernatant (i.e. avoid the beads) load and run on a 12-well 4-20% gradient SDS-PAGE gel.</li>
 <li> Run gel at no higher than 150 volt, transfer to membrane using Towbin buffer and constant current of 100 W.</li>
 <li> Block membrane with 5% milk/TBST solution and probe with RhoC-specfic antibody.</li>
</ol>

3. Representative Results
Good results should produce a single band at approximately 22 kDa. Bad results produce multiple bands or high background. This is indicative of protein degradation, incomplete washing of samples or the use of old GST-RBD.

<img src="/files/ftp_upload/2083/2083fig1.jpg" alt="Figure 1" /> 
Figure 1. Three separate cell lines expressing different levels of RhoC GTPase are shown to demonstrate low levels of active and total RhoC, high levels of active and total RhoC or no RhoC. The blot was then stripped and reprobed with an antibody to the house keeping gene actin to serve as a loading control for the total RhoC protein.
<img src="/files/ftp_upload/2083/2083fig2.jpg" alt="Figure 2" /> 
Figure 2. Multiple bands or high background results from protein degradation, incomplete washing of the samples or the use of old GST-RBD.

<ol>
	<li>van Golen, K. L., Wu, Z. F., Qiao, X. T., Bao, L. W., Merajver, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoC+GTPase,+a+novel+transforming+oncogene+for+human+mammary+epithelial+cells+that+partially+recapitulates+the+inflammatory+breast+cancer+phenotype.">RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype.</a> Cancer Res. 60 (20), 5832-5838 (2000).</li><li>van Golen, K. L., Wu, Z. F., Qiao, X. T., Bao, L., Merajver, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoC+GTPase+overexpression+modulates+induction+of+angiogenic+factors+in+breast+cells.">RhoC GTPase overexpression modulates induction of angiogenic factors in breast cells.</a> Neoplasia. 2 (5), 418-425 (2000).</li><li>Golen, K. L. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversion+of+RhoC+GTPase-induced+inflammatory+breast+cancer+phenotype+by+treatment+with+a+farnesyl+transferase+inhibitor.">Reversion of RhoC GTPase-induced inflammatory breast cancer phenotype by treatment with a farnesyl transferase inhibitor.</a> Mol Cancer Ther. 1 (8), 575-583 (2002).</li><li>Golen, K. L. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogen+activated+protein+kinase+pathway+is+involved+in+RhoC+GTPase+induced+motility,+invasion+and+angiogenesis+in+inflammatory+breast+cancer.">Mitogen activated protein kinase pathway is involved in RhoC GTPase induced motility, invasion and angiogenesis in inflammatory breast cancer.</a> Clin Exp Metastasis. 19 (4), 301-311 (2002).</li><li>Yao, H., Dashner, E. J., van Golen, C. M., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoC+GTPase+is+required+for+PC-3+prostate+cancer+cell+invasion+but+not+motility.">RhoC GTPase is required for PC-3 prostate cancer cell invasion but not motility.</a> Oncogene. 25 (16), 2285-2296 (2006).</li><li>Hall, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+I+Collagen+receptor+(alpha2beta1)+signaling+promotes+prostate+cancer+cell+invasion+through+RhoC+GTPase.">Type I Collagen receptor (alpha2beta1) signaling promotes prostate cancer cell invasion through RhoC GTPase.</a> Neoplasia. 10 (8), 797-803 (2008).</li><li>Sequeira, L., Dubyk, C. W., Riesenberger, T. A., Cooper, C. R., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+GTPases+in+PC-3+prostate+cancer+cell+morphology,+invasion+and+tumor+cell+diapadesis.">Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell diapadesis.</a> Clin Exp Metastasis. 25, 569-579 (2008).</li><li>van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL2+Induces+Prostate+Cancer+Transendothelial+Cell+Migration+via+Activation+of+the+Small+GTPase+Rac.">CCL2 Induces Prostate Cancer Transendothelial Cell Migration via Activation of the Small GTPase Rac.</a> J Cell Biochem. 104, 1587-1597 (2008).</li><li>Chatterjee, M., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Int J Cancer. , (2010).</li><li>Sander, E. E., ten Klooster, J. P., van Delft, S., van der Kammen, R. A., Collard, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac+downregulates+Rho+activity:+reciprocal+balance+between+both+GTPases+determines+cellular+morphology+and+migratory+behavior.">Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior.</a> J Cell Biol. 147 (5), 1009-1022 (1999).</li></ol>

No conflicts of interest declared.

The most critical step of the procedure is the generation of fresh GST-RBD. Failure to pay close attention to this step is main reason for failure. All the steps, from generation of the GST-RBD (1.2) to the overnight incubation with the cell lysate (2.5), should be done in a single day. Another crucial step is to make certain that there are enough cells to produce the cell lysates. This is of particular importance when looking at RhoC GTPase, which tends to be present in low levels in the cell.

<table><tbody><tr><td>Gluthione-sepharose 4B beads</td><td>GE Healthcare</td><td>17-0756-01</td><td></td></tr><tr><td>Criterion 4-20% Tris-HCl gels</td><td>Bio-Rad</td><td>345-0032</td><td></td></tr></tbody></table>

rhoc gtpase activation assay

RhoC GTPase 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.

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

RhoC GTPase Activation Assay

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

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

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12.4K Views.  University of Delaware. This protocol utilizes a pull down assay to determine the levels of active RhoC GTPase within cells.

Video: RhoC GTPase Activation Assay

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

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a major pharmacological target for multiple indications, including analgesia, blood pressure regulation, and the treatment of psychiatric disease. Upon ligand binding, GPCRs catalyze the activation of intracellular G-proteins by stimulating the incorporation of guanosine triphosphate (GTP). Activated G-proteins then stimulate signaling pathways that elicit cellular responses. GPCR signaling can be monitored by measuring the incorporation of a radiolabeled and non-hydrolyzable form of GTP, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), into G-proteins. Unlike other methods that assess more downstream signaling processes, [35S]GTP&#947;S binding measures a proximal event in GPCR signaling and, importantly, can distinguish agonists, antagonists, and inverse agonists. The present protocol outlines a sensitive and specific method for studying GPCR signaling using crude membrane preparations of an archetypal GPCR, the &#181;-opioid receptor (MOR1). Although alternative approaches to fractionate cells and tissues exist, many are cost-prohibitive, tedious, and/or require non-standard laboratory equipment. The present method provides a simple procedure that enriches functional crude membranes. After isolating MOR1, various pharmacological properties of its agonist, [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO), and antagonist, naloxone, were determined.

Guanosine triphosphate (GTP) binding is one of the earliest events in G-Protein-Coupled Receptor (GPCR) activation. This protocol describes how to pharmacologically characterize specific GPCR-ligand interactions by monitoring the binding of the radio-labeled GTP analog, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), in response to a ligand of interest.

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

Biochemistry

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.

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

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

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

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.

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

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

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

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

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

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

Medicine

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).

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

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.

This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

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

Competition Binding Assay to Study Competing GTPase-Binding Protein Partners

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

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

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Protein-protein interactions

Small GTPases - Ras and Rho

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

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

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KEY TERMS AND CONCEPTS

Activation and Inactivation of G Proteins

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

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

RhoC GTPase Activation Assay

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