Name: a bw reporter system for studying receptor-ligand interactions
Uploaded: 2019-01-07T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A BW Reporter System for Studying Receptor-Ligand Interactions at JoVE.com

The authors thank Esther Singer for language editing. This study was supported by the European Research Council under the European Union&#39;s Seventh Framework Programme (FP/2007-2013) / ERC Grant Agreement number 320473-BacNK. Further support was provided by the I-CORE Program of the Planning and Budgeting Committee and the Israel Science Foundation and by the I-Core on Chromatin and RNA in Gene Regulation, the GIF Foundation, the Lewis Family Foundation, the ICRF professorship grant, the Helmholtz Israel grant and the Rosetrees Trust (all to O.M.). This study was also supported by the Israel Science Foundation (grant 502/15), the Kass Medical Research Award and a research grant from the Israel Society of Hematology and Transfusion Medicine (to S.E). O.M is a Crown professor of Molecular Immunology.

1. Generation of a Plasmid that Expresses the Chimeric Construct
NOTE: The aim is to generate a plasmid that expresses the extracellular domain of the receptor of interest fused to the transmembrane and intracellular domains of the mouse CD3&#950; chain (Figure 2A).
<ol>
	<li>Retrieve the sequence of the extracellular domain of the receptor of interest including the signal peptide. Obtain DNA material which is expected to express this receptor (e.g., cDNA or a plasmid).</li>
	<li>Obtain DNA material that expresses the transmembrane and the intracellular domains of the mouse CD3&#950; chain (e.g., cDNA or a plasmid).</li>
	<li>Design the sequence of the fusion protein based on the general structure shown in Figure 2A. Make sure that the whole sequence is within the same codon reading frame. This sequence is used to design appropriate primers and verify the final product.</li>
	<li>Design two PCR reactions to amplify each of the fragments of the fusion proteins separately (Figure 2B, 2C)10. 
	NOTE: The specific conditions for the PCR reactions (e.g., elongation time and annealing temperature) depend on the nature of the primers, which are designed for a specific receptor.
	<ol>
		<li>Amplify the extracellular segment of the receptor.
		<ol>
			<li>Design a 5&#39; primer that includes part of the signal peptide of the receptor, a Kozak consensus sequence, and an appropriate restriction site (Figure 2B).</li>
			<li>Design a 3&#39; primer that flanks both parts of the fusion protein (Figure 2B). For example, for the initial design this 3&#39; primer can include the last 20 base pairs of the extracellular segment of the receptor and the first 9 base pairs of CD3&#950; segment. 
			NOTE: There is no need to add a restriction site to the 3&#39; primer since this primer will be used to generate the fused sequence, and is not used for ligation.</li>
		</ol>
		</li>
		<li>Amplify the CD3&#950; segment.
		<ol>
			<li>Design a 5&#39; primer which flanks both parts of the fusion protein (Figure 2C). For example, for the initial design the 5&#39; primer can include the last 9 base pairs of the extracellular segment of the receptor and the first 20 base pairs of the CD3&#950; segment. 
			NOTE: There is no need to add a restriction site to the 5&#39; primer since this primer will be used to generate the fused sequence, and is not used for ligation.</li>
			<li>Design a 3&#39; primer which includes the end of the CD3&#950; sequence as well as an appropriate restriction site (Figure 2C).</li>
		</ol>
		</li>
		<li>Correct the primer sequences in each of these two reactions so that the annealing temperature is similar in each pair (note that the primer, which includes sequences of the two parts of the fusion protein, only anneals with part of the sequence in each reaction).</li>
	</ol>
	</li>
	<li>Perform an additional PCR in which a mixture of the PCR products of the first two reactions is used as the DNA template with the 5&#39; primer of the extracellular receptor segment (Step 1.4.1.1) and the 3&#39; of the CD3&#950; segment (Step 1.4.2.2) (Figure 2D)10. This reaction should generate the final fused sequence.</li>
	<li>Proceed as usual for cloning.
	<ol>
		<li>Ligate the final PCR product to the target cut vector (the target vector is usually pcDNA3 which expresses G418 and ampicillin resistance).</li>
		<li>Transform the ligated vector into competent bacteria11.
		<ol>
			<li>Thaw 50 &#181;L of the competent bacteria on ice.</li>
			<li>Add the ligated vector to the bacteria and incubate on ice for 20 min.</li>
			<li>Incubate at 42 &#176;C for 45 s.</li>
			<li>Add 200 &#181;L of LB without antibiotics.</li>
			<li>Incubate for 1 h at 37 &#176;C with continuous shaking.</li>
			<li>Seed on an LB plate with the appropriate antibiotics (e.g., ampicillin solution with a final concentration of 0.1 mg/mL).</li>
		</ol>
		</li>
		<li>Collect and grow several bacterial colonies in 5 mL of LB (in 15 mL tubes).</li>
		<li>The next day, extract plasmids with a mini prep kit.</li>
		<li>Analyze the gene insert with restriction enzymes.</li>
		<li>Sequence the relevant colonies and verify that the sequence and the reading frame are correct (as indicated in Step 1.3).</li>
	</ol>
	</li>
	<li>Transform the verified plasmid into competent bacteria11.
	<ol>
		<li>Thaw 20 &#181;L of the competent bacteria on ice.</li>
		<li>Add 1 &#181;L of the plasmid to the bacteria and incubate on ice for 20 min.</li>
		<li>Incubate at 42 &#176;C for 45 s.</li>
		<li>Add 200 &#181;L of LB without antibiotics.</li>
		<li>Incubate for 1 h at 37 &#176;C with continuous shaking.</li>
		<li>Seed on a bacterial growth plate with the appropriate antibiotics (e.g., ampicillin solution with a final concentration of 0.1 mg/mL).</li>
	</ol>
	</li>
	<li>The next day, grow a bacterial colony in a large LB container with 0.1 mg/mL ampicillin (~250 mL).</li>
	<li>The next day, perform maxi prep according to the specific instructions provided by the kit. 
	NOTE: For the electroporation procedure, a large quantity of plasmid is required, as detailed below.</li>
</ol>
2. Transfection of the Plasmid that Expresses the Chimeric Protein into BW Cells
NOTE: Different methods can also be employed for transfection (e.g., by lentiviral infection). Prior to the electroporation, perform ethanol precipitation of the DNA as described below. The next steps require sterile conditions.
<ol>
	<li>The day before, prepare the BW5147 cells (&#34;BW cells&#34;) for electroporation. Plate 10 plates (of 10 cm) with 10 mL of 100,000 BW5147 cells/mL (i.e., a total of 10 x 106 cells) with RPMI supplemented with 10% fetal calf serum (FCS), 1% sodium pyruvate, 1% L-glutamine, 1% non-essential amino acids and 1% penicillin-streptomycin (&#34;complete medium&#34;).</li>
	<li>Place 100 &#181;g of the pcDNA3 plasmid that expresses the fusion protein in a 1.5-2 mL tube and add 3 M sodium acetate at pH 5.3-5.5 to it. The volume of the sodium acetate should correspond to 0.1 (10%) of the volume of the 100 &#181;g plasmid; titrate the pH of the sodium acetate with a pH meter by adding NaOH or HCl.</li>
	<li>Add 2.5 volumes of 100% ethanol to the mixture of the plasmid and the sodium acetate as described in Step 2.2 (2.5x the total volume of plasmid and sodium acetate). Incubate overnight at -20 &#176;C or for 2 h at 70 &#176;C.</li>
	<li>2.4. 24 h after the BW cells have been plated, collect all the BW cells (~100 mL) in 2 50 mL tubes. Centrifuge the cells for 5 min at 515 x g and discard the supernatant.</li>
	<li>Re-suspend the pellet from both tubes in 25 mL of RPMI- in a single 50 mL tube. For example, re-suspend the pellet of one tube with 25 mL of RPMI- and then use this fluid to re-suspend the fluid in the other 50 mL tube so that the total pellet from the BW cells is re-suspended in 25 mL of medium in a single tube. 
	NOTE: The medium should be without additions, since the serum might interfere with electroporation.</li>
	<li>Centrifuge the cells again for 5 min at 515 x g. Re-suspend the pellet in 1 mL of RPMI, and transfer the contents to a 0.4 cm cuvette on ice.</li>
	<li>Centrifuge the plasmid that underwent ethanol precipitation (Step 2.3) for 30 min at 16,000 x g and 4 &#176;C.</li>
	<li>Wash once with 1 mL of 70% ethanol and centrifuge for another 20 min at 16,000 x g and 4 &#176;C (to wash out the salt).</li>
	<li>Remove all the ethanol and let the pellet dry slightly (i.e., in an open tube in the tissue culture hood). Re-suspend the pellet with 100 &#181;L of RPMI- previously heated to 60 &#176;C.</li>
	<li>Add the re-suspended plasmid (Step 2.8) to the cells in the cuvette and incubate for 5 min on ice.</li>
	<li>Electroporate the cells at 0.23 kV, 250 capacitation. This should take ~4 ms.</li>
	<li>Transfer the electroporated cells to a 50 mL tube, add 50 mL of complete medium and centrifuge for 5 min at 515 x g. </li>
	<li>Discard the supernatant and re-suspend the cells with 50 mL of complete medium.</li>
	<li>Plate the electroporated cells in 24-well culture dishes (1 mL per well, for a total of ~50 wells) and incubate at 37 &#176;C and 5% CO2 for 48 h.</li>
	<li>Select the transfected cells with antibiotics. After 48 h add 1 mL of complete medium supplemented with 10 mg/mL G418 to each well (at this stage the final volume in each well is 2 mL and the final concentration of G418 in each well is 5 mg/mL). 
	NOTE: If the plasmid contains a different antibiotic resistance, determine the antibiotic concentration required to kill the untransfected BW cells prior to this step.</li>
	<li>Every 48 h, carefully discard 1 mL of the upper volume of each well without stirring the medium in the well (the BW cells tend to be at the bottom of the well). Add complete medium supplemented with 5 mg/mL G418 to each well, so that the final volume in each well is again 2 mL.</li>
	<li>Examine the culture plates for cell growth in all wells regularly. 
	NOTE: A change in the color of the medium to yellow may help identify growing cells; however, at the beginning the medium will be yellow because of the G418 itself. Identify positive wells (wells with growing cells). These wells are G418 resistant and therefore are expected to express the fusion protein. This process usually takes ~3 weeks.</li>
</ol>
3. Verification of the Expression of the Transfected Receptor in Cells in the Positive Wells.
<ol>
	<li>Stain the transfected BW cells with a specific antibody against the receptor which was transfected.</li>
	<li>Compare the expression level of the receptor by flow cytometry to the expression of the control cells (untransfected BW cells).</li>
	<li>After verification of one or more wells, use cells immediately for experimental purposes, grow in culture, or freeze for future applications.</li>
	<li>Verify the expression of the transfected receptor before performing a new experiment. After a few weeks of growth, the cells with G418 with a stable receptor expression, G418 can be omitted from the culture medium.</li>
</ol>
4. Incubation of the Transfected BW Cells with Targets
NOTE: When using the transfected BW cells for the first time, it is preferable to test them on targets that express a known ligand of the receptor of interest or on plate-bound antibodies specifically directed against the receptor of interest (cross-linking experiments; see below, section 4.2.1.3).
<ol>
	<li>Preferably, split the BW cells 24 h before the experiment (for example, by adding 10 mL of complete medium to 2 mL of cells in culture in a new 10 cm culture plate).</li>
	<li>Incubate the transfected BW cells with their targets. Perform the experiment simultaneously on control BW cells that express an empty vector (or parental BW cells). 96F plates are preferable (but 96U plates can also be used). Perform the experiment in triplicate. Suspend all the cells in the complete medium.
	<ol>
		<li>Prepare the targets. The type of target varies as a function of the objective, and can be cells, cells that have been pre-incubated with antibodies, or antibodies alone. This system has also been used with bacteria as targets5.
		<ol>
			<li>If the targets are dividing cells (i.e., cell lines), irradiate them at 6,000 rad prior to the assay. Place 50,000 of the target cells in a single well of a 96 plate (in a volume of 100 &#181;L).</li>
			<li>For experiments with antibodies and BW cells that express human Fc&#947;Rs, incubate the target cells with an antibody on ice in a 96 well plate (for example, 50 &#181;L of the target cells and 50 &#181;L of the antibody; different antibody doses can be tested).</li>
			<li>If using antibodies alone as targets (cross-linking experiments), they should be plate- bound. For this purpose, first incubate the antibodies in a complete medium in a 96F plate for 1-2 h at 37 &#176;C and 5% CO2 (a typical starting dose is 0.5 &#956;g of a specific antibody in 50 &#956;L). Wash the plate to remove unbound antibodies. 
			NOTE: In this case, a 96F plate will enable binding of the antibody to the plate, which after addition of the transfected BW cells will lead to activation of the transfected receptor.</li>
		</ol>
		</li>
		<li>Add the BW cells (effector cells). Place 50,000 BW cells in a single well of a 96 plate (in a volume of 100 &#181;L).</li>
		<li>Complete the volume in each well to 200 &#181;L if necessary.</li>
		<li>Incubate the plates at 37 &#176;C and 5% CO2 for 48 h (this time period can be calibrated).</li>
	</ol>
	</li>
	<li>After 48 h, freeze the plates at -20&#176;C and thaw prior to ELISA, or use immediately for ELISA (see next steps).</li>
</ol>
5. ELISA
<ol>
	<li>Coat an ELISA plate with an anti-mouse IL-2 antibody (anti-mIL-2). Place 0.05 &#181;g of anti-mIL-2 in a volume of 50 &#181;L of 1x PBS per well. Incubate the coated ELISA plate with the anti-mIL-2 antibody at 4 &#176;C overnight or at 37 &#176;C for 2 h. 
	NOTE: To perform ELISA directly after the incubation step, the ELISA plate should be coated 24 h before the end of the incubation period of the BW cells.</li>
	<li>Discard the fluid in the ELISA plate and add a blocking solution (200 &#181;L per well) which is composed of 1x PBS and 1% bovine serum albumin (BSA). Incubate the ELISA plate for 2 h at room temperature.</li>
	<li>Wash the ELISA plate three times with 0.05% PBS Tween solution (i.e., 0.5 mL of Tween-20 in 1 liter of 1x PBS).</li>
	<li>Take the 96 plate which has the BW cells that were incubated with their targets (4.3). If the plate was frozen, completely thaw it (e.g., by short incubation at 37 &#176;C). Centrifuge the 96 plate (5 min, 515 x g) and carefully transfer 100 &#181;L of the supernatant in each well to the pre-coated and blocked ELISA plate. 
	NOTE: The supernatant should be collected from the sides of each well to avoid taking the cells.</li>
	<li>If desired, add recombinant mIL-2 with defined concentrations to one of the empty rows of the coated ELISA plate to generate a standard curve of mIL-2. For example, start from a mIL-2 concentration of 2,500 pg/mL and then decrease by 50% in each subsequent well; do not add mIL-2 to the last well.</li>
	<li>Incubate the ELISA plate at 4 &#176;C overnight or at 37 &#176;C for 2 h.</li>
	<li>Wash the ELISA plate four times with PBS Tween.</li>
	<li>Add biotin anti-mouse IL-2 to the ELISA plate. Use a concentration of 1 &#181;g antibody in 1 mL of 1x PBS with 1% BSA and divide into 100 &#181;L per well.</li>
	<li>Incubate at room temperature for 1 h.</li>
	<li>Wash the ELISA plate six times with PBS Tween.</li>
	<li>Add HRP conjugated streptavidin. Use a concentration of 1 &#181;L streptavidin in 1 mL of PBSX1 with 1% BSA and divide it into 100 &#181;L per well.</li>
	<li>Incubate at room temperature for 30 min.</li>
	<li>Wash the ELISA plate six times with PBS Tween.</li>
	<li>Add 100 &#181;L per well of TMB substrate solution. Complete this stage quickly to minimize differences between the wells due to time lags when adding the TMB.</li>
	<li>Read the ELISA plate with an ELISA plate reader at 650 nm (the reading can be repeated if the signal is weak).</li>
</ol>

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The authors declare no competing financial interests.

Here we present a protocol for generating a reporter system to investigate receptor-ligand interactions (see an abbreviated form of this protocol in1). The protocol is composed of three main parts: cloning of a chimeric protein, which includes the extracellular domain of a specific receptor fused to the intracellular domain of CD3&#950;, transfection of a plasmid that expresses the fusion protein to BW cells (e.g., by electroporation), and quantitative detection of mouse IL-2 by ELISA. The final product of each of these parts should be verified independently: the cloning process should be verified by complete sequencing of the fusion protein in the target plasmid, the transfection of the BW cells should be verified by examining the expression of the receptor of interest in the BW cells (e.g., by flow cytometry), and the ELISA process should be verified by general positive controls (e.g., recombinant mIL-2) as well as with specific controls for the transfected BW cells; i.e., targets that express a known ligand of the receptor of interest (e.g., cells that express CD48 can be used as a positive control for BW cells that express the NK cell receptor 2B4). For instance, the cloning process may be successful, but the fusion protein is not expressed by the BW cells. In this case, the electroporation should be repeated. Alternatively, if there is no signal above the background level of the ELISA plate (especially for the positive controls) the transfected BW cells may have failed to express the receptor (or the expression has been lost) or a technical problem may have occurred during the ELISA process.
Several modifications can be made to this protocol while preserving the general principles of the procedure. These modifications include the vector used to express the fusion protein, the method for transfecting the plasmid to BW cells and specific parameters related to the incubation and ELISA such as the type of target (e.g., cells, antibodies, etc.), number of target cells or the antibody concentration if relevant (we use 50,000 cells per well and an antibody starting dose of 0.5 &#181;g/well), incubation time (we use an incubation time of 48 h) and the supernatant volume which is transferred to the ELISA plate.
This method is sensitive, specific and can be easily quantified. It is ideal for screening for the presence of ligands for specific receptors (especially if the ligand itself has not been recognized). After preparing transfected BW cells that express a specific receptor, these cells can be frozen and used when needed for additional experiments. We and others have used this system successfully in previous studies to explore receptor-ligand interactions (for example2,3,5,12,13,14,15,16) and the results obtained by this system have been verified by other techniques. This method can also be used to study the activation of different human Fc&#947;R receptors by antibodies, as has been done for anti-viral antibodies in serum6,7,8 or with monoclonal antibodies against CD209.
Since this is a reporter system in which only the extracellular segment of the receptor is expressed, the findings obtained by this system should be complemented by additional experiments with the endogenous receptor, such as cytotoxicity assays with natural killer (NK) cells to study interactions with NK cell receptors. Moreover, certain ligand/receptor interactions may not result in a mIL-2 secretion by the reporter system, and therefore might be overlooked. In addition, as in any experimental setup, the results should be verified for various parameters as described above. This is especially important in cases where a comparison of two conditions is required (e.g., activation of the same Fc receptor by different antibodies). It is also important to verify that the sensitivity of a specific ligand-receptor interaction is within the linear range of the system. Otherwise this could lead to saturation values which may preclude a valid comparison across conditions. This can be achieved by using a mIL-2 standard curve as well as by generating a dose-response curve with the tested ligand (in the case of saturating values, the experimental parameters should be modified; e.g., a smaller volume of supernatant should be analyzed by ELISA). Another possible limitation of this system is the use of target cells with endogenous secretions of mIL-2 (e.g., mouse T cells) which would mask the IL-2 secretion of BW cells. To overcome these highly unusual occurrences which are confined to mouse cells, this reporter system could be improved by using a different reporter (e.g., GFP) which is not expressed by the target cells.

The BW reporter system is a technique for studying receptor-ligand interactions1. This system is especially advantageous when the ligand of a specific receptor is unknown or experiments with the endogenous ligand are technically difficult. It can also be used for studying the binding of monoclonal antibodies to human Fc&#947;Rs. The method is based on expressing a chimeric protein in mouse BW cells. This chimeric protein is composed of the extracellular domain of the receptor of interest fused to the transmembrane and intracellular domains of the mouse &#950; chain. Binding of appropriate ligands of the receptor leads to secretion of mouse IL-2, which can be easily detected by ELISA, as illustrated in Figure 1. This method is sensitive and specific to an individual receptor, easy to operate, and highly reproducible. It can thus complement additional tools for studying receptor-ligand interactions. For example, it can be used to screen several cell lines for the presence of a ligand for a specific receptor (even if the ligand itself has not been identified) or to detect the activation of human Fc&#947;Rs by monoclonal antibodies or by human serum containing anti-viral antibodies. Although primary immune cells express endogenous Fc&#947;Rs, they are generally difficult to handle and usually express several Fc receptors.
We have used this system successfully in many of our studies to characterize receptor-ligand interactions. These include identifying haemagglutinin as the ligand of the NK cell receptor NKp462, identifying PVR and nectin-2 as ligands for human and mouse TIGIT3,4, and showing that the fusobacterium nucleatum bacterium binds and activates human TIGIT5. In addition, BW cells that express Fc receptors have been used successfully to detect anti-viral antibodies in patients&#39; sera6,7,8. Specifically, we recently established BW cells that express human Fc&#947;Rs to detect differential FcR activation of anti-CD20 antibodies used for the treatment of chronic lymphocytic leukemia (CLL)9. Importantly, the results of the BW reporter system have been validated in complementary experiments.

<table><tbody><tr><td>AccuPrep, Plasmid Mini Extraction Kit</td><td>Bioneer</td><td>K-3030</td><td></td></tr><tr><td>Anti-mouse IL-2</td><td>BioLegend</td><td>503702</td><td></td></tr><tr><td>Biotin anti-mouse IL-2</td><td>BioLegend</td><td>503804</td><td></td></tr><tr><td>ELISA plates</td><td>De-groot</td><td>60-655061</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F7524-500ml</td><td></td></tr><tr><td>G418</td><td>Mercury</td><td>MBS3458105GM</td><td></td></tr><tr><td>Gene Pulser II</td><td>Bio-Rad</td><td>165-2105, 165-2106, 165-2107, 165-2108, 165-2109, 165-2110</td><td></td></tr><tr><td>L-Glutamine</td><td>Biological industry</td><td>03-020-1B</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution</td><td>Biological industry</td><td>01-340-1B</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Biological industry</td><td>03-031-1B</td><td></td></tr><tr><td>peroxidase streptavidin</td><td>Jackson ImmunoResearch</td><td>016-030-084</td><td></td></tr><tr><td>PureLink HiPure Plasmid Filter Maxiprep Kit</td><td>ThermoFisher Scientific</td><td>K210016</td><td></td></tr><tr><td>RPMI-1640 Medium</td><td>Biological industry</td><td>30-2001</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Biological industry</td><td>03-042-1B</td><td></td></tr><tr><td>TMB</td><td>SouthernBiothech</td><td>0410-01</td><td></td></tr></tbody></table>

a bw reporter system for studying receptor-ligand interactions

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native receptor and the ligand are challenging since the ligand of a specific receptor may be unknown and experimental procedures with the native ligand can be technically complicated. To address these obstacles, we describe a reporter system to detect the binding and activation of a specific receptor by a ligand of interest. In this reporter system, the extracellular domain of a specific receptor is conjugated to mouse CD3&#950; and this chimeric protein is then expressed in mouse BW cells. These transfected BW cells can then be incubated with different targets (e.g., cells or antibodies). Activation of a transfected receptor leads to the secretion of mouse interleukin-2 (mIL-2) which can be detected by enzyme-linked immunosorbent assay (ELISA). This reporter system has the advantages of being sensitive and specific to a single receptor. In addition, the activation level of a specific receptor can easily be quantified and can be used even in cases where the ligand of the receptor is unknown. This system has been implemented successfully in many of our studies to characterize receptor-ligand interactions. We have recently employed this system to study the activation of human Fc&#947; receptors (Fc&#947;Rs) by different monoclonal anti-CD20 antibodies in clinical use.

Figure 3 illustrates the results of an experiment with a BW reporter system. In this experiment, CLL cells were pre-incubated with different anti-CD20 antibodies (rituximab and obinutuzumab) and then co-incubated with transfected BW cells which express CD16a-CD3&#950;. Similar experiments were conducted in our study9. Figure 3A presents an image of a raw ELISA plate where the color intensity corresponds to a concentration of mIL-2. The experiment was performed in triplicate. This experiment included several controls: CLL cells incubated with parental untransfected BW cells (upper row) and CLL cells incubated with BW cells which express CD16a-CD3&#950; but without an antibody or with a control antibody (six wells in the second row on the left). The incubation of BW cells that expressed CD16 with CLL cells that were pre-incubated with anti-CD20 antibodies induced a significant secretion of mIL-2 compared to the mIL-2 level of the control wells. Importantly, pre-incubation of the CLL cells with the anti-CD20 obinutuzumab activated CD16 more strongly than rituximab, a known finding which was recapitulated by our system. The last row shows descending pre-defined concentrations of recombinant mIL-2 (without the addition of cells). The right well in the last row does not have mIL-2 and represents the background reading, which appears similar to the control wells. Figure 3B presents the quantification of the results of the ELISA plate shown in Figure 3A. The optical density (OD) levels were converted into mIL-2 concentrations based on the standard curve with recombinant mIL-2. Figure 3C presents a dose response experiment where different doses of antibodies were pre-incubated with CLL cells and then incubated with BW cells expressing CD16.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58685/58685fig1.jpg" /> 
Figure 1: Schematic representation of the BW reporter system. BW5147 cells are stably transfected with the extracellular portion of different receptors fused to the transmembrane and cytoplasmic domains of the mouse CD3&#950; chain (chimeric recptor-CD3&#950;). Activation of a specific receptor-CD3&#950; by a ligand results in a secretion of mIL-2 which can be detected by ELISA. <a href="https://www.jove.com/files/ftp_upload/58685/58685fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58685/58685fig2.jpg" /> 
Figure 2: The structure of the chimeric receptor-CD3&#950; and the design of PCR primers. (A) General structure of an extracellular receptor fused to the transmembrane and intracellular domains of mouse CD3&#950;. (B) The extracellular domain of the receptor was amplified with a 3&#39; primer which also included nucleotides of CD3&#950;. (C) The intracellular and transmembrane domains of CD3&#950; were amplified with a 5&#39; primer that also included receptor nucleotides. (D) In the final PCR reaction, both segments, which have overlapping sequences, were used as the DNA template. In all figure panels, different protein domains are color-coded and boxed in blue rectangles (CD3&#950; sequence) and red rectangles (the receptor sequence). <a href="https://www.jove.com/files/ftp_upload/58685/58685fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58685/58685fig3.jpg" /> 
Figure 3: Illustration of the final product of the BW reporter system. (A, B, C) CLL cells were pre-incubated with or without two anti-CD20 antibodies (rituximab and obinutuzumab) or a control antibody. Afterwards, the antibody-bound cells were incubated with parental BW cells or BW cells which express CD16a-CD3&#950;. The level of mIL-2 in the supernatant was determined by ELISA. (A) An image of a raw ELISA plate. Color intensity indicates the concentration of mIL-2 in the supernatant. The experiment was performed in triplicate. The lower row shows the ELISA readout in decreasing concentrations of recombinant mIL-2 which were analyzed in the same plate. (B) Quantification of the ELISA reading shown in (A). The mIL-2 levels were calculated using a standard curve of mil-2. (C) A dose response experiment where different doses of antibodies were pre-incubated with CLL cells and then incubated with BW cells expressing CD16a-CD3&#950;. ***, P &#60; 0.001; Student&#39;s t test (B) or ANOVA with multiple comparisons (C). The only comparison in (C) which was not significantly different was for rituximab and the control Ab in the first concentration (0.01 &#181;g/mL). Error bars represent the standard deviation of the triplicates. Similar experiments were conducted as part of one of our recent study9. <a href="https://www.jove.com/files/ftp_upload/58685/58685fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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A BW Reporter System for Studying Receptor-Ligand Interactions

This protocol describes how to establish a reporter system that can be used to identify and quantify receptor-ligand interactions.

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native ...

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JoVE Journal

Biology

7.6K Views.  Hadassah - Hebrew University Medical Center. This protocol describes how to establish a reporter system that can be used to identify and quantify receptor-ligand interactions.

Video: A BW Reporter System for Studying Receptor-Ligand Interactions

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

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

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

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

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

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

Monitoring GPCR-&#946;-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. Currently, the characterization of novel drugs towards their interactions with &#946;-arrestins and other cytosolic proteins is extremely valuable in the field of GPCR drug discovery particularly during the study of GPCR biased agonism. Here, we show the application of a novel structural complementation assay to accurately monitor receptor-&#946;-arrestin interactions in real time living systems. This method is simple, accurate and can be easily extended to any GPCR of interest and also it has the advantage that it overcomes unspecific interactions due to the presence of a low expression promoter present in each vector system. This structural complementation assay provides key features that allow an accurate and precise monitoring of receptor-&#946;-arrestin interactions, making it suitable in the study of biased agonism of any GPCR system as well as GPCR c-terminus &#8216;phosphorylation codes&#8217; written by different GPCR-kinases (GRKs) and post-translational modifications of arrestins that stabilize or destabilize the receptor-&#946;-arrestin complex.

GPCR-&#946;-arrestin interactions are an emerging field in GPCR drug discovery. Accurate, precise and easy to set up methods are necessary to monitor such interactions in living systems. We show a structural complementation assay to monitor GPCR-&#946;-arrestin interactions in real time living cells, and it can be extended to any GPCR.

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Bioengineering

A High-Throughput Screening Approach for Evaluating the Binding Affinity of Environmental Pollutants to Nuclear Receptors

Increasing levels of compounds have been detected in the environment, causing widespread pollution and posing risks to human health. However, despite their high environmental occurrence, there is very limited information regarding their toxicological effects. It is urgent to develop high-throughput screening (HTS) methods to guide toxicological studies. In this study, a receptor-ligand binding assay using an HTS system was developed to determine the binding potency of environmental pollutants on nuclear receptors. The test is conducted using a microplate reader (i.e., a 96-well plate containing various chemicals) by measuring the fluorescence polarization (FP) of a specific fluorescent probe. This assay consists of four parts: the construction and transformation of recombinant vectors, the expression and purification of the receptor protein (ligand-binding domain), receptor-probe binding, and competitive binding of chemicals with the receptor. The binding potency of two environmental pollutants, perfluorooctanesulfonic acid (PFOS) and triphenyl phosphate (TPHP), with peroxisome proliferator-activated receptor gamma (PPAR&#947;) was determined to illustrate the assay procedure. Finally, the advantages and disadvantages of this method and its potential applications were also discussed.

This protocol describes a high-throughput screening system that uses fluorescence polarization of a specific fluorescent probe binding to a nuclear receptor as a readout for screening environmental pollutants.

Increasing levels of compounds have been detected in the environment, causing widespread pollution and posing risks to human health. However, despite ...

Environment

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

Competitive Binding Assay to Identify Compounds Disrupting Receptor-Ligand Interactions

Source: Schoofs, G., et al. <a href="https://app.jove.com/v/57271">A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4.</a> J. Vis. Exp. (2018).
In this video, we describe a flow cytometry-based competitive binding assay to detect the interactions between the CXC Chemokine Receptor 4 (CXCR4) and its fluorescently-labeled natural ligand CXC Chemokine Ligand 12 (CXCL12), in the presence of a CXCR4-targeting small molecule. Incubating CXCR4-expressing cells with lower small molecule concentrations allows CXCR4-CXCL12 binding to some extent, which progressively declines upon a gradual increase in small molecule concentrations.

Source: Schoofs, G., et al. A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to ...

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-protein interactions

ELISA-Based Reporter Assay to Study the Receptor-Ligand Interaction in BW Cells

Source: Elias, S. et al., <a href="https://app.jove.com/v/58685">A BW Reporter System for Studying Receptor-Ligand Interactions.</a> J. Vis. Exp. (2019)
This video demonstrates the reporter assay-based detection of the receptor-ligand interaction. The successful receptor-ligand interaction activates the downstream signaling pathway, causing the generation and release of cytokines as secretory reporter molecules in the solution, which is confirmed using ELISA.