Name: co-immunoprecipitation assay for studying functional interactions between receptors and enzymes
Uploaded: 2018-09-28T15:00:00
Description: Watch this Scientific Journal Video about Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes at JoVE.com

This project was funded by NIH Grants 1R01AI125640-01 and the Rheumatology Research Foundation.

1. Transfection of Cells
<ol>
	<li>Seed HEK 293T cells into twelve 10 cm plates (5 x 106 cells per plate) the day before transfection (Figure 1). Perform the cell counting using a hemocytometer. For each plate, use 10 mL of DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Incubate the cells at 37 &#176;C in 5% CO2.</li>
	<li>Once the cells are 80-90% confluent, transfect 5 HEK 293T plates using a lipid-based transfection reagent with one of the following...

<ol>
	<li>Montor, W. R., Salas, A., Melo, F. H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+tyrosine+kinases+and+downstream+pathways+as+druggable+targets+for+cancer+treatment:+the+current+arsenal+of+inhibitors.">Receptor tyrosine kinases and downstream pathways as druggable targets for cancer treatment: the current arsenal of inhibitors.</a> Molecular Cancer. 17 (1), (2018).</li><li>Ngoenkam, J., Schamel, W. W., Pongcharoen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selected+signalling+proteins+recruited+to+the+T-cell+receptor-CD3+complex.">Selected signalling proteins recruited to the T-cell receptor-CD3 complex.</a> Immunology. 153 (1), 42-50 (2018).</li><li>Azoulay-Alfaguter, I., Strazza, M., Pedoeem, A., Mor, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+coreceptor+programmed+death+1+inhibits+T-cell+adhesion+by+regulating+Rap1.">The coreceptor programmed death 1 inhibits T-cell adhesion by regulating Rap1.</a> Journal of Allergy and Clinical Immunology. 135 (2), 564-567 (2015).</li><li>Chemnitz, J. M., Parry, R. V., Nichols, K. E., June, C. H., Riley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHP-1+and+SHP-2+associate+with+immunoreceptor+tyrosine-based+switch+motif+of+programmed+death+1+upon+primary+human+T+cell+stimulation,+but+only+receptor+ligation+prevents+T+cell+activation.">SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation.</a> The Journal of Immunology. 173 (2), 945-954 (2004).</li><li>Patsoukis, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+effects+of+PD-1+on+Akt+and+Ras+pathways+regulate+molecular+components+of+the+cell+cycle+and+inhibit+T+cell+proliferation.">Selective effects of PD-1 on Akt and Ras pathways regulate molecular components of the cell cycle and inhibit T cell proliferation.</a> Science Signaling. 5 (230), 46 (2012).</li><li>Pentcheva-Hoang, T., Chen, L., Pardoll, D. M., Allison, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+death-1+concentration+at+the+immunological+synapse+is+determined+by+ligand+affinity+and+availability.">Programmed death-1 concentration at the immunological synapse is determined by ligand affinity and availability.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (45), (2007).</li><li>Yokosuka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+cell+death+1+forms+negative+costimulatory+microclusters+that+directly+inhibit+T+cell+receptor+signaling+by+recruiting+phosphatase+SHP2.">Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2.</a> The Journal of Experimental Medicine. 209 (6), 1201-1217 (2012).</li><li>Hui, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+costimulatory+receptor+CD28+is+a+primary+target+for+PD-1-mediated+inhibition.">T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition.</a> Science. 355 (6332), 1428-1433 (2017).</li><li>Sheppard, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-1+inhibits+T-cell+receptor+induced+phosphorylation+of+the+ZAP70/CD3zeta+signalosome+and+downstream+signaling+to+PKCtheta.">PD-1 inhibits T-cell receptor induced phosphorylation of the ZAP70/CD3zeta signalosome and downstream signaling to PKCtheta.</a> FEBS Letters. 574 (1-3), 37-41 (2004).</li><li>Okazaki, T., Maeda, A., Nishimura, H., Kurosaki, T., Honjo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-1+immunoreceptor+inhibits+B+cell+receptor-mediated+signaling+by+recruiting+src+homology+2-domain-containing+tyrosine+phosphatase+2+to+phosphotyrosine.">PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine.</a> Proceedings of the National Academy of Sciences of the United States of America. 98 (24), 13866-13871 (2001).</li><li>Sun, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonism+between+binding+site+affinity+and+conformational+dynamics+tunes+alternative+cis-interactions+within+Shp2.">Antagonism between binding site affinity and conformational dynamics tunes alternative cis-interactions within Shp2.</a> Nature Communications. 4, 2037 (2013).</li><li>Peled, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+purification+mass+spectrometry+analysis+of+PD-1+uncovers+SAP+as+a+new+checkpoint+inhibitor.">Affinity purification mass spectrometry analysis of PD-1 uncovers SAP as a new checkpoint inhibitor.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (3), E468-E477 (2018).</li><li>Jang, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protein+tyrosine+phosphatase+inhibitor,+pervanadate,+inhibits+angiotensin+II-Induced+beta-arrestin+cleavage.">A protein tyrosine phosphatase inhibitor, pervanadate, inhibits angiotensin II-Induced beta-arrestin cleavage.</a> Molecules and Cells. 28 (1), 25-30 (2009).</li><li>Pluskey, S., Wandless, T. J., Walsh, C. T., Shoelson, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+stimulation+of+SH-PTP2+phosphatase+activity+by+simultaneous+occupancy+of+both+SH2+domains.">Potent stimulation of SH-PTP2 phosphatase activity by simultaneous occupancy of both SH2 domains.</a> The Journal of Biological Chemistry. 270 (7), 2897-2900 (1995).</li><li>McAvoy, T., Nairn, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serine/threonine+protein+phosphatase+assays.">Serine/threonine protein phosphatase assays.</a> Current Protocols in Molecular Biology. , (2010).</li><li>Shlapatska, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD150+association+with+either+the+SH2-containing+inositol+phosphatase+or+the+SH2-containing+protein+tyrosine+phosphatase+is+regulated+by+the+adaptor+protein+SH2D1A.">CD150 association with either the SH2-containing inositol phosphatase or the SH2-containing protein tyrosine phosphatase is regulated by the adaptor protein SH2D1A.</a> The Journal of Immunology. 166 (9), 5480-5487 (2001).</li></ol>

Receptor-enzyme interactions are crucial for intracellular signaling. Many enzymes are recruited to receptors through SH2 domains binding to phosphorylated tyrosines that decorate tails of the same receptors. However, enzymes are often folded into closed inactive conformations, and activation requires a conformational change11 that can be mediated by other domains of the same receptor. The assay described here measures the interactions between receptors and enzymes as well as the activity induced ...

Catalytic receptors and receptor tyrosine kinases are transmembrane proteins in which binding of an extracellular ligand causes enzymatic activity on the intracellular side1. Some receptors possess both receptor and enzymatic functions, while others recruit specific enzymes such as kinases and phosphatases to their cytoplasmic tails. Recruitment of an enzyme to the receptor&#39;s tail and the subsequent catalytic action of this enzyme are two separate processes that are not always regulated by the same protein domains2. Unfortunately, there are no specific tools to assess both the interaction and enzymatic activity simultaneously. The functional co-immunoprecipitation assay described here is a useful method to dissect the recruitment of an enzyme to the tail of a receptor from its activation. This assay utilizes immunoprecipitation of tagged receptors by antibody-coated beads. Subsequently, both an enzymatic activity assay and western blot analysis on beads are performed. The overall goal of this method is to uncover which protein domains are necessary for interactions between receptors and enzymes (assessed by western blot analysis) and which domains are obligatory for complete activation of the enzymes (measured by on-bead enzymatic activity assay). It is significant to develop tools for studying the separate functions of receptor-associated enzymes due to their involvement in the pathogenesis of human diseases. Moreover, further understanding the mechanisms of action of these proteins may help the design of novel therapeutic interventions.
Programmed death-1 (PD-1) is an inhibitory receptor on the surface of T cells and is required for limiting excessive T cell responses. In recent years, anti-PD-1 antibodies have been implicated in the treatment of multiple malignancies1,2. PD-1 ligation restrains numerous T cell functions, including proliferation, adhesion, and secretion of multiple cytokines3,4,5. PD-1 is localized to the immunological synapse, the interface between T cells and antigen-presenting cells6, where it colocalizes with the T cell-receptor (TCR)7. Subsequently, the tyrosine phosphatase SHP2 [Src homology 2 (SH2) domain containing tyrosine phosphatase 2] is recruited to the cytoplasmic tail of PD-1, leading to dephosphorylation of key tyrosine residues within the TCR complex and its associated proximal signaling molecules3,4,5,8,9. The cytoplasmic tail of PD-1 contains two tyrosine motifs, an immunoreceptor tyrosine-based inhibitory motif (ITIM), and an immunoreceptor tyrosine based-switch motif (ITSM)10. Both motifs are phosphorylated upon PD-1 ligation9,10. Mutagenesis studies have revealed a primary role for the ITSM in SHP2 recruitment, as opposed to the ITIM, whose role in PD-1 signaling and function is not clear4.
SHP2 adopts either a closed (folded), inhibited conformation or an open (extended), active conformation11. The contribution of each tail domain of PD-1 to SHP2 binding or activation has not yet been elucidated. To answer this question, we developed an assay that enables parallel testing of the recruitment of SHP2 to the tail of PD-1 and its activity12. We employed co-immunoprecipitation and an on-bead phosphatase activity assay to test both the interaction and enzymatic activity in parallel. Using this assay, we show that the ITSM of PD-1 is sufficient to recruit SHP2 to the tail of PD-1, while the ITIM of PD-1 is needed to fully extend and activate the enzyme.
There are many receptors that have several adjacent domains in their cytoplasmic tails. The functional co-immunoprecipitation assay can uncover the role of specific domains that are necessary for either protein recruitment or enzymatic activation.

<table>
	<tbody>
		<tr>
			<td>PBS</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td>Phosphate Buffered Saline</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5424-R</td>
			<td>1.5 ml</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%) Phenol Red</td>
			<td>Gibco</td>
			<td>25200114</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Inactivated FBS</td>
			<td>Denville</td>
			<td>FB5001-H</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>Penicillin / Streptomycin</td>
			<td>Fisher</td>
			<td>BP295950</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose without L-glutamine</td>
			<td>Lonza</td>
			<td>BE12-614F</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFect Transfection Reagent</td>
			<td>Qiagen</td>
			<td>301305</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-SHP2</td>
			<td>Santa Cruz</td>
			<td>SC-280</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti&#8211;GFP-agarose</td>
			<td>MBL</td>
			<td>D153-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP</td>
			<td>Roche</td>
			<td>118144600</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Actin</td>
			<td>Santa Cruz</td>
			<td>SC-1616</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK-293 cells</td>
			<td>ATCC</td>
			<td>CRL-1573</td>
			<td></td>
		</tr>
		<tr>
			<td>Orthovanadate</td>
			<td>Sigma</td>
			<td>S6508</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Sigma</td>
			<td>216763</td>
			<td>30%</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td>EDTA-free</td>
		</tr>
		<tr>
			<td>Tris-Glycine SDS Sample Buffer (2X)</td>
			<td>Invitrogen</td>
			<td>LC2676</td>
			<td>Modified Laemmli buffer</td>
		</tr>
		<tr>
			<td>4-20% Tris-Glycine Mini Gels</td>
			<td>Invitrogen</td>
			<td>XP04205BOX</td>
			<td>15-well</td>
		</tr>
		<tr>
			<td>Nitrocellulose membranes</td>
			<td>General Electric</td>
			<td>10600004</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>Sodium chloride</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td>N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Invitrogen</td>
			<td>D1532</td>
			<td>Dithiothreitol</td>
		</tr>
		<tr>
			<td>pNPP</td>
			<td>Sigma</td>
			<td>20-106</td>
			<td>p-Nitrophenyl Phosphate</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>S8045</td>
			<td>Sodium hydroxide</td>
		</tr>
		<tr>
			<td>BCA</td>
			<td>Fisher</td>
			<td>23225</td>
			<td>Bi Cinchoninic Acid assay</td>
		</tr>
	</tbody>
</table>

co-immunoprecipitation assay for studying functional interactions between receptors and enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

While the contribution of the ITSM of PD-1 to SHP2 binding is established, the role of the ITIM of PD-1 is less clear. Because SHP2 has two SH2 domains that can bind to two sequential phosphotyrosines on PD-1 (one tyrosine in the ITSM and another in the ITIM), we hypothesized that the ITSM of PD-1 anchors SHP2 to PD-1, while the ITIM of PD-1 facilitates SHP2 activity by stabilizing its open conformational state11,14. To test this,...

Watch this Scientific Journal Video about Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes at JoVE.com

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

This method can help answer key questions in the cell-signaling field such as the importance of different protein domains in receptor enzyme interaction and enzymatic activation. The main advantage of this technique is that it allows concomitant testing of receptor enzyme interactions and the functional consequences of this interaction on the enzyme activity. For each plate, use 10 milliliters of DMEM supplemented with 10%FBS and 1%penicillin and streptomycin.The day before transfection, seed human embryonic kidney 293-T cells into 12 10-centimeter plates using a hemocytometer to count the cells. Incubate at 37 degrees Celsius in 5%C02. Once the cells are 80 to 90%confluent, use a lipid-based transfection agent to transfect five of the plates.Perform the transfection following the manufacturer's instructions for adherence cells in 10-centimeter plates. Then transfect an identical second set of five plates along with one additional plate serving as a nontransfected control for the measurement of expressed protein amount before the immunoprecipitation. Incubate the transfected plates in a tissue culture incubator at 37 degrees Celsius in 5%CO2 for 48 hours.Use a fluorescent microscope to examine the cells for GFP expression and ensure the transfection has successfully occurred. Mix 15 microliters of 100-millimolar sodium orthovanadate with 50 microliters of 30%hydrogen peroxide to prepare a fresh pervanadate mixture. To phosphorylate the tagged versions of PD-1, remove the media from the PD-1-GFP transfected cells.Add 10 milliliters of plain DMEM and 10 microliters of pervanadate to each plate. Incubate in the dark at room temperature for 15 minutes. After this, wash the cells three times in ice-cold PBS using five milliliters of PBS per wash.While performing immunoprecipitation, all steps should be performed either on ice or at four degrees Celsius. Supplement the lysis buffer with protease inhibitors by dissolving one tablet of the inhibitors in 10 milliliters of buffer. Also add one millimolar of sodium orthovanadate to the buffer that will be used on the PD-1-GFP-transfected plates.Add 500 microliters of ice-cold lysis buffer to the cells, making sure to add the lysis buffer containing the sodium orthovanadate to only the plates containing PD-1-GFP-transfected cells. Use a cell scraper to immediately remove and collect the cells from the plates. Transfer the lysates into 1.5-milliliter cold tubes and rotate them at 0.005 times gravity and at four degrees Celsius for 30 minutes.To collect the post-nuclei supernatant from the lysates, spin them down at 10, 000 times gravity and four degrees Celsius for 10 minutes. Transfer the supernatants into new tubes and discard the pellet. Store the supernatants for the second set of six plates on ice for later WCL analysis.To begin preparing anti-GFP beads, gently shake the bottle containing the beads before opening to prevent settling. Remove 40 microliters of the anti-GFP beads from the slurry per each condition. Centrifuge at 500 times gravity and four degrees Celsius for three minutes.Remove the supernatant to wash the beads, making sure to minimize contact between the pipette and the beads to prevent loss. Resuspend the beads in 80 microliters of lysis buffer per sample. Add the washed beads directly to the cell lysate from the PD-1-GFP-expressing cells of the first set of four plates.Rotate at 0.005 times gravity for 30 minutes at four degrees Celsius to immunoprecipitate the GFP-tagged proteins. Wash the beads three times using one milliliter of cold lysis buffer that does not contain orthovanadate for each wash. Then centrifuge at 2, 500 times gravity for 10 seconds.Divide the lysate of the active SHP2 from the first set of plates into three equal portions and add a portion to each of the three tubes containing the washed PD-1-GFP-containing beads. Add 1/3 of the lysate volume from the nontransfected cells to the second tube of the wild-type, PD-1-GFP beads. Discard the remaining 2/3.Incubate the beads for four hours at four degrees Celsius with gentle rotation at 0.005 times gravity. After this, wash the beads twice using one milliliter of cold lysis buffer for each wash. Add 80 microliters of lysis buffer to each sample, ensuring that the total volume in each tube is 100 microliters.Using a cut pipette tip, pipette gently up and down to mix. Then transfer 50 microliters from each tube into two, fresh, 1.5 milliliter tubes. Wash the beads once with phosphatase wash buffer.Then remove the supernatant completely. Add 100 microliters of assay buffer to the beads and incubate at 30 degrees Celsius for 30 minutes under gentle agitation. When the buffer turns yellow, terminate the reaction by adding 50 microliters of one molar solution of sodium hydroxide.Centrifuge at 2, 500 times gravity for 10 seconds. After this, transfer the supernatant into two wells of half area in a 96-well plate. Read the absorbents at 405 nanometers and express the results as relative optical density over the control wild-type version of PD-1-GFP.First spin down the beads at 2, 000 times gravity and four degrees Celsius for 30 seconds and remove the supernatant. Add 20 microliters of two times Laemmli buffer and boil at 95 degrees Celsius for five minutes. Using a BCA kit, measure the protein concentration of the input controls.Transfer 30 microliters of the most-diluted sample to a new tube. Use lysis buffer to dilute the rest of the input controls to the same concentration as the most-diluted sample. Then transfer 30 microliters of each of the diluted input controls to a new tube.Add equal volumes of two times Laemmli buffer to the lysates and boil them 95 degrees Celsius for five minutes. After this, spin down the beads at 2, 000 times gravity and four degrees Celsius for 30 seconds before performing Western Blot analysis as outlined in the text protocol. In this study, a combined co-immunoprecipitation and enzymatic activity assay is developed for the parallel assessment of receptor enzyme interactions and activation.Unsurprisingly, SHP2 failed to bind to PD-1 when ITSM was mutated. Remarkably, the mutant version of the ITIM inhibited SHP2 binding only to a limited extent. Nevertheless, the SHP2-phosphatase activity assay reveals that ITIM and ITSM are equally indispensable for the enzymatic activity.This reveals a two-step activation model in which SHP2 is folded into an autoinhibited conformation under resting conditions. Upon activation of PD-1, SHP2 is recruited to the phosphorylated ITSM. However, the ITIM must also be phosphorylated to unfold SHP2 into its active conformation.After its development, this technique can pave the way for researchers in the field of cell signaling to explore the function of protein domains in receptor-enzyme interactions.

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