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 plasmids: a SHP2-expressing vector, a PD-1-GFP-expressing vector [a wild type (WT) version of PD-1], an ITIM-mutated version (Y223F) of a PD-1-GFP-expressing vector, and an ITSM-mutated version (Y248F) of a PD-1-GFP-expressing vector (Figure 1). 
	NOTE: Two plates will be transfected with the WT PD-1-GFP-expressing vector. One additional plate will serve as a non-transfected cells control (Figure 1).</li>
	<li>Perform the transfection as per the manufacturer&#39;s protocol for adherent cells in 10 cm plates. 
	NOTE: In the mutated versions of PD-1, the tyrosine (Y) in each motif was replaced by phenylalanine (F) that cannot be phosphorylated.</li>
	<li>Transfect a second identical set of five plates and one additional plate serving as a non-transfected control for the measurement of expressed protein amount [input; also known as whole cell lysate (WCL)] before the immunoprecipitation (Figure 1).</li>
	<li>Incubate the transfected cells for 48 h in 37 &#176;C, 5% CO2 in a tissue culture incubator. 
	NOTE: To ensure that transfection has successfully occurred, examine the cells for GFP expression using a fluorescent microscope.</li>
</ol>
2. Promoting Phosphorylation in Transfected Cells
<ol>
	<li>Following incubation, prepare fresh pervanadate by mixing 50 &#181;L of sodium-orthovanadate (from 100 mM stock) with 50 &#181;L of 30% H2O2. 
	NOTE: The mixture should change into a yellowish color. Pervanadate is a cell-permeable phosphatase inhibitor that promotes phosphorylation of tyrosine residues13. In this assay, it is used to robustly induce phosphorylation of the tails of PD-1.</li>
	<li>To phosphorylate the tagged versions of PD-1, remove the media from the PD-1-GFP-transfected cells and add 10 mL of plain DMEM (without serum or antibiotics) together with 10 &#181;L of pervanadate (step 2.1) to each 10 cm plate (resulting in eight plates expressing different versions of PD-1) (Figure 1). Place the plates at room temperature in the dark for 15 min. 
	NOTE: The SHP2-transfected cells (two plates; Figure 1) and the two non-transfected control plates are not treated with pervanadate, since these plates will serve as the source for the active SHP2 enzyme.</li>
	<li>Wash the cells with 5 mL of ice-cold 1x PBS. Repeat this step twice.</li>
</ol>
3. Immunoprecipitation
NOTE: The following steps should be performed on ice or at 4 &#176;C.
<ol>
	<li>Supplement the lysis buffer (50 mM Tris-HCl at pH 7.2, 250 mM NaCl, 0.1% NP-40, 2 mM EDTA, 10% glycerol) with protease inhibitors (dissolve 1 tablet in 10 mL of lysis buffer) and with 1 mM sodium orthovanadate. Add 500 &#181;L of ice-cold lysis buffer to the cells and remove and collect the cells from the plates immediately using a cell scraper. 
	NOTE: It is important to add the sodium orthovanadate only to the lysis buffer that will be used for the PD-1-GFP-transfected plates, since the SHP2-transfected plates and non-transfected control plates must retain phosphatase activity.</li>
	<li>Transfer the lysates into 1.5 mL cold tubes and rotate them at 0.005 x g, 4 &#176;C for 30 min.</li>
	<li>To collect the post nuclei supernatant (PNS) from the lysates, spin down the lysates for 10 min at 10,000 x g at 4 &#176;C and transfer the supernatants into new tubes. Discard the pellet. Store the supernatants of the second set of six plates (Figure 1) on ice for later WCL analysis.</li>
	<li>Preparation of the anti-GFP beads for immunoprecipitation of PD-1-GFP from the PD-1-GFP-transfected lysates of the first set (four plates) (Figure 1).
	<ol>
		<li>To prevent settling of the beads, gently shake the bottle with the beads before opening. Remove 40 &#181;L of the anti-GFP beads from the slurry per each condition.</li>
		<li>Spin at 500 x g for 3 min at 4 &#176;C and remove the supernatant to wash the beads. 
		NOTE: It is important to minimize the contact between the pipette plastic tips and agarose beads to prevent loss. It is recommended to cut the edge of a 200 &#181;L tip before transferring the beads to another tube.</li>
		<li>Resuspend the beads in 80 &#181;L of lysis buffer (per sample).</li>
	</ol>
	</li>
	<li>Add the washed beads directly to the cell lysate (PNS) from the PD-1-GFP-expressing cells of the first set (four plates) (step 3.3). Rotate at 0.005 x g for 30 min at 4 &#176;C to immunoprecipitate the GFP-tagged proteins.</li>
	<li>Wash the beads with 1 mL of cold lysis buffer (without orthovanadate) 3 times. Centrifuge the tube at 2,500 x g for 10 s. 
	NOTE: When adding the lysis buffer to the beads, add it directly onto the beads without touching them with the tips. There is no need to pipette up and down during the washes.</li>
	<li>Equally divide the lysate of the active SHP2 from the first set (one plate; step 3.3) and add it to three tubes of the washed PD-1-GFP-containing beads (the WT PD-1-GFP and the two different phosphdeficient mutations, Y223F and Y248F). Add one-third of the volume from the lysate of the non-transfected cells to the second tube of the WT PD-1-GFP beads. Discard the remaining two-thirds.</li>
	<li>Incubate the beads for 4 h at 4 &#176;C with gentle rotation (0.005 x g).</li>
	<li>Wash the beads twice with 1 mL of cold lysis buffer (without pervanadate or orthovanadate), as reported in step 3.6.</li>
	<li>Add 80 &#181;L of lysis buffer (without pervanadate or orthovanadate) per sample ensuring that the total volume in each tube is 100 &#181;L (20 &#181;L of beads and 80 &#181;L of lysis buffer). Mix gently and transfer 50 &#181;L from each tube into two fresh 1.5 mL tubes. 
	NOTE: Following this step, there will be two tubes filled with a mixture that contains beads and lysis buffer: one tube will be used for testing the co-immunoprecipitated SHP2 by western blotting, and the second will be used for the phosphatase activity assay.</li>
</ol>
4. Phosphatase Activity Assay
<ol>
	<li>Wash the beads once with phosphatase wash buffer (30 mM Hepes at pH 7.4 and 120 mM NaCl). Remove the supernatant completely.</li>
	<li>Add 100 &#181;L of assay buffer (30 mM Hepes at pH 7.4, 120 mM NaCl, 5 mM DTT, 10 mM p-nitrophenylphosphate) to the beads, and incubate at 30 &#176;C for 30 min under gentle agitation. NOTE: Incubation time may vary, but wait until the buffer turns yellow upon dephosphorylation.</li>
	<li>To terminate the reaction, add 50 &#181;L of 1 M NaOH when the buffer turns yellow.</li>
	<li>Spin down at 2,500 x g for 10 s and transfer 50 &#181;L of the supernatant to two wells (duplicates with 50 &#181;L per well) of half-area in a 96-well plate. Read the absorbance at 405 nm.</li>
	<li>Express the results as relative optical density (OD) over the control wild-type version of PD-1-GFP.</li>
</ol>
5. SHP2 Western Blot Analysis
<ol>
	<li>Spin down the beads and remove the supernatant.</li>
	<li>Add 20 &#181;L of 2x Laemmli buffer (see Materials Table) to the beads and boil at 95 &#176;C for 5 min.</li>
	<li>Measure the protein concentration of the input controls (second set) using a BCA kit (see Materials Table). Transfer 30 &#181;L of the most diluted sample to a new tube. Dilute the rest of the input controls with lysis buffer to the same concentration as the most diluted sample, and transfer 30 &#181;L from each of them to a new tube. 
	NOTE: Following this step, there will be 4 tubes with 30 &#181;L each, at similar protein concentrations, representing the input controls lysates.</li>
	<li>Add equal volumes of 2x Laemmli buffer to the lysates and boil at 95 &#176;C for 5 min. Spin down the beads and load the supernatant on 4&#8211;20% SDS-PAGE Tris-based gel (see Materials Table) for western blot analysis. In addition, load 50 &#181;L from each sample of the second set.</li>
	<li>Continue western blot analysis according to the protocol previously described12.</li>
</ol>

<ol><li>Montor, W. R., Salas, A., Melo, F. H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Receptor+tyrosine+kinases+and+downstream+pathways+as+druggable+targets+for+cancer+treatment%3A+the+current+arsenal+of+inhibitors%5BTitle%5D)+AND+%22Molecular+Cancer%22%5BJournal%5D)">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/pubmed?term=((Selected+signalling+proteins+recruited+to+the+T-cell+receptor-CD3+complex%5BTitle%5D)+AND+%22Immunology%22%5BJournal%5D)">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/pubmed?term=((The+coreceptor+programmed+death+1+inhibits+T-cell+adhesion+by+regulating+Rap1%5BTitle%5D)+AND+%22Journal+of+Allergy+and+Clinical+Immunology%22%5BJournal%5D)">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/pubmed?term=((SHP-1+and+SHP-2+associate+with+immunoreceptor+tyrosine-based+switch+motif+of+programmed+death+1+upon+primary+human+T+cell+stimulation%2C+but+only+receptor+ligation+prevents+T+cell+activation%5BTitle%5D)+AND+%22The+Journal+of+Immunology%22%5BJournal%5D)">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/pubmed?term=((Selective+effects+of+PD-1+on+Akt+and+Ras+pathways+regulate+molecular+components+of+the+cell+cycle+and+inhibit+T+cell+proliferation%5BTitle%5D)+AND+%22Science+Signaling%22%5BJournal%5D)">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/pubmed?term=((Programmed+death-1+concentration+at+the+immunological+synapse+is+determined+by+ligand+affinity+and+availability%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((Programmed+cell+death+1+forms+negative+costimulatory+microclusters+that+directly+inhibit+T+cell+receptor+signaling+by+recruiting+phosphatase+SHP2%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Medicine%22%5BJournal%5D)">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/pubmed?term=((T+cell+costimulatory+receptor+CD28+is+a+primary+target+for+PD-1-mediated+inhibition%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((PD-1+inhibits+T-cell+receptor+induced+phosphorylation+of+the+ZAP70%2FCD3zeta+signalosome+and+downstream+signaling+to+PKCtheta%5BTitle%5D)+AND+%22FEBS+Letters%22%5BJournal%5D)">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/pubmed?term=((PD-1+immunoreceptor+inhibits+B+cell+receptor-mediated+signaling+by+recruiting+src+homology+2-domain-containing+tyrosine+phosphatase+2+to+phosphotyrosine%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((Antagonism+between+binding+site+affinity+and+conformational+dynamics+tunes+alternative+cis-interactions+within+Shp2%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((Affinity+purification+mass+spectrometry+analysis+of+PD-1+uncovers+SAP+as+a+new+checkpoint+inhibitor%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((A+protein+tyrosine+phosphatase+inhibitor%2C+pervanadate%2C+inhibits+angiotensin+II-Induced+beta-arrestin+cleavage%5BTitle%5D)+AND+%22Molecules+and+Cells%22%5BJournal%5D)">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/pubmed?term=((Potent+stimulation+of+SH-PTP2+phosphatase+activity+by+simultaneous+occupancy+of+both+SH2+domains%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pubmed/20890902">Serine/threonine protein phosphatase assays.</a> Current Protocols in Molecular Biology. , Chapter 18, Unit18 18 (2010).</li>
<li>Shlapatska, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD150+association+with+either+the+SH2-containing+inositol+phosphatase+or+the+SH2-containing+protein+tyrosine+phosphatase+is+regulated+by+the+adaptor+protein+SH2D1A%5BTitle%5D)+AND+%22The+Journal+of+Immunology%22%5BJournal%5D)">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>

The authors have nothing to disclose.

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&amp;ndash;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>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</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 ...

co-immunoprecipitation-assay-for-studying-functional-interactions

Research

JoVE Journal

Immunology and Infection

15.1K Views.  The Chaim Sheba Medical Center. 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.

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

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and ...

Chemistry

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Biochemistry

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Biology

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Protein Interaction Analysis in HEK-293 Cells Using Co-Immunoprecipitation

Immunoprecipitation with an Anti-Epitope Tag Affinity Gel to Study Protein-Protein Interactions

Protein-protein interactions (PPIs) play a pivotal role in biological phenomena, such as cellular organization, intracellular signal transduction, and transcriptional regulation. Therefore, understanding PPIs is an important starting point for further investigation of the function of the target protein. In this study, we propose a simple method to determine the binding of two target proteins by introducing mammalian expression vectors into HEK-293 cells using the polyethylenimine method, lysing the cells in homemade protein lysis buffer, and pulling down the target proteins on an epitope tag affinity gel. In addition, the PPI between the various epitope tag fused proteins can be confirmed by using affinity antibodies against each tag instead of the epitope tag affinity gel. This protocol could also be used to verify various PPIs, including nuclear extracts, from other cell lines. Therefore, it can be used as a basic method in a variety of PPI experiments. Proteins degrade by extended time course and repeated freeze-thaw cycles. Therefore, cell lysis, immunoprecipitation, and immunoblotting should be performed as seamlessly as possible.

Author Spotlight: Unraveling the Molecular Mechanisms of Brown and Beige Adipocyte Regulation

Protein-protein interactions are important for elucidating the function of target proteins, and co-immunoprecipitation (co-IP) can easily confirm PPIs. We transiently transfected a plasmid encoding an epitope-tagged protein into HEK-293 cells and developed an immunoprecipitation method to easily confirm the binding of two target proteins.

Protein-protein interactions (PPIs) play a pivotal role in biological phenomena, such as cellular organization, intracellular signal transduction, and ...

Co-Immunoprecipitation and Pull-Down Assays

Co-immunoprecipitation (CoIP) and pull-down assays are closely related methods to identify stable protein-protein interactions. These methods are related to immunoprecipitation, a method for separating a target protein bound to an antibody from unbound proteins. In CoIP, an antibody-bound protein is itself bound to another protein that does not bind with the antibody, this is followed by a separation process that preserves the protein-protein complex. The difference in pull-down assays is that affinity-tagged bait proteins replace antibodies, and affinity chromatography is used to isolate protein-protein complexes.
This video explains CoIP, pull-down assays, and their implementation in the laboratory. A step-by-step protocol for each technique is covered, including the reagents, apparatus, and instruments used to purify and analyze bound proteins. Additionally, the applications section of this video describes a procedure to study how myxovirus proteins inhibit influenza nucleoprotein, an investigation into the role of calcium ions in calmodulin via a pull-down assay, and a modified pull-down assay for characterizing transient protein interactions.
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Protein-protein interactions play a significant role in a wide variety of biological functions. The majority of protein-protein interactions and their biological effects have yet to be identified. Co-immunoprecipitation, or CoIP, and pull-down assays are two closely related methods for the identification of stable protein-protein interactions. This video will cover the principles of the two assays, their general laboratory procedures, and applications of these techniques.
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CoIP and pull-down assays are variants of immunoprecipitation, a method for selectively isolating a protein species from a complex solution. In an immunoprecipitation experiment, an antibody specific to a target protein is allowed to form an immune complex with that target in the sample. The complex is then captured on a solid support, typically protein A bound to a sepharose bead. Any proteins not captured are removed by centrifugation steps. The protein is then released from the antibody and solid support by boiling in reducing SDS-PAGE sample-loading buffer.
Co-immunoprecipitation is conducted in the same manner, except that intact protein complexes are captured onto the solid support. The antibody binds to the target protein, which, in turn, is bound to another protein that is not targeted by the antibody. As in immunoprecipitation, the protein complex is released from the antibody and solid support by boiling in reducing SDS-PAGE sample loading buffer.
Pull-down assays are similar to co-immunoprecipitation, differing only in the use of a &#34;bait&#34; protein, as opposed to an antibody. Through molecular biology techniques, this bait protein is engineered with an affinity tag, such as a series of histidine residues. These affinity tags are described in &#34;Chromatography-based Biomolecule Separations.&#34; The protein is then captured on an immobilized affinity ligand specific for the tag. The captured protein is then incubated with a sample that contains proteins that form complexes with the &#34;bait&#34;. The protein complex is released from the affinity support by washing with a solution containing a competitive analyte specific for the tag on the &#34;bait&#34; protein. Pull-down assays are useful for confirming protein interactions predicted by co-immunoprecipitation, and for discovering unknown protein interactions.
Now that the principles of co-immunoprecipitation and pull-down assays have been discussed, let&#39;s look at their laboratory procedures.
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First let&#39;s discuss co-immunoprecipitation. To a series of microfuge tubes, the following is added: PBS buffer, and a 50% solution of protein A-sepharose, a resin-protein complex that binds to the antibody. The microfuge tubes are rotated to ensure proper distribution, and then the resin is washed with additional PBS buffer. Cell lysate, containing the desired protein, and 2 &#956;g of antibody are added to the microfuge tubes, and the mixture is rotated for 1 h at 4 &#176;C. The beads are pelleted by centrifugation, the supernatant is discarded, and the beads re-washed three times with buffer to remove non-bound protein. The beads containing the antibody-protein complex are in reducing SDS-PAGE sample-loading buffer, for removal of the complex from the antibody and for analysis by SDS-PAGE and immunoblotting.
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Now we will discuss the procedure for pull-down assays: The &#34;bait&#34; protein is expressed in a plasmid with the appropriate affinity tag. After reaching log-phase growth, the cells are lysed, and then centrifuged. Suspended streptavidin-sepharose beads, which capture biotin-tagged &#34;bait&#34; protein, are pipetted into a microfuge tube. The beads are then centrifuged and the supernatant carefully removed by aspiration. The beads are then washed with buffer, centrifuged, and the supernatant removed.
Cells containing the putative &#34;prey&#34; protein, which has an affinity for the &#34;bait&#34; protein, are harvested by centrifugation. The supernatant is then added to the microfuge tube containing the resin, and incubated at 4 &#176;C for 3 h on a shaker. The resin is then centrifuged, the supernatant removed, and the resin washed to remove non-bound protein. Elution buffer is added to the resin, and the mixture is incubated at room temperature for 30 min on a shaker. The resin is then centrifuged, and the supernatant containing the desired complex is analyzed by immunoblotting.
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Now that we&#39;ve reviewed the procedures, let&#39;s look at some of the useful applications of co-immunoprecipitation and pull-down assays.
Co-immunoprecipitation can be useful in better understanding of enzymes&#39; mechanism of action. Myxovirus-, or Mx-, resistance proteins inhibit a wide range of viruses, including influenza A, for which the mechanism is poorly understood. Co-immunoprecipitation was used to study the interaction between mouse Mx1 protein and influenza nucleoprotein.
Pull-down assays have proven useful in studying the effects of second messengers, which are proteins that communicate a signal from the cellular environment. They are a component of a signaling pathway where multiple proteins interact in response to environmental cues. Calcium ions act as secondary messengers by binding to calmodulin, which, in turn, binds to a wide variety of proteins, mediating many types of biological responses. Without the calcium, the protein can&#39;t bind to the calmodulin. A pull-down assay was conducted to test the ability of proteins to bind to calmodulin in the presence or absence of calcium ions.
Co-immunoprecipitation and pull-down assays are generally used for analyzing stable or strong protein interactions, but not transient ones. A recent development in pull-down assays, the HaloTag, has simplified the study of transient protein interactions; HaloTag is a genetically-encoded protein fusion tag, fused to the protein of interest, capable of chemically reacting with a haloalkane solid support. If functional analysis is desired, the protein complex, minus the tag, in its entirety could then be isolated by incubating with tobacco etch virus protease.
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Co-immunoprecipitation (CoIP) and pull-down assays are closely related methods to identify stable protein-protein interactions. These methods are related ...

Education

JoVE Science Education

On-Membrane Protein Digestion to Prepare Co-Immunoprecipitated Proteins for Interaction Studies

Source: Obama, T. et al., <a href="https://app.jove.com/v/59733">Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique.</a>&#160;J. Vis. Exp.&#160; (2019)
This video demonstrates co-immunoprecipitated protein complexes on a PVDF membrane for protein-protein interaction analysis. The reduced proteins from complexes were treated with trypsin to cleave the individual proteins at the smaller peptides, then, the remaining peptides were extracted from the PVDF membrane. The pulled peptide derived from both proteins in the complex was then dried and resuspended in a low concentration of formic acid for further analysis.

Source: Obama, T. et al., Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique.&#160;J. Vis. Exp.&#160; (2019)
This ...

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-protein interactions

Enzyme-Linked Immunosorbent Assay to Verify Chemically-Coupled Antibody-Antigen Conjugates

Source: Volckmar, J. et al., <a href="https://app.jove.com/v/62018">Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo.</a> J. Vis. Exp. (2021)
This video demonstrates the detection of chemically conjugated antibody-antigen complexes via the sandwich enzyme-linked immunosorbent assay, or ELISA, technique. The captured antibody binds to the specific epitope of the antigen of the antibody-antigen conjugate, which can be later visualized using an enzyme-linked secondary antibody.

Source: Volckmar, J. et al., Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In ...

Assay Techniques

Immunoassays

Tandem Affinity Purification Assay to Study Protein-Protein Interactions

Source: Ma, Z., et al. <a href="https://app.jove.com/v/55236">Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.</a> J. Vis. Exp. (2017).
This video demonstrates tandem affinity purification &#8212; a technique to purify protein complexes from eukaryotic cells to study protein-protein interaction. The complex contains two proteins labeled with different epitope tags. Upon the purification of the complex using two resins with an affinity for the two different tags in a sequential manner, the presence of both proteins in the elute confirms the interaction between the two.

Source: Ma, Z., et al. Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells. J. Vis. Exp. (2017).
This video demonstrates tandem ...

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

Summary

Explore More Videos

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

Evaluation of Protein–Protein Interactions using an On-Membrane Digestion Technique

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Imaging Protein-protein Interactions in vivo

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Immunoprecipitation with an Anti-Epitope Tag Affinity Gel to Study Protein-Protein Interactions

Co-Immunoprecipitation and Pull-Down Assays

On-Membrane Protein Digestion to Prepare Co-Immunoprecipitated Proteins for Interaction Studies

Enzyme-Linked Immunosorbent Assay to Verify Chemically-Coupled Antibody-Antigen Conjugates

Tandem Affinity Purification Assay to Study Protein-Protein Interactions