This work was supported by grants from the Research Program (NRF- 2015M3A9E7029172) of the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning.

1. Primer design strategy
<ol>
	<li>Design primers to introduce genes of interest into pBiT1.1-C [TK/LgBiT], pBiT2.1-C [TK/SmBiT], pBiT1.1-N [TK/LgBiT] and pBiT2.1-N [TK/SmBiT] Vectors.</li>
	<li>Select at least one of these three sites as one of the two unique restriction enzymes needed for directional cloning due to the presence of an in-frame stop codon that divides the multicloning site as shown in Figure 111.</li>
	<li>Incorporate nucleotide sequence into the primers as shown in Table 1 to encode the linker residues shown in red in Table 211.</li>
	<li>For pBiT1.1-C [TK/LgBiT] and pBiT2.1-C [TK/SmBiT] vectors, make sure that the 5 &#769; primer contains an ATG codon and a potent Kozak consensus sequence (GCCGCCACC).</li>
	<li>For pBiT1.1-N [TK/LgBiT] and pBiT2.1-N [TK/SmBiT] vectors, ensure that the 3 &#769; primer contains a stop codon. 
	NOTE: Each vector contains the HSV-TK promoter to minimize nonspecific association and reduce experimental artifacts, also each vector has an expression cassette for ampicillin resistance in bacteria.</li>
</ol>
2. PCR
<ol>
	<li>Set up and run PCR reactions to amplify the insert DNA of the gene of interest using the primers designed from step 1. It is important to use a high-fidelity DNA polymerase to minimize mutations.</li>
	<li>Use exactly the following order to prepare 50 &#181;L of PCR reaction. Add 35.5 &#181;L of distilled water, 5 &#181;L of 10x Polymerase buffer, 5 &#181;L of dNTP mixture (2.5 mM each), 1 &#181;L of plasmid template (200 ng/&#181;L), 1.25 &#181;L of forward and reverse primers (10 &#956;M) and 1 &#181;L of high-fidelity polymerase (5 U/&#181;L).</li>
	<li>Using a thermocycler set up the following DNA amplification program.
	<ol>
		<li>Denature at 95 &#176;C for 5 min.</li>
		<li>Repeat 25 times the following thermal cycle: 95 &#176;C for 30 s, 60 &#176;C for 1 min, 72 &#176;C for 2 min per 1 kbp to be amplified.</li>
		<li>Run a final extension at 72 &#176;C for 10 min.</li>
		<li>Hold the samples at 4 &#176;C within the thermocycler. 
		NOTE: It is highly recommended to use a high-fidelity polymerase in order to minimize point mutations particularly those occurring during the amplification of long sequences. As the amplicon becomes longer, the degree of accuracy in the replication of the DNA decreases. For the PCR reaction, choose the annealing temperature based on the melting temperature of the region where the oligos directly hybridize with the DNA template and not with all the sequence of the primer.</li>
	</ol>
	</li>
	<li>PCR product purification
	<ol>
		<li>Isolate the PCR product from the rest of the PCR reaction using a kit from a manufacturer of preference12. The PCR product is now ready for restriction digestion.</li>
	</ol>
	</li>
</ol>
3. DNA digestion
<ol>
	<li>For the PCR product digestion prepare 50 &#956;L of digestion reaction as follows.
	<ol>
		<li>Using a 1.5 mL tube, add 12 &#956;L of distilled water.</li>
		<li>Add 5 &#956;L of 10x buffer with the best compatibility with both restriction enzymes.</li>
		<li>From step 2.4 add 30 &#956;L of PCR product</li>
		<li>Finally, add 1.5 &#956;L of each restriction enzyme.</li>
		<li>Briefly mix by vortex and incubate at 37.5 &#176;C overnight.</li>
	</ol>
	</li>
	<li>For the recipient plasmid digestion prepare 50 &#956;L of digestion reaction as follows:
	<ol>
		<li>Using a 1.5 mL tube, add 23 &#956;L of distilled water.</li>
		<li>Add 5 &#956;L of 10x buffer with the best compatibility with both restriction enzymes.</li>
		<li>Add 15 &#956;L of recipient plasmid (200 ng/&#956;L).</li>
		<li>Add 1.5 &#956;L of each restriction enzyme.</li>
		<li>Briefly mix by vortex and incubate at 37.5 &#176;C overnight. 
		NOTE: It is important to use 3 &#956;g of recipient plasmid in order to obtain sufficient material after DNA agarose gel purification. It is also relevant to leave DNA digestions overnight using both enzymes to obtain high cloning efficiency.</li>
	</ol>
	</li>
</ol>
4. DNA agarose gel purification and cloning
<ol>
	<li>Prepare a 1% agarose gel to run the digested DNA plasmid and inserts and proceed to cut the corresponding bands. Once the corresponding vector and insert bands have been purified12, determine the DNA concentration using a spectrophotometer.</li>
	<li>Perform DNA ligation to fuse the insert to the recipient plasmid.</li>
	<li>Prepare ligation reactions of around 100 ng of total DNA including 50 ng of plasmid vector.</li>
	<li>Set up recipient plasmid-insert ratio of approximately 1:3; it can be calculated using a vector-insert calculator13.</li>
	<li>Set up negative controls in parallel. For instance, a ligation of the recipient plasmid DNA without any insert will provide information about how much background of undigested or self-ligating recipient plasmid is present.</li>
</ol>
5. Transformation of clones
<ol>
	<li>Place a tube of DH5&#945; competent cells from the freezer at -80 &#176;C and immediately transfer it on ice for 20 min.</li>
	<li>After that time, take 55 &#181;L of DH5&#945; competent cells and add 4 &#181;L of ligation reaction and mix by flicking the tube and store on ice for 45 min.</li>
	<li>Place the tube in a water bath previously warmed at 42 &#176;C for exactly 48 s and immediately get the tubes back on ice for another 3 min.</li>
	<li>Add 600 &#181;L of Luria Broth (LB) medium previously warmed at 37.5 &#176;C and incubate with shaking for 1 hour at 200 rpm.</li>
	<li>Transfer 200 &#181;L into an agar plate containing ampicillin 100 &#956;g/mL and gently spread over the surface with the liquid is mostly absorbed.</li>
	<li>Incubate the plates overnight to see the colonies next morning. The recipient plasmid on the insert plate should have significantly more colonies than the recipient plasmid alone plate.</li>
</ol>
6. Isolation of the finished plasmid
<ol>
	<li>Pick 3-10 individual bacterial colonies and transfer into 1 mL of LB medium containing ampicillin (100 &#956;g/mL) and incubate for 6 h.</li>
	<li>Take 200 &#181;L of bacterial suspension and transfer to 5 mL of LB medium containing the same concentration of ampicillin as in step 6.1 and incubate overnight at 37.5 &#176;C with shaking at 200 rpm.</li>
	<li>Using a miniprep DNA kit purification, perform miniprep DNA purifications using 5 mL of LB grown overnight following the manufacturer instructions14.</li>
	<li>To identify successful ligations, set up PCR reactions in the same way as in section 2 using the DNA obtained from step 6.3 as a template with the same primers as in section 2 during the first PCR. Positive clones will produce PCR products with the corresponding size.</li>
	<li>After large prep DNA purification of the positive clones, conduct a diagnostic restriction digestion of 500 ng of purified DNA with the enzymes used during the cloning step and run the digested products on an 1% agarose gel. There should be two bands: one the size of the vector and one the size of the new insert.</li>
	<li>Verify the construct sequence by sequencing using the following primers: Forward 5&#8217;- aaggtgacgcgtgtggcctcgaac-3&#8217; and reverse 5&#8217;-gcatttttttcactgcattctagtt-3&#8217;. 
	NOTE: When DNA is replicated using PCR, there is always the possibility of errors during the amplification even when using a high-fidelity polymerase, therefore is very important to sequence the final constructs.</li>
</ol>
7. Transfection and protein expression
<ol>
	<li>In a previously poly-L-lysine-coated white 96 well plate, perform cell seeding one day before transfection at 2.5 x 104 cells per well using Dulbecco&#8217;s modified Eagle&#8217;s medium supplemented with 10% of Fetal Bovine Serum, 100 U/mL penicillin G, and 100 &#956;g/mL streptomycin.</li>
	<li>Use only the 60 inner wells to minimize the potential for thermal gradients across the plate and edge effects from evaporation. Add 200 &#956;L of sterile distilled water to the 36 outside wells and 150 &#956;L in the spaces between wells and incubate overnight at 37.5 &#176;C and 5% of CO2.</li>
	<li>The following morning perform transfection using 100 ng of total DNA (50 ng each construct).</li>
	<li>Set up four different plasmid combinations (receptor:&#946;-arrestin) according to Figure 2b.</li>
	<li>For each plasmid combination use 20 &#181;L of modified Eagle&#39;s Minimum Essential Media buffered with HEPES using 0.3 &#181;L of lipidic transfection reagent per well.</li>
	<li>Add 20 &#956;L of lipidic transfection reagent-DNA mixture to each well and mix the plate in circles for 10 s.</li>
	<li>Change fresh medium after 6 hr incubation at 37.5 &#176;C and 5% CO2.</li>
	<li>Incubate the plate for 24 h at 37.5 &#176;C and 5% CO2.</li>
</ol>
8. Monitoring receptor-&#946;-arrestin1/2 interactions in HEK293 cells
<ol>
	<li>Aspirate medium and add 100 &#956;L of modified Eagle&#39;s Minimum Essential Media buffered with HEPES to each well and let the plate stabilize at RT for 10 min.</li>
	<li>Prepare the furimazine substrate by combining 1 volume of 100x substrate with 19 volumes of LCS Dilution Buffer (a 20-fold dilution)11, creating a 5x stock to mix with cell culture medium.</li>
	<li>Add 25 &#956;L of 5x furimazine to each well and gently mix in circles for 10 s.</li>
	<li>Measure luminescence for 10 min for signal stabilization at RT. 
	NOTE: By using this baseline signal to normalize the response of each well it will help to reduce variability caused by differences in the number of cells plated per well, also differences in transfection efficiency, etc. Once calculated, average the normalized response from replicate wells for a given drug treatment.</li>
	<li>Prepare 13.5x ligand solution in modified Eagle&#39;s Minimum Essential Media buffered with HEPES.</li>
	<li>For experiments at room temperature with 10 &#181;L of 13.5x ligand addition, add the compounds using injectors or a multichannel pipette and mix the plate by hand or using an orbital shaker (20 s at 200 rpm).</li>
	<li>For experiments at 37.5 &#176;C with 10 &#181;L of 13.5x ligand addition, use injectors to dispense compounds and mix by using the instrument orbital shaker. In case of not using injectors, remove the plate from the luminometer, add the ligands and mix the plate by hand or using an orbital shaker (20 s at 200 rpm). 
	NOTE: Use injectors and a shaker within the detection instrument to minimize temperature fluctuations associated with removing the plate from the luminometer. Standard benchtop luminometers can be used for this assay. Use an integration time of 0.25&#8211;2 s.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pharmacology+of+GPCRs.">Molecular pharmacology of GPCRs.</a> British Journal of Pharmacology. 175 (21), 1754005-1754008 (2018).</li><li>Lohse, M. J., Hoffmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arrestin+Interactions+with+G+Protein-Coupled+Receptors.">Arrestin Interactions with G Protein-Coupled Receptors.</a> Handbook of Experimental Pharmacology. 219, 15-56 (2014).</li><li>Kang, D. S., 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=Structure+of+an+arrestin2-clathrin+complex+reveals+a+novel+clathrin+binding+domain+that+modulates+receptor+trafficking.">Structure of an arrestin2-clathrin complex reveals a novel clathrin binding domain that modulates receptor trafficking.</a> Journal of Biological Chemistry. 284, 29860-29872 (2009).</li><li>Park, S. 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=Effects+of+&#946;-Arrestin-Biased+Dopamine+D2+Receptor+Ligands+on+Schizophrenia-Like+Behavior+in+Hypoglutamatergic+Mice.">Effects of &#946;-Arrestin-Biased Dopamine D2 Receptor Ligands on Schizophrenia-Like Behavior in Hypoglutamatergic Mice.</a> Neuropsychopharmacology. 41 (3), 704-715 (2016).</li><li>Zhu, L., Cui, Z., Zhu, Q., Zha, X., Xu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Opioid+Receptor+Agonists+with+Reduced+Morphine-like+Side+Effects.">Novel Opioid Receptor Agonists with Reduced Morphine-like Side Effects.</a> Mini-Reviews in Medicinal Chemistry. 18 (19), 1603-1610 (2018).</li><li>Smith, J. S., Lefkowitz, R. J., Rajagopal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biased+signalling:+from+simple+switches+to+allosteric+microprocessors.">Biased signalling: from simple switches to allosteric microprocessors.</a> Nature Reviews Drug Discovery. 17 (4), 243-260 (2018).</li><li>Dixon, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoLuc+Complementation+Reporter+Optimized+for+Accurate+Measurement+of+Protein+Interactions+in+Cells.">NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells.</a> ACS Chemical Biology. 11 (2), 400-408 (2016).</li><li>Reyes-Alcaraz, A., Lee, Y. N., Yun, S., Hwang, J. I., Seong, J. 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The authors declare no competing interests.

Using the method presented here, interactions between any GPCR and &#946;-arrestin1/2 can be monitored in real time living systems using this GPCR-&#946;-arrestin structural complementation assay. In this regard, we were able to observe differential &#946;-arrestin recruitment between the two &#946;-arrestin isoforms by the GLP-1r (A prototypical Class B GPCR), we also observed a dissociation of the receptor-&#946;-arrestin complex a few minutes after reaching the maximum luminescent signal.
I...

GPCRs represent the target of nearly 35% of current drugs in the market1,2 and a clear understanding of their pharmacology is crucial in the development of novel therapeutic drugs3. One of the key aspects in GPCR drug discovery, particularly during the development of biased agonists is the characterization of novel ligands towards receptor-&#946;-arrestin interactions4 and &#946;-arrestin interactions with other cytosolic proteins such as clathrin5.
It has been documented that &#946;-arrestin dependent signaling plays a key role in neurological disorders such as bipolar disorder, major depression, and schizophrenia6 and also severe side effects in some medications such as morphine7.
Current methods used to monitor these interactions usually do not represent actual endogenous levels of the proteins in study, in some cases they show weak signal, photobleaching and depending of the GPCR it might be technically challenging to set up8. This novel structural complementation assay uses low expression promoter vectors in order to mimic endogenous physiological levels and provides high sensitivity compared to current methods9. Using this approach, it was possible to easily characterize Galanin receptor-&#946;-arrestin1/2 and also &#946;-arrestin2-clathrin interactions10. This methodology can be widely used to any GPCR of particular interest where &#946;-arrestins play a key physiological function or their signaling is relevant in some diseases.

<table>
	<tbody>
		<tr>
			<td>Antibiotics penicillin streptomycin</td>
			<td>Welgene</td>
			<td>LS202-02</td>
			<td>Penicillin/Streptomycin</td>
		</tr>
		<tr>
			<td>Bacterial Incubator</td>
			<td>JEIO Tech</td>
			<td>IB-05G</td>
			<td>Incubator (Air-Jacket), Basic</td>
		</tr>
		<tr>
			<td>Cell culture medium</td>
			<td>Welgene</td>
			<td>LM 001-05</td>
			<td>DMEM Cell culture medium</td>
		</tr>
		<tr>
			<td>Cell culture transfection medium</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td>Optimem 1X cell culture medium</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>NUAIRE</td>
			<td>NU5720</td>
			<td>Direct Heat CO2 Incubator</td>
		</tr>
		<tr>
			<td>Digital water bath</td>
			<td>Lab Tech</td>
			<td>LWB-122D</td>
			<td>Digital water bath lab tech</td>
		</tr>
		<tr>
			<td>DNA Polymerase proof reading</td>
			<td>ELPIS Biotech</td>
			<td>EBT-1011</td>
			<td>PfU DNA polymerase</td>
		</tr>
		<tr>
			<td>DNA purification kit</td>
			<td>Cosmogenetech</td>
			<td>CMP0112</td>
			<td>miniprepLaboPass Purificartion Kit Plasmid Mini</td>
		</tr>
		<tr>
			<td>DNA Taq Polymerase</td>
			<td>Enzynomics</td>
			<td>P750</td>
			<td>nTaq DNA polymerase</td>
		</tr>
		<tr>
			<td>Enzyme restriction BglII</td>
			<td>New England Biolabs</td>
			<td>R0144L</td>
			<td>BglII</td>
		</tr>
		<tr>
			<td>Enzyme restriction buffer</td>
			<td>New England Biolabs</td>
			<td>B72045</td>
			<td>CutSmart 10X Buffer</td>
		</tr>
		<tr>
			<td>Enzyme restriction EcoRI</td>
			<td>New England Biolabs</td>
			<td>R3101L</td>
			<td>EcoRI-HF</td>
		</tr>
		<tr>
			<td>Enzyme restriction NheI</td>
			<td>New England Biolabs</td>
			<td>R01315</td>
			<td>NheI</td>
		</tr>
		<tr>
			<td>Enzyme restriction XhoI</td>
			<td>New England Biolabs</td>
			<td>R0146L</td>
			<td>XhoI</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco Canada</td>
			<td>12483020</td>
			<td>Fetal Bovine Serum</td>
		</tr>
		<tr>
			<td>Gel/PCR DNA MiniKit</td>
			<td>Real Biotech Corporation</td>
			<td>KH23108</td>
			<td>HiYield Gel/PCR DNA MiniKit</td>
		</tr>
		<tr>
			<td>Ligase</td>
			<td>ELPIS Biotech</td>
			<td>EBT-1025</td>
			<td>T4 DNA Ligase</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Olympus</td>
			<td>CKX53SF</td>
			<td>CKX53 Microscope Olympus</td>
		</tr>
		<tr>
			<td>lipid transfection reagent</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td>Lipofectamine 2000</td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Biotek/Fisher Scientific</td>
			<td>12504386</td>
			<td>Synergy 2 Multi-Mode Microplate Readers</td>
		</tr>
		<tr>
			<td>NanoBiT System</td>
			<td>Promega</td>
			<td>N2014</td>
			<td>NanoBiT PPI MCS Starter System</td>
		</tr>
		<tr>
			<td>Nanoluciferase substrate</td>
			<td>Promega</td>
			<td>N2012</td>
			<td>Nano-Glo Live Cell assay system</td>
		</tr>
		<tr>
			<td>PCR Thermal cycler</td>
			<td>Eppendorf</td>
			<td>6336000015</td>
			<td>Master cycler Nexus SX1</td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma Aldrich</td>
			<td>P4707-50ML</td>
			<td>Poly-L-lysine solution</td>
		</tr>
		<tr>
			<td>Trypsin EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>Trysin EDTA 10X</td>
		</tr>
		<tr>
			<td>White Cell culture 96 well plates</td>
			<td>Corning</td>
			<td>3917</td>
			<td>Assay Plate 96 well plate</td>
		</tr>
	</tbody>
</table>

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.

Using the procedure presented here, interactions between a prototypical GPCR and two &#946;-arrestin isoforms were monitored. Glucagon like peptide receptor (GLP-1r) constructs were made using primers containing NheI and EcoRI enzyme restriction sites and cloned into the vectors pBiT1.1-C [TK/LgBiT] and pBiT2.1-C [TK/SmBiT] while in the case of &#946;-arrestins, two additional vectors were used pBiT1.1-N [TK/LgBiT] and pBiT2.1-N [TK/SmBiT] using enzyme restriction sites BgIII and EcoRI in the case of &#946;-arrestin2 and...

Watch this Scientific Journal Video about Monitoring GPCR-ÃŽÂ²-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery at JoVE.com

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

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. ...

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Research

JoVE Journal

Biochemistry

6.8K Views.  Korea University. GPCR-β-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-β-arrestin interactions in real time living cells, and it can be extended to any GPCR.

Video: Monitoring GPCR-β-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical probe that targets the enzyme&#39;s active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo.

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Real-time Tracking of DNA Fragment Separation by Smartphone

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

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 ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) and captures double-stranded DNA (dsDNA) non-specifically to interrogate it for a homologous sequence. After encountering homology, Rad51 catalyzes DNA strand exchange to mediate pairing of the ssDNA with the complementary strand of the dsDNA. This reaction is highly regulated by numerous accessary proteins in vivo. Although conventional biochemical assays have been successfully employed to examine the role of such accessory protein in vitro, kinetic analysis of intermediate formation and its progression into a final product has proven challenging due to the unstable and transient nature of the reaction intermediates. To observe these reaction steps directly in solution, fluorescence resonance energy transfer (FRET)-based real-time observation systems of this reaction were established. Kinetic analysis of real-time observations shows that the DNA strand exchange reaction mediated by Rad51 obeys a three-step reaction model involving the formation of a three-strand DNA intermediate, maturation of this intermediate, and the release of ssDNA from the mature intermediate. The Swi5-Sfr1 complex, an accessary protein conserved in eukaryotes, strongly enhances the second and third steps of this reaction. The FRET-based assays presented here enable us to uncover the molecular mechanisms through which recombination accessary proteins stimulate the DNA strand exchange activity of Rad51. The primary goal of this protocol is to enhance the repertoire of techniques available to researchers in the field of homologous recombination, particularly those working with proteins from species other than Schizosaccharomyces pombe, so that the evolutionary conservation of the findings presented herein can be determined.

Fluorescence resonance energy transfer-based real-time observation systems of the DNA strand exchange reaction mediated by Rad51 were developed. Using the protocols presented here, we are able to detect the formation of reaction intermediates and their conversion into products, while also analyzing the enzymatic kinetics of the reaction.

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.

We present a protocol to screen anti-hepatitis B virus (HBV) compounds targeting pre- and post-viral entry lifecycle stages, using isothermal titration calorimetry to measure binding affinity (KD) with host sodium taurocholate cotransporting polypeptide. Antiviral efficacy was determined through the suppression of viral lifecycle markers (cccDNA formation, transcription, and viral assembly).

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load ...

Coating CryoEM Grids with Monolayer Graphene to Improve Cryo-Sample Preparation

Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice, suspended across roughly 1 &#181;m wide foil holes. The resulting sample is imaged using cryogenic transmission electron microscopy, and after image processing using suitable software, near-atomic resolution structures can be determined. Despite cryoEM&#39;s widespread adoption, sample preparation remains a severe bottleneck in cryoEM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice. Recently, methods have been developed to modify cryoEM grids with a single continuous layer of graphene, which acts as a support surface that often increases particle density in the imaged area and can reduce interactions between particles and the air-water interface. Here, we provide detailed protocols for the application of graphene to cryoEM grids and for rapidly assessing the relative hydrophilicity of the resulting grids. Additionally, we describe an EM-based method to confirm the presence of graphene by visualizing its characteristic diffraction pattern. Finally, we demonstrate the utility of these graphene supports by rapidly reconstructing a 2.7 &#197; resolution density map of a Cas9 complex using a pure sample at a relatively low concentration.

Author Spotlight: Enhancing CryoEM Sample Preparation Using Graphene Monolayer on Microscopy Grids 

The application of support layers to cryogenic electron microscopy (cryoEM) grids can increase particle density, limit interactions with the air-water interface, reduce beam-induced motion, and improve the distribution of particle orientations. This paper describes a robust protocol for coating cryoEM grids with a monolayer of graphene for improved cryo-sample preparation.

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to ...