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 p...

<ol>
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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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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-Î²-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery at JoVE.com

Monitoring GPCR-&amp;#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.

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. ...

This protocol has a high impact in the field of drug discovery and development, especially in the development of novel, first-in-class drugs with minimal side effects. The main advantage of this technique is that it can be applied to any GPCR system, and used to obtain accurate information about receptor pharmacology in real-time living systems. The implications of this technique are closely related to new therapies particularly where beta-arrestins are relevant.These include opiod receptors in pain and dopamine receptors in schizophrenia. This method can be useful in many medical scientific research areas from neurology to cardiopulmonary disease. PCR demonstration of our method is important, because it allows for a detailed presentation of the critical steps in the protocol, which would not be possible with conventional publication.Begin by designing primers to introduce genes of interest to the pBiT vectors. Due to the inframe stop codon that divides the multi-cloning site, select at least one of the three sites as one of the two unique restriction enzymes needed for directional cloning. Refer to the manuscript for nucleotide sequences to encode the linker residues and incorporate them into the primers.For the pBiT1.1-C and pBiT2.1-C make sure that the forward primer contains an ATG codon and a potent Kozak consensus sequence. For the pBiT1.1-N and pBiT2.1-N make sure that the reverse primer contains a stop codon. Use the designed primers to amplify the insert DNA of the gene of interest.Combine the PCR reagents according to manuscript directions, making sure to use a high-fidelity DNA polymerase to minimize mutations. Program the thermocycler and start the reaction. After completing PCR, digest the product in a 50-microliter digestion reaction.In a 1.5-milliliter tube, combine 12 microliters of distilled water, five microliters of the appropriate 10X restriction digestion buffer, and 30 microliters of the PCR product. Then add 1.5 microliters of each restriction enzyme. Vortex the reaction mixture.And incubate overnight at 37.5 degrees Celsius. Prepare a second reaction to digest the recipient plasmid. In a 1.5-milliliter tube, combine 23 microliters of water, five microliters of the appropriate 10X restriction digestion buffer, 15 microliters of the recipient plasmid, and 1.5 microliters of each restriction enzyme.Briefly mix the tube. And incubate overnight at 37.5 degrees Celsius. On the day following the cloning and transformation, pick three to 10 individual bacterial colonies and transfer them to one milliliter of LB medium with ampicillin.Incubate the cells for six hours. And then transfer 200 microliters of bacterial suspension to five milliliters of fresh LB medium with ampicillin. Incubate the culture at 37.5 degrees Celsius with shaking at 200 rpm overnight.On the next day, isolate the plasmid with a mini-prep DNA purification kit. Screen the purified plasmid for successful ligation with PCR. One day before transfection, seed the cells on a poly-L-lysine coated 96-well plate using Dulbecco's modified Eagle's medium supplemented with 10%fetal bovine serum, 100 units per milliliter of penicillin G, and 100 microliters per milliliter of streptomycin.Only add cells to the 60 inner wells to minimize potential for thermal gradients across the plate and edge effects from evaporation. Add 200 microliters of sterile distilled water to the 36 outside wells, and 150 microliters into the spaces between the wells. Then incubate the plate overnight at 37.5 degrees Celsius and 5%carbon dioxide.On the next day, perform transfection using a total of 100 nanograms DNA. Set up four different plasmid combinations according to manuscript directions. Using 20 microliters of modified Eagle's minimum essential media buffered with HEPES, and 0.3 microliters of lipidic transfection reagent per well.Add 20 microliters of lipidic transfection reagent and DNA mixture to each well, and mix the plate in circles for 10 seconds. Incubate the plate at 37.5 degrees Celsius and 5%carbon dioxide for six hours. Refresh the medium.And incubate for another 24 hours. After the incubation, aspirate the medium and add 100 microliters of modified Eagle's minimum essential media buffered with HEPES to each well. Allow the plate to stabilize at room temperature for 10 minutes.Meanwhile, prepare the furimazine substrate by combining one volume of the 100X substrate with 19 volumes of LCS dilution buffer, creating a 5X stock to mix with the cell culture medium. When the cells are ready, add 25 microliters of the stock to each well, and gently mix the plate for 10 seconds. Then measure the luminescence for 10 minutes at room temperature for signal stabilization.For experiments with ligand addition, prepare the 13.5X ligand solution in modified Eagle's minimum essential media buffered with HEPES, and add 10 microliters per well using a multichannel pipette. Then mix the plate by hand or with an orbital shaker. This protocol has been successfully used to study interactions between a prototypical GPCR and two beta-arrestin isoforms.Four different plasmid combinations were screened, and the one with the highest luminescent signal was selected for further experiments. The EC50 values for each beta-arrestin isoform were determined using dose-response curves, which were obtained from the maximum response of each concentration from kinetic studies. It's critical to pay special attention when designing the PCR primers.The development of the right constructs is essential to the success of these assays. Following this methodology, you will be able to monitor receptor-beta-arrestin interactions, as well as receptor interactions with other cytosolic proteins. This methodology will help to answer fundamental questions in the field of GPCR pharmacology by studying how GPCR-beta-arrestin interactions can be modulated by modifications in the ligand structure.

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Research

JoVE Journal

Biochemistry

6.7K vistas.  Korea University. Este protocolo tiene un alto impacto en el campo del descubrimiento y desarrollo de fármacos, especialmente en el desarrollo de nuevos fármacos de primera clase con efectos secundarios mínimos. La principal ventaja de esta técnica es que se puede aplicar a cualquier sistema GPCR, y se utiliza para obtener información precisa sobre la farmacología de los receptores en sistemas de vida en tiempo real. Las implicaciones de esta técnica están estrechamente relacionadas con nuevas terapias...