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>

<ol>
	<li>Sriram, K., Insel, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPCRs+as+targets+for+approved+drugs:+How+many+targets+and+how+many+drugs?.">GPCRs as targets for approved drugs: How many targets and how many drugs?.</a> Molecular Pharmacology. 93 (4), 251-258 (2018).</li><li>Hauser, A. S., Attwood, M. M., Rask-Andersen, M., Schi&#246;th, H. B., Gloriam, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+GPCR+drug+discovery:+new+agents,+targets+and+indications.">Trends in GPCR drug discovery: new agents, targets and indications.</a> Nature Reviews Drug Discovery. 16 (12), 829-842 (2017).</li><li>Langmead, C. J., Summers, R. 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. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+signatures+in+&#946;-arrestin2+reveal+natural+biased+agonism+at+a+G-protein-coupled+receptor.">Conformational signatures in &#946;-arrestin2 reveal natural biased agonism at a G-protein-coupled receptor.</a> Communications Biology. 3, 1-128 (2018).</li><li>Promega. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nanobit Protein Protein Interaction System Protocol. , (2019).</li><li>Life Biomedical. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> HiYield Gel/PCR Fragments Extraction Kit. , (2019).</li><li>New England BioLabs. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ligation Calculator. , (2019).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cosmo Genetech. , (2019).</li><li>Baggio, L. L., Drucker, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+incretins:+GLP-1+and+GIP.">Biology of incretins: GLP-1 and GIP.</a> Gastroenterology. 132, 2131-2157 (2007).</li><li>ProMega. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> NanoGLO Endurazine and Vivazine Live Cell Substrates Technical Manual. , (2019).</li><li>Ali, R., Ramadurai, S., Barry, F., Nasheuer, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+fluorescent+protein+expression+for+quantitative+fluorescence+microscopy+and+spectroscopy+using+herpes+simplex+thymidine+kinase+promoter+sequences.">Optimizing fluorescent protein expression for quantitative fluorescence microscopy and spectroscopy using herpes simplex thymidine kinase promoter sequences.</a> FEBS Open Bio. 8 (6), 1043-1060 (2018).</li></ol>

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-Î²-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. ...

monitoring-gpcr-arrestin12-interactions-real-time-living-systems-to

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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

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

Immunology and Infection

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Bioengineering

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent &#946;-arrestin2 recruitment assay, the universality of &#946;-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango&#39;s sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient &#946;-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

cAMP Analysis with cADDis: Single-Cell to High-Throughput Screening

Real-Time cAMP Dynamics in Live Cells Using the Fluorescent cAMP Difference Detector In Situ

cAMP Difference Detector In Situ (cADDis) is a novel biosensor that allows for the continuous measurement of cAMP levels in living cells. The biosensor is created from a circularly permuted fluorescent protein linked to the hinge region of Epac2. This creates a single fluorophore biosensor that displays either increased or decreased fluorescence upon binding of cAMP. The biosensor exists in red and green upward versions, as well as green downward versions, and several red and green versions targeted to subcellular locations. To illustrate the effectiveness of the biosensor, the green downward version, which decreases in fluorescence upon cAMP binding, was used. Two protocols using this sensor are demonstrated: one utilizing a 96-well plate reading spectrophotometer&#160;compatible with high-throughput screening and another utilizing single-cell imaging on a fluorescent microscope. On the plate reader, HEK-293 cells cultured in 96-well plates were stimulated with 10 &#181;M forskolin or 10 nM isoproterenol, which induced rapid and large decreases in fluorescence in the green downward version. The biosensor was used to measure cAMP levels in individual human airway smooth muscle (HASM) cells monitored under a fluorescent microscope. The green downward biosensor displayed similar responses to populations of cells when stimulated with forskolin or isoproterenol. This single-cell assay allows visualization of the biosensor location at 20x and 40x magnification. Thus, this cAMP biosensor is sensitive and flexible, allowing real-time measurement of cAMP in both immortalized and primary cells, and with single cells or populations of cells. These attributes make cADDis a valuable tool for studying cAMP signaling dynamics in living cells.

Author Spotlight: Advancing Real-Time cAMP Detection in Cells Using cADDis Biosensor

The protocol presented in this study illustrates the effectiveness of the cAMP Difference Detector In Situ in measuring cAMP through two methods. One method utilizes a 96-well plate reading spectrophotometer with HEK-293 cells. The other method demonstrates individual HASM cells under a fluorescent microscope.

cAMP Difference Detector In Situ (cADDis) is a novel biosensor that allows for the continuous measurement of cAMP levels in living cells. The biosensor is ...

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

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

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

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

Biology

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include well-validated targets for therapeutic intervention. Discovery of lead compounds, including small synthetic molecules, that specifically inhibit the receptor&#39;s function, is an important initial step in drug development and relies on sensitive, specific, and robust cell-based assays. Here, we describe a kinetic cellular assay with a fluorescent readout primarily designed to identify receptor-specific antagonists that inhibit the intracellular Ca2+ release evoked upon the activation of the CXC chemokine receptor 4 (CXCR4) by its endogenous ligand, the CXC chemokine ligand 12 (CXCL12). A key advantage of this method is that it also enables screening of compounds endowed with intrinsic agonistic properties (i.e., compounds eliciting an increase in intracellular Ca2+ concentration in the absence of CXCL12) or compounds modulating the receptor&#39;s function via interaction with allosteric binding sites (i.e., positive and negative allosteric modulators (PAMs and NAMs, respectively)). On the down side, autofluorescent compounds might interfere with the assay&#39;s readout, thereby hampering reliable data interpretation. Most likely this assay can be implemented, with minimal adaptations, as a generic drug discovery assay for many other GPCRs of which the activation leads to a release of intracellular Ca2+.

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

The described cellular assay is designed for the identification of CXC chemokine receptor 4 (CXCR4)-interacting agents that inhibit or stimulate, either competitively or allosterically, the intracellular Ca2+ release initiated by CXCR4 activation.

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include ...

Medicine

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between G&#945; subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the &#946;2-adrenergic receptor and the G&#945;s C-terminus peptide upon treatment with 100 &#181;M isoproterenol hydrochloride as previously characterized1. Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a G&#945; C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.

Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, F&#1255;rster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group ...

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

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Real-time Imaging of Leukotriene B₄ Mediated Cell Migration and BLT1 Interactions with β-arrestin