The authors of this paper would like to thank Prof. Jiayan Xie for technical assistance and equipment usage. This work was financially supported by the National Natural Science Foundation of China (41406137).

1. Cell Preparation
<ol>
	<li>Cell recovery
	<ol>
		<li>Transfer the cryopreserved human embryonic kidney 293 (HEK293) cells from a liquid nitrogen tank to a 37 &#176;C water bath and shake continuously for 1 - 2 min.</li>
		<li>Add 10 mL of equilibrated growth medium (Dulbecco&#39;s modified Eagle&#39;s medium (DMEM), 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin solution) to a 10-cm cell culture dish. Gently add the thawed cells to the equilibrated growth medium.</li>
		<li>Incubate the cells in a 37 &#176;C water-jacketed CO2 incubator with a humidified atmosphere containing 95% air and 5% CO2 for 2 - 4 h.</li>
		<li>After 2 - 4 h, check that the cells are attached. Replace the equilibrated growth medium and culture for another 24 - 48 h. 
		NOTE: Changing the equilibrated growth medium is useful because it removes dimethyl sulfoxide (DMSO) and protects against cell damage.</li>
	</ol>
	</li>
	<li>Cell passage
	<ol>
		<li>After 24 - 48 h, when the cells have grown to near confluence, discard the equilibrated growth medium and wash the cells with 2 mL of phosphate-buffered saline (PBS). Swirl the dish gently.</li>
		<li>Discard the PBS and add 1 mL of Trypsin-EDTA (saline, 0.25% trypsin, and 0.02% EDTA). To completely digest the cells, swirl the dish carefully and aspirate off the Trypsin-EDTA solution. Put the dish in the 37 &#176;C water-jacketed CO2 incubator for 3 min. 
		NOTE: During this period, prepare three new 10-cm cell culture dishes and add 10 mL of equilibrated growth medium to each.</li>
		<li>Using a light microscope, determine if the cells have lifted from the bottom of the dish. Add 2 mL of equilibrated growth medium to the dish. Mix by pipetting and immediately pipet 0.6 mL of the cell suspension to each of the new dishes. Gently mix by pipetting and allow the cells to incubate at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
</ol>
2. Cell Seeding
<ol>
	<li>After 24 - 48 h, when the passaged cells have grown to near confluence, discard the equilibrated growth medium and wash the cells with 2 mL of PBS. Swirl the dish gently.</li>
	<li>Discard the PBS and add 1 mL of Trypsin-EDTA. To completely digest the cells, swirl the dish carefully and aspirate off the Trypsin-EDTA. Put the dish in the 37 &#176;C water-jacketed CO2 incubator for 3 min. 
	NOTE: During this period, prepare a 6-well plate by adding 2 mL of equilibrated growth medium to each well.</li>
	<li>Add 1 mL of equilibrated growth medium to the dish, if, under microscopic observation, the cells to have lifted from the dish bottom. Mix by pipetting and immediately pipet 0.1 mL of cell suspension (the volume is adjusted according to the cell growth density) to each well of the prepared 6-well plate (cell-less 6-well plate with 2 mL of equilibrated growth medium). Gently mix by pipetting and allow the cells to incubate at 37 &#176;C and 5% CO2.</li>
</ol>
3. Cell Transfection
<ol>
	<li>Transfection method 1, with liposome formulations 
	NOTE: Please see the Table of Materials for transfection reagent 1.
	<ol>
		<li>The following day, ensure that cells should be 85 - 95% confluent.</li>
		<li>Add 3 &#181;g of plasmid DNA (500 ng/&#181;L) and 100 &#181;L of reduced serum media to a sterile microcentrifuge tube, and mix gently. Incubate for 5 min at room temperature.</li>
		<li>Prepare 9 &#181;L of Reagent 1 and 100 &#181;L of reduced serum media in another microcentrifuge tube, and mix gently. Incubate for 5 min at room temperature.</li>
		<li>Combine the diluted plasmid mixture with diluted Reagent 1 (total volume = 215 &#181;L). Mix gently and incubate for 20 min at room temperature.</li>
		<li>Aspirate medium off cells-cultured plate, and add 1.5 mL fresh DMEM without FBS.</li>
		<li>Add 215 &#181;L of DNA-transfection reagent mixture to each well dropwise, and mix gently by swirling the plate.</li>
		<li>Incubate at 37 &#176;C and 5% CO2 for 4 - 8 h. Replace with fresh DMEM with 10% FBS.</li>
		<li>Incubate cells at 37 &#176;C and 5% CO2 for another 24 - 48 h before running any experiments.</li>
	</ol>
	</li>
	<li>Transfection method 2, with liposome formulations 
	NOTE: Please see the Table of Materials for transfection reagent 2.
	<ol>
		<li>Add 2 &#181;g of plasmid DNA (500 ng/&#181;L), 4 &#181;L of Reagent 2, and 100 &#181;L reduced serum media into a sterile microcentrifuge tube. Mix gently and incubate for 15 min at room temperature.</li>
		<li>Add the transfection mixture to the cells in a dropwise manner.</li>
		<li>After transfection, incubate cells for 24 - 48 h at 37 &#176;C and 5% CO2, before running any experiments.</li>
	</ol>
	</li>
</ol>
4. Cell Culture on Cover Slips
<ol>
	<li>Remove the growth medium from the culture 6-well plate.</li>
	<li>Wash the cell monolayer with 1 mL of PBS without calcium and magnesium.</li>
	<li>Add 0.5 mL Trypsin-EDTA. Swirl the dish carefully and aspirate off Trypsin-EDTA. Incubate the culture plate for 3 min at 37 &#176;C and 5% CO2. The actual incubation time varies with the cell line used.&#160; 
	NOTE: During this period, prepare a new 12-well plate and add 15 mm diameter sterile cover slips to each well. Cover slips coated with lysine, laminin, or collagen may improve cell attachment for HEK293 cells that easily detach.</li>
	<li>When cells are detached, add 1 mL pre-warmed complete growth medium to each well. Mix by pipetting and immediately transfer&#160; the required number of cells to the 12-well plate with cover slips. Add fresh complete growth medium to each&#160; well to adjust the total volume to 1 mL</li>
	<li>Incubate the cells for 16 - 24 h at 37 &#176;C and 5% CO2. 
	NOTE: After 30 - 60 min, the majority of cells will have adhered to the sterile cover slips.</li>
</ol>
5. Confocal Microscopy Detection
<ol>
	<li>Cellular localization
	<ol>
		<li>After 16 - 24 h, when the cells have grown to near confluence on the sterile cover slips in the 12-well plate, add the membrane probe, DiI, to each well, giving a final concentration of 5 &#181;M. Allow the cells to incubate for 30 min at 37 &#176;C and 5% CO2.</li>
		<li>After 30 min, remove the equilibrated growth medium and wash the cells twice in cold PBS (2 - 8 &#176;C).</li>
		<li>Fix the cells by adding 1 mL of a 4% paraformaldehyde solution (in PBS) to each well. Gently agitate for 15 min. 
		Caution: Paraformaldehyde is moderately toxic by skin contact or inhalation, and designated as a probable human carcinogen. Chemicals fume hoods, vented balance enclosures or other protective measures should be used during the weighing and handling of paraformaldehyde.</li>
		<li>Wash the fixed cells twice in PBS. Add 200-300 &#181;L of DAPI (4&#39;,6-diamidino-2-phenylindole) solution (0.5 &#181;g/mL) to each well and incubate for 10 min at room temperature.</li>
		<li>Wash the cells twice with PBS. Carefully remove the cover slips from the plate and wick off excess PBS. Invert onto microscope slides loaded with one drop of mounting medium (50% v/v glycerol/water). Remove excess mounting medium from the slides.</li>
	</ol>
	</li>
	<li>Cellular internalization
	<ol>
		<li>After 16 - 24 h, when the cells have grown to near confluence on the sterile cover slips in the 12-well plate, remove the equilibrated growth medium and add fresh, warmed DMEM medium (37 &#176;C). Incubate the cells for 1 h at 37 &#176;C and 5% CO2.</li>
		<li>Treat the cells with different concentrations (0, 10-12, 10-9, and 10-6 M) of ligand for 30 min and treat them with 10-6 M ligand for different times (0, 10, 30, and 60 min).</li>
		<li>Wash the cells twice in cold PBS (2 - 8 &#176;C). Fix the cells by adding 1 mL of a 4% paraformaldehyde solution (in PBS) to each well. Gently agitate for 15 min.</li>
		<li>Wash the cells twice in PBS. Carefully remove the cover slips from the plate and wick off the excess PBS. Invert onto microscope slides loaded with one drop of mounting medium (50% v/v glycerol/water). Remove excess mounting medium from the slides.</li>
	</ol>
	</li>
	<li>Confocal microscopy
	<ol>
		<li>Turn on the hardware of confocal microscope, including the mercury lamp power, PC microscope, scanner, and laser. Login to operating system account, launch the software, check the configuration and select laser. Turn on the different units of confocal microscopy system in the order according to instructions.</li>
		<li>Place the prepared slides on the microscope stage. Position the sample over the objective lens using the stage controller. Add one drop of cedar oil onto coverslip when the oil immersion lens is selected. Place the cover slips on slides facing the lens.</li>
		<li>Find the cells of interest under the fluorescent microscopy.</li>
		<li>Switch to scan mode. Choose the laser power and the spectral range of the emission fluorophore. Capture the high quality confocal images by tuning laser intensity and other parameters.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Mellman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis+and+molecular+sorting.">Endocytosis and molecular sorting.</a> Annu. Rev. Cell Dev. Biol. 12, 575-625 (1996).</li><li>Hausott, B., Klimaschewski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+turnover+and+receptor+trafficking+in+regenerating+axons.">Membrane turnover and receptor trafficking in regenerating axons.</a> Eur J Neuro. 43, 309-317 (2016).</li><li>Kallal, L., Benovic, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+green+fluorescent+proteins+to+study+G-protein-coupled+receptor+localization+and+trafficking.">Using green fluorescent proteins to study G-protein-coupled receptor localization and trafficking.</a> Trends Pharmacol Sci. 21, 175-180 (2000).</li><li>Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., Cormier, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+structure+of+the+Aequorea+victoria+green-fluorescent+protein.">Primary structure of the Aequorea victoria green-fluorescent protein.</a> Gene. 111, 229-233 (1992).</li><li>Spector, D. L., Goldman, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constructing+and+Expressing+GFP+Fusion+Proteins.">Constructing and Expressing GFP Fusion Proteins.</a> CSH protocols. 2006, (2006).</li><li>Drew, D. E., von Heijne, G., Nordlund, P., de Gier, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+as+an+indicator+to+monitor+membrane+protein+overexpression+in+Escherichia+coli.">Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli.</a> FEBS Lett. 507, 220-224 (2001).</li><li>von Arnim, A. G., Deng, X. W., Stacey, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+vectors+for+the+expression+of+green+fluorescent+protein+fusion+proteins+in+transgenic+plants.">Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants.</a> Gene. 221, 35-43 (1998).</li><li>Webb, C. D., Decatur, A., Teleman, A., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+green+fluorescent+protein+for+visualization+of+cell-specific+gene+expression+and+subcellular+protein+localization+during+sporulation+in+Bacillus+subtilis.">Use of green fluorescent protein for visualization of cell-specific gene expression and subcellular protein localization during sporulation in Bacillus subtilis.</a> J Bacteriol. 177, 5906-5911 (1995).</li><li>Kain, S. R., 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=Green+fluorescent+protein+as+a+reporter+of+gene+expression+and+protein+localization.">Green fluorescent protein as a reporter of gene expression and protein localization.</a> BioTechniques. 19, 650-655 (1995).</li><li>Fukunaga, S., Setoguchi, S., Hirasawa, A., Tsujimoto, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+ligand-mediated+internalization+of+G+protein-coupled+receptor+as+a+novel+pharmacological+approach.">Monitoring ligand-mediated internalization of G protein-coupled receptor as a novel pharmacological approach.</a> Life Sci. 80, 17-23 (2006).</li><li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agonist-Activated+Bombyx+Corazonin+Receptor+Is+Internalized+via+an+Arrestin-Dependent+and+Clathrin-Independent+Pathway.">Agonist-Activated Bombyx Corazonin Receptor Is Internalized via an Arrestin-Dependent and Clathrin-Independent Pathway.</a> Biochemistry. 55, 3874-3887 (2016).</li><li>Kohout, T. A., Lefkowitz, 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=Regulation+of+G+protein-coupled+receptor+kinases+and+arrestins+during+receptor+desensitization.">Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization.</a> Mol Pharmacol. 63, 9-18 (2003).</li><li>Ferguson, S. 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=Role+of+beta-arrestin+in+mediating+agonist-promoted+G+protein-coupled+receptor+internalization.">Role of beta-arrestin in mediating agonist-promoted G protein-coupled receptor internalization.</a> Science. 271, 363-366 (1996).</li><li>Stojilkovic, S. S., Reinhart, J., Catt, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadotropin-releasing+hormone+receptors:+structure+and+signal+transduction+pathways.">Gonadotropin-releasing hormone receptors: structure and signal transduction pathways.</a> Endocr Rev. 15, 462-499 (1994).</li><li>Yan, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+Expression+Profile+of+the+Gonadotropin-Releasing+Hormone+Receptor+in+Common+Chinese+Cuttlefish,+Sepiella+japonica.">Identification and Expression Profile of the Gonadotropin-Releasing Hormone Receptor in Common Chinese Cuttlefish, Sepiella japonica.</a> J Exp Zool A Ecol Genet Physiol. , (2016).</li><li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+activation+of+the+G+protein-coupled+receptor+BNGR-A21+by+the+neuropeptide+corazonin+from+the+silkworm,+Bombyx+mori,+dually+couples+to+the+G(q)+and+G(s)+signaling+cascades.">Specific activation of the G protein-coupled receptor BNGR-A21 by the neuropeptide corazonin from the silkworm, Bombyx mori, dually couples to the G(q) and G(s) signaling cascades.</a> J Biol Chem. 288, 11662-11675 (2013).</li><li>Lu, Z. 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=Cloning,+Characterization,+and+Expression+Profile+of+Estrogen+Receptor+in+Common+Chinese+Cuttlefish,+Sepiella+japonica.">Cloning, Characterization, and Expression Profile of Estrogen Receptor in Common Chinese Cuttlefish, Sepiella japonica.</a> J Exp Zool A Ecol Genet Physiol. 325, 181-193 (2016).</li></ol>

The authors declare no conflicts of interest.

The protocol presented here provides an efficient and easily interpretable method to detect the subcellular localization and internalization of expressed GPCR fusion protein in HEK293 cells. The technique can be easily adapted for many different genes and cell types, such as for the cellular localization and internalization of the corazonin receptor from Bombyx mori in HEK293 and BmN cells16.
The successful application of this powerful assay relies on a few key...

Cells possess cargo trafficking machinery to transport extracellular materials-such as ligands, microorganisms, nutrients, and transmembrane proteins-into the cell for information, energy, and other purposes1,2. It is critical for cellular homeostasis, tissue function, and overall cell survival. The cell-based expression of fluorescent fusion proteins is a powerful way to study the localization and internalization of transmembrane receptors, such as G protein-coupled receptors (GPCR), in signaling pathways3.
The expression of fusion proteins tagged with green fluorescent protein (GFP) from jellyfish Aequorea victoria, combined with direct fluorescence detection techniques, has gained wide acceptance in protein-targeting research4,5,6. GFP-based detection has the advantage of allowing the real-time imaging of living cells while avoiding fixation artifacts7. A series of versatile cloning vectors has been constructed to facilitate the expression of protein fusions to GFP in various cells8,9. The pEGFP-N1 vector is a widely used and commercialized plasmid vector encoding an enhanced green fluorescent protein (EGFP), which has been optimized from wild-type GFP for brighter fluorescence and higher expression in mammalian cells. To observe the fluorescent signal of EGFP expressed in mammalian cells, the fluorescence microscopy should be conducted with the excitation at 488 nm and the emission at 507 nm, preferably with confocal microscopy.
Monitoring the subcellular localization and ligand-mediated internalization of receptors is a common technique to investigate the signal transduction and function of GPCRs10,11. In most cases, internalization leads to desensitization of the GPCRs (the attenuation of the stimulus response in spite of the presence of agonists)12,13. The internalized receptors can be incorporated into the lysosome and degraded, or they can be recycled back to the cell surface, depending on the nature of the receptors and the cell lines used in the experiments.
The gonadotropin-releasing hormone receptors (GnRHRs) belong to the GPCR family and have a typical GPCR protein structure, with an extracellular amino terminal, an intracellular -COOH terminal, and seven transmembrane (TM) domains14. The transfection of recombinant plasmid SjGnRHR-EGFP (with the GnRHR gene from Sepiella japonica) and the confocal microscopy detection of EGFP-tagged SjGnRHR (expressed in HEK293 cells) was reported previously, and GFP fluorescence was established as a reporter for transmembrane protein localization assays15. Now, the internalization of SjGnRHR, activated by S. japonica gonadotropin-releasing hormone (SjGnRH), was demonstrated in time- and dose-dependent manners by confocal microscopy.

<table><tbody><tr><td>HEK293 cell line&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>DMEM/High glucose</td><td>Hyclone</td><td>SH30022.01</td><td>High glucose 4.0 mM L-glutamine</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>PuFei</td><td>1101-100</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Genom</td><td>GNM10010</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Genom</td><td>GNM15140</td><td></td></tr><tr><td>0.25% Trypsin &amp;amp; 0.02% EDTA</td><td>Genom</td><td>GNM25200</td><td></td></tr><tr><td>Opti-MEM (1x)</td><td>GIBCO, by Life Technologies</td><td>31985-062</td><td>reduced serum media</td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>Invitrogen</td><td>11668027</td><td>Reagent 1</td></tr><tr><td>X-tremeGENE HP DNA Transfection Reagent</td><td>Roche</td><td>6366236001</td><td>Reagent 2</td></tr><tr><td>DAPI Staining Solution</td><td>Beyotime</td><td>C1006</td><td></td></tr><tr><td>DiI</td><td>Beyotime</td><td>C1036</td><td></td></tr><tr><td>Antifade Mounting Medium</td><td>Beyotime</td><td>P0126</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sinopharm Chemical Reagent Co.(SCRC)</td><td>80096618</td><td></td></tr><tr><td>100 mm cell culture</td><td>CORNING</td><td>430167</td><td></td></tr><tr><td>6-well plate&amp;nbsp;</td><td>ExCell Bio</td><td>CS016-0092</td><td></td></tr><tr><td>12-well plate&amp;nbsp;</td><td>ExCell Bio</td><td>CS016-0093</td><td></td></tr><tr><td>Cover Glass</td><td>NEST</td><td>801007</td><td></td></tr><tr><td>Microscope Slides</td><td>Citoglas</td><td>10127105P-G</td><td></td></tr><tr><td>Thermo Scientific Forma CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo</td><td>3111</td><td></td></tr><tr><td>TCS SP5II laser scanning confocal microscope</td><td>Leica&amp;nbsp;</td><td>5100001311</td><td></td></tr></tbody></table>

detection of ligand-activated g protein-coupled receptor internalization by confocal microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

Figure 1 shows an example of a confocal microscope system. Figure 2 presents the expression of pEGFP-N1 in HEK293 cells. The GFP signal was detected in the cytoplasm and nucleus. Figure 3 shows the subcellular localization of GnRHR from S. japonica in HEK293 cells, consistent with our previously published results15. The fusion protein with the EGFP tag at the C-terminus of SjGnRHR (SjGnRH...

Watch this Scientific Journal Video about Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy at JoVE.com

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...

detection-ligand-activated-g-protein-coupled-receptor-internalization

Research

JoVE Journal

Biology

10.7K Views.  Zhejiang Ocean University.  Dit protocol beschrijft confocale microscopie detectie van G-eiwit-gekoppelde receptor (GPCR) internalisatie in zoogdiercellen. Het omvat de basis-celkweek, transfectie, en confocale microscopie procedure en zorgt voor een efficiënte en gemakkelijk te interpreteren methode om de subcellulaire lokalisatie en de internalisering van-fusion uitgedrukt GPCR detecteren. 

Video: Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

Chemistry

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

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Neuroscience

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma membrane and the numerous factors that influence protein behavior in live cells. Giant unilamellar vesicles (GUVs) are a biomimetic and highly tunable in vitro model system for investigating protein-membrane interactions and probing protein behavior in a precise, stimulus-dependent manner. In this protocol, we present an inexpensive and effective method for fabricating GUVs with the human serotonin 1A receptor (5-HT1AR) stably integrated in the membrane. We fabricate GUVs using a modified hydrogel swelling method; by depositing a lipid film on top of a mixture of agarose and 5-HT1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT1AR incorporated into the membrane. These GUVs can then be used to examine protein-membrane interactions and localization behavior via microscopy. Ultimately, this protocol can advance our understanding of the functionality of integral membrane proteins, providing profound physiological insight.

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors GPCRs

This protocol utilizes agarose swelling as a powerful and generalizable technique for incorporating integral membrane proteins (IMPs) into giant unilamellar lipid vesicles (GUVs), as described here for the reconstitution of the human 1A serotonin receptor protein (5-HT1AR), one of the classes of pharmacologically important G protein-coupled receptors.

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

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 ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

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