Name: detection of ligand-activated g protein-coupled receptor internalization by confocal microscopy
Uploaded: 2017-04-09T14:00:00
Description: Watch this Scientific Journal Video about Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy at JoVE.com

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 th...

<ol>
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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. 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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. 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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 border="0" cellpadding="0" cellspacing="0" width="857">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">HEK293 cell line&#160;</td>
			<td style="width:236px;"></td>
			<td style="width:119px;"></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">DMEM/High glucose</td>
			<td style="width:236px;">Hyclone</td>
			<td style="width:119px;">SH30022.01</td>
			<td>High glucose 4.0 mM L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Foetal Bovine Serum (FBS)</td>
			<td style="width:236px;">PuFei</td>
			<td style="width:119px;">1101-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:236px;">Genom</td>
			<td style="width:119px;">GNM10010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Penicillin-Streptomycin</td>
			<td style="width:236px;">Genom</td>
			<td style="width:119px;">GNM15140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">0.25% Trypsin &#38; 0.02% EDTA</td>
			<td style="width:236px;">Genom</td>
			<td style="width:119px;">GNM25200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Opti-MEM (1X)</td>
			<td style="width:236px;">GIBCO, by Life Technologies</td>
			<td style="width:119px;">31985-062</td>
			<td>reduced serum media</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:300px;">Lipofectamine 2000 Transfection Reagent</td>
			<td style="width:236px;">Invitrogen</td>
			<td style="width:119px;">11668027</td>
			<td>Reagent 1</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:300px;">X-tremeGENE HP DNA Transfection Reagent</td>
			<td style="width:236px;">Roche</td>
			<td style="width:119px;">6366236001</td>
			<td>Reagent 2</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:300px;">DAPI Staining Solution</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">C1006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">DiI</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">C1036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Antifade Mounting Medium</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">P0126</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:300px;">Paraformaldehyde</td>
			<td style="width:236px;">Sinopharm Chemical Reagent Co.(SCRC)</td>
			<td style="width:119px;">80096618</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">100 mm cell culture</td>
			<td style="width:236px;">CORNING</td>
			<td style="width:119px;">430167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">6-well plate&#160;</td>
			<td style="width:236px;">ExCell Bio</td>
			<td style="width:119px;">CS016-0092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">12-well plate&#160;</td>
			<td style="width:236px;">ExCell Bio</td>
			<td style="width:119px;">CS016-0093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Cover Glass</td>
			<td style="width:236px;">NEST</td>
			<td style="width:119px;">801007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Microscope Slides</td>
			<td style="width:236px;">Citoglas</td>
			<td style="width:119px;">10127105P-G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Thermo Scientific Forma CO2 Incubator</td>
			<td style="width:236px;">Thermo</td>
			<td style="width:119px;">3111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">TCS SP5II laser scanning confocal microscope</td>
			<td style="width:236px;">Leica&#160;</td>
			<td style="width:119px;">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 (Video) | JoVE 

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.

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 ...

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