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. Preparazione delle cellule <ol><li> il recupero delle cellule <ol><li> Trasferire le cellule renali 293 crioconservate embrionali umane (HEK293) da un serbatoio di azoto liquido bagnomaria C 37 ° e agitare continuamente per 1 - 2 min. </li><li> Aggiungere 10 ml di mezzo di crescita Equilibrated (mezzo di Dulbecco modificato da Eagle (DMEM), 10% siero bovino (FBS), e 1% di soluzione di penicillina-streptomicina fetale) a 10 cm piatto di coltura cellulare. Delicatamente aggiungere le cellule scongel...

<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>

Il protocollo presentato qui offre un metodo efficiente e facilmente interpretabili per rilevare la localizzazione subcellulare e l&#39;interiorizzazione della proteina di fusione GPCR espresso in cellule HEK293. La tecnica può essere facilmente adattato per molti geni differenti e tipi di cellule, come per la localizzazione cellulare e internalizzazione del recettore corazonin da Bombyx mori in cellule HEK293 e Bmn 16. L&#39;applicazione di successo di ques...

Cellule possiedono macchine traffico merci da trasportare extracellulare materiali, ad esempio leganti, microrganismi, nutrienti e proteine transmembrana-nella cella per informazioni, energia e altri scopi 1, 2. È fondamentale per l&#39;omeostasi cellulare, la funzione dei tessuti, e la sopravvivenza delle cellule complessiva. L&#39;espressione a base di cellule di proteine di fusione fluorescenti è un potente strumento per studiare la localizzazione e internalizzazione di recettori transmembrana, come G recettori accoppiati alle proteine (GPCR), in vie di segnalazione 3. L&#39;espressione di proteine di fusione contrassegnate con proteina fluorescente verde (GFP) dalla medusa Aequorea victoria, combinato con tecniche di rivelazione di fluorescenza diretta, ha guadagnato un largo consenso nel proteina-targeting di ricerca 4, 5, 6. detecti GFP-basedsulla presenta il vantaggio di consentire l&#39;imaging in tempo reale delle cellule viventi evitando artefatti fissaggio 7. Una serie di vettori di clonaggio versatili è stato costruito per facilitare l&#39;espressione di proteine per fusioni GFP in varie cellule 8, 9. Il vettore pEGFP-N1 è un vettore plasmidico utilizzato e commercializzato codificante una maggiore proteina fluorescente verde (EGFP), che è stato ottimizzato da wild-type per GFP luminosa fluorescenza e più alta espressione in cellule di mammifero. Per osservare il segnale fluorescente EGFP espresso in cellule di mammifero, la microscopia a fluorescenza deve essere condotta con l&#39;eccitazione a 488 nm e l&#39;emissione a 507 nm, preferibilmente con microscopia confocale. Monitoraggio della localizzazione subcellulare e internalizzazione ligando mediata da recettori è una tecnica comune per indagare la trasduzione del segnale e funzione della GPCR 10 </sup&gt;, 11. Nella maggior parte dei casi, porta alla internalizzazione desensibilizzazione dei GPCR (l&#39;attenuazione della risposta stimolo nonostante la presenza di agonisti) 12, 13. I recettori internalizzati possono essere incorporati nella lisosoma e degradati, oppure possono essere riciclati alla superficie cellulare, a seconda della natura dei recettori e le linee cellulari utilizzate negli esperimenti. I recettori dell&#39;ormone di rilascio delle gonadotropine (GnRHRs) appartengono alla famiglia GPCR e hanno una struttura tipica proteina GPCR, con un terminale ammino extracellulare, un terminale -COOH intracellulare, e sette transmembrana (TM) 14 domini. La trasfezione di plasmidico ricombinante Sj GnRHR-EGFP (con il gene GnRHR da Sepiella japonica) e la rilevazione di microscopia confocale GnRHR Sj EGFP-tag (espressi in cellule HEK293) è stata riportata Precemalizioso, e la fluorescenza GFP è stato stabilito come reporter per saggi di localizzazione della proteina transmembrana 15. Ora, l&#39;interiorizzazione di Sj GnRHR, attivato da S. japonica gonadotropina-releasing hormone (GnRH Sj), è stata dimostrata in maniere tempo-e dose-dipendenti mediante microscopia confocale.

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			<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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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.

La figura 1 mostra un esempio di un sistema di microscopia confocale. La figura 2 presenta l&#39;espressione di pEGFP-N1 in cellule HEK293. Il segnale GFP è stato rilevato nel citoplasma e nucleo. La Figura 3 mostra la localizzazione subcellulare di GnRHR da S. japonica in cellule HEK293, coerente con i risultati precedentemente pubblicati 15. La proteina di fusione con il tag EGFP al C-terminale di ...

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

Questo protocollo descrive rilevamento microscopia confocale del recettore accoppiato alla proteina G (GPCR) internalizzazione in cellule di mammifero. Esso comprende la coltura di cellule di base, trasfezione, e la procedura microscopia confocale e fornisce un metodo efficiente e facilmente interpretabili per rilevare la localizzazione subcellulare e internalizzazione del GPCR fusione espressa.

Rilevamento di Ligand-activated proteina G-recettore accoppiato internalizzazione da microscopia confocale

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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Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

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