Name: g protein-selective gpcr conformations measured using fret sensors in a live cell suspension fluorometer assay
Uploaded: 2016-09-10T15:00:00
Description: Watch this Scientific Journal Video about G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay at JoVE.com

R.U.M was funded by the American Heart Association Pre-doctoral Fellowship (14PRE18560010). Research was funded by the American Heart Association Scientist Development Grant (13SDG14270009) &#38; the NIH (1DP2 CA186752-01 &#38; 1-R01-GM-105646-01-A1) to S.S.

1. DNA Preparation
<ol>
	<li>Design sensor constructs using a modular cloning scheme. Please reference the &#946;2-AR sensor design detailed previously1.</li>
	<li>Prepare DNA according to commercial miniprep kit protocol and elute in 2 mM Tris-HCl solution, pH 8, at concentration &#8805; 750 ng/&#181;l, A260/A280 of 1.7 - 1.9, A260/A230 of 2.0 - 2.29.</li>
</ol>
2. Cell Culture Preparation
<ol>
	<li>Culture HEK-293T-Flp-n cells...

<ol>
	<li>Malik, R. U., 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=Detection+of+G+Protein-selective+G+Protein-coupled+Receptor+(GPCR)+Conformations+in+Live+Cells.">Detection of G Protein-selective G Protein-coupled Receptor (GPCR) Conformations in Live Cells.</a> J. Biol. Chem. 288, 17167-17178 (2013).</li><li>Oldham, W. M., Hamm, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotrimeric+G+protein+activation+by+G-protein-coupled+receptors.">Heterotrimeric G protein activation by G-protein-coupled receptors.</a> Nat. Rev. Mol. Cell Bio. 9, 60-71 (2008).</li><li>Rasmussen, S. G., 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=Crystal+structure+of+the+&#946;2+adrenergic+receptor-Gs+protein+complex.">Crystal structure of the &#946;2 adrenergic receptor-Gs protein complex.</a> Nature. 477, 549-555 (2011).</li><li>Alexander, N. 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=Energetic+analysis+of+the+rhodopsin-G-protein+complex+links+the+&#945;5+helix+to+GDP+release.">Energetic analysis of the rhodopsin-G-protein complex links the &#945;5 helix to GDP release.</a> Nat. Struct. Mol. Biol. 21 (1), 56-63 (2014).</li><li>Conklin, B. R., Farfel, Z., Lustig, K. D., Julius, D., Bourne, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substitution+of+three+amino+acids+switches+receptor+specificity+of+Gq&#945;+to+that+of+Gi&#945;.">Substitution of three amino acids switches receptor specificity of Gq&#945; to that of Gi&#945;.</a> Nature. 363, 274-276 (1993).</li><li>Conklin, B. 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=Carboxyl-Terminal+Mutations+of+Gq&#945;+and+Gs&#945;+That+Alter+the+Fidelity+of+Receptor+Activation.">Carboxyl-Terminal Mutations of Gq&#945; and Gs&#945; That Alter the Fidelity of Receptor Activation.</a> Mol. Pharmacol. 50, 855-890 (1996).</li><li>Onaran, H. O., Costa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Where+have+all+the+active+receptor+states+gone.">Where have all the active receptor states gone.</a> Nat. Chem. Bio. 8, 674-677 (2012).</li><li>Jares-Erijman, E. A., Jovin, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET+imaging.">FRET imaging.</a> Nat Biotechnol. 21 (11), 1387-1395 (2003).</li><li>Lohse, M. J., Nuber, S., Hoffman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence/bioluminescence+resonance+energy+transfer+techniques+to+study+G-protein-coupled+receptor+activation+and+signaling.">Fluorescence/bioluminescence resonance energy transfer techniques to study G-protein-coupled receptor activation and signaling.</a> Pharmacol Rev. 64 (2), 299-336 (2012).</li><li>Vilardaga, J. P., B&#252;nemann, M., Krasel, C., Castro, M., Lohse, 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=Measurement+of+the+millisecond+activation+switch+of+G+protein-coupled+receptors+in+living+cells.">Measurement of the millisecond activation switch of G protein-coupled receptors in living cells.</a> Nat. Biotechnol. 21, 807-812 (2003).</li><li>B&#252;nemann, M., Frank, M., Lohse, 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=Gi+protein+activation+in+intact+cells+involves+subunit+rearrangement+rather+than+dissociation.">Gi protein activation in intact cells involves subunit rearrangement rather than dissociation.</a> Proc. Natl. Acad. Sci. USA. 100 (26), 16077-16082 (2003).</li><li>Stumpf, A. D., Hoffman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+probes+based+on+G+protein-coupled+receptors+-+added+work+or+added+value.">Optical probes based on G protein-coupled receptors - added work or added value.</a> Brit. J. Pharmacol. 173, 255-266 (2016).</li><li>Sivaramakrishnan, S., Spudich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+control+of+protein+interaction+using+a+modular+ER/K+&#945;-helix+linker.">Systemic control of protein interaction using a modular ER/K &#945;-helix linker.</a> Proc. Natl. Acad. Sci. USA. 108, 20467-20472 (2011).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat. Methods. 2 (12), 905-909 (2005).</li></ol>

The tight dynamic range of FRET measurements in this system reinforces the necessity of sensitive quality control in every step of this protocol. The most important steps to ensure a successful FRET experiment are 1) cell culturing, 2) transfection 3) protein expression and 4) timely, precise coordination during the assay execution.
Cell health and maintenance/plating quality can have a significant impact on the signal-to-noise of the experimental system and poor cell health can make it imposs...

G-protein-coupled receptors (GPCRs) are seven-transmembrane receptors. The human genome alone contains approximately 800 genes coding for GPCRs, which are activated by a variety of ligands including light, odorants, hormones, peptides, drugs and other small molecules. Nearly 30% of all pharmaceuticals currently on the market target GPCRs because they play a large role in many disease states2. Despite decades of extensive work done on this receptor family, there remain significant outstanding questions in the field, particularly with regards to the molecular mechanisms that drive GPCR-effector interactions. To date, only one high-resolution crystal structure has been published, providing insight into the interaction between the &#946;2-adrenergic receptor (&#946;2-AR) and the Gs protein3. Together with extensive research in the last three decades, it reiterates one specific structural component that is critical in this interaction: the G&#945; subunit C-terminus. This structure is important for both G protein activation by the GPCR4 and G protein selection5-6. Hence, the G&#945; C-terminus provides a crucial link between ligand stimulation of the GPCR and selective G protein activation.
Research over the last decade suggests that GPCRs populate a broad conformational landscape, with ligand-binding stabilizing subsets of GPCR conformations. While several techniques, including crystallography, NMR and fluorescence spectroscopy, and mass spectrometry are available to examine the GPCR conformational landscape, there is a paucity of approaches to elucidate their functional significance in effector selection7. Here, we outline a F&#1255;rster resonance energy transfer (FRET)-based approach to detect G protein-selective GPCR conformations. FRET relies on the proximity and parallel orientation of two fluorophores with overlapping emission (donor) and excitation (acceptor) spectra8. As the donor and acceptor fluorophores come closer together as a result of either conformational change in the protein or a protein-protein interaction, the FRET between them increases, and can be measured using a range of methods8. FRET-based biosensors have been employed extensively in the GPCR field9. They have been used to probe conformation changes in the GPCR by inserting donor and acceptor in the third intracellular loop and GPCR C-terminus; sensors have been designed to probe GPCR and effector interactions by separately labeling the GPCR and effector (G protein subunits/arrestins) with a FRET pair10; some sensors also detect conformational changes in the G protein11. These biosensors have enabled the field to ask a multitude of outstanding questions including conformational changes in the GPCR and effector, GPCR-effector interaction kinetics, and allosteric ligands12. Our group was particularly interested in creating a biosensor that could detect G protein-specific GPCR conformations under agonist-driven conditions. This biosensor relies on a recently developed technology named SPASM (systematic protein affinity strength modulation)13. SPASM involves tethering interacting protein domains using an ER/K linker, which controls their effective concentrations. Flanking the linker with a FRET pair of fluorophores creates a tool which can report the state of the interaction between proteins12. Previously1 the SPASM module was used to tether the G&#945; C-terminus to a GPCR and monitor their interactions with FRET fluorophores, mCitrine (referred to in this protocol by its commonly known variant, Yellow Fluorescent Protein (YFP), excitation/emission peak at 490/525 nm) and mCerulean (referred to in this protocol by its commonly known variant Cyan Fluorescent Protein (CFP), excitation/emission peak 430/475 nm). From N- to C-terminus, this genetically encoded single polypeptide contains: a full length GPCR, FRET acceptor (mCitrine/YFP), 10 nm ER/K linker, FRET donor (mCerulean/CFP), and the G&#945; C-terminus peptide. In this study, sensors are abbreviated as GPCR-linker length-G&#945; peptide. All components are separated by an unstructured (Gly-Ser-Gly)4 linker which enables free rotation of each domain. The detailed characterization of such sensors was previously performed using two prototypical GPCRs: &#946;2-AR and opsin1.
This sensor is transiently transfected into HEK-293T cells and fluorometer-based live cell experiments measure fluorescence spectra of the FRET pair in arbitrary units of counts per second (CPS) in the presence or absence of ligand. These measurements are used to calculate a FRET ratio between the fluorophores (YFPmax/CFPmax). A change in FRET (&#916;FRET) is then calculated by subtracting the average FRET ratio of untreated samples from the FRET ratio of ligand treated samples. &#916;FRET can be compared across constructs (&#946;2-AR-10 nm-G&#945;s peptide versus &#946;2-AR-10 nm-no peptide). Here, we detail the protocol to express these sensors in live HEK-293T cells, monitor their expression, and the setup, execution, and analysis of the fluorometer-based live cell FRET measurement for untreated versus drug treated conditions. While this protocol is specific for the &#946;2-AR-10 nm-G&#945;s peptide sensor treated with 100 &#181;M isoproterenol bitartrate, it can be optimized for different GPCR-G&#945; pairs and ligands.

<table border="0" cellpadding="0" cellspacing="0" width="1151">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">B2-AR-10 nm- Gas peptide sensor</td>
			<td style="width:144px;">Addgene</td>
			<td style="width:148px;">47438</td>
			<td style="width:547px;">https://www.addgene.org/Sivaraj_Sivaramakrishnan/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">GeneJET Plasmid Miniprep Kit</td>
			<td style="width:144px;">Fermentas/Fisher Sci</td>
			<td style="width:148px;">FERK0503</td>
			<td>Elute in 2 mM Tris elution buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">HEK-293T-Flp-n cells</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">R78007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Trypsin (0.25%)</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">DMEM- high glucose</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">11960-044</td>
			<td>Warm in&#160; 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">FBS, certified, Heat inactivated, US origin</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">10082147</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">Glutamax I 100x</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">HEPES</td>
			<td style="width:144px;">Corning</td>
			<td style="width:148px;">MT25060CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Opti-MEM</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">31985-070</td>
			<td>Reduced serum media; Bring to room temperature before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">XtremeGene HP transfection reagenet</td>
			<td style="width:144px;">Roche</td>
			<td style="width:148px;">6366236001</td>
			<td>Highly recommended for its consistency. Bring to room temperature before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">FluoroMax 4</td>
			<td style="width:144px;">Horiba</td>
			<td style="width:148px;"></td>
			<td>Use with FluorEssence V3.8 software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">3-mm path length quartz cuvette</td>
			<td style="width:144px;">Starna</td>
			<td style="width:148px;">NC9729944(16.45F-Q-3/z8.5)</td>
			<td>May require cuvette holder/adaptor for use in Fluorometer, available from Starna</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Sc100-S3 Heated Circulating water bath pump</td>
			<td style="width:144px;">Fisher Scientific</td>
			<td style="width:148px;">13-874-826</td>
			<td>Warm to 37 &#176;C before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">Thermomixer Heat Block</td>
			<td style="width:144px;">Eppendorf</td>
			<td style="width:148px;">22670000</td>
			<td>Warm to 37 &#176;C before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Ultrapure DNA/RNAse free water</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">10977015</td>
			<td>Use at room temperature</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">D(+)-glucose, anhydrous</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">G5767</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">aprotinin from bovine lung</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">A1153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">leupeptin hemisulfate</td>
			<td style="width:144px;">EMD</td>
			<td style="width:148px;">10-897</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">L-ascorbic acid, reagent grade</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">A0278</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(-)-isoproterenol (+)-bitartrate</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">I2760</td>
			<td>Use fresh aliquot each experiment</td>
		</tr>
	</tbody>
</table>

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.

A generalized schematic of the experiment set up and execution is detailed in Figure 3.
In order to detect a FRET change in the narrow dynamic range of the sensor, it is critical to adhere to the nuances of the system be adhered to. Cell quality is imperative to protein expression as well as consistency in sampling. Figure 1 features images of cultured cells growing in a consistent monolayer (10X) that is optimal for six-well plating and transfection F...

Watch this Scientific Journal Video about G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay at JoVE.com

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

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 ...

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