Name: genetically-encoded molecular probes to study g protein-coupled receptors
Uploaded: 2013-09-13T16:30:00
Description: Watch this Scientific Journal Video about Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors at JoVE.com

We thank the generous support of several foundations and philanthropic donors (see SakmarLab.org).

1. Site-specific Genetic Incorporation of Unnatural Amino Acids into GPCRs
<ol>
	<li>Maintain HEK293T cells in DMEM (4.5 g/L of glucose, 2 mM glutamine) supplemented with 10% fetal bovine serum (FBS) at 37 &#176;C in a 5% CO2 atmosphere.</li>
	<li>Transfect the cells grown to 60-80% confluence in a 10-cm plate using Lipofectamine Plus reagent.
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		<li>To 750 &#956;l DMEM, add 10 &#956;l Plus reagent, 3.5 &#956;g of GPCR cDNA (pMT4. Rho or pcDNA 3.1. CCR5) containing the amber stop codon at a desired posi...

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+ligand-binding+site+on+a+G+protein-coupled+receptor+(GPCR)+using+genetically+encoded+photocrosslinkers.">Mapping the ligand-binding site on a G protein-coupled receptor (GPCR) using genetically encoded photocrosslinkers.</a> Biochemistry. 50, 3411-3413 (2011).</li><li>Grunbeck, A., 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=Genetically+encoded+photo-cross-linkers+map+the+binding+site+of+an+allosteric+drug+on+a+G+protein-coupled+receptor.">Genetically encoded photo-cross-linkers map the binding site of an allosteric drug on a G protein-coupled receptor.</a> ACS chemical biology. 7, 967-972 (2012).</li><li>Dunham, T. D., Farrens, D. 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J. 84, 2078-2100 (2004).</li><li>Okada, T., 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=The+retinal+conformation+and+its+environment+in+rhodopsin+in+light+of+a+new+2.2+&#197;+crystal+structure.">The retinal conformation and its environment in rhodopsin in light of a new 2.2 &#197; crystal structure.</a> J. Mol. Biol. 342, 571-583 (2004).</li><li>Huber, T., Tian, H., Ye, S., Naganathan, S., Kazmi, M. A., Duarte, T., Sakmar, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioorthogonal+labeling+of+functional+G+protein-coupled+receptors+at+genetically+encoded+azido+groups.">Bioorthogonal labeling of functional G protein-coupled receptors at genetically encoded azido groups.</a> , (2013).</li><li>Naganathan, S., Ye, S., Sakmar, T. P., Huber, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+epitope+tagging+of+GPCRs+by+bioorthogonal+modification+of+a+genetically-encoded+unnatural+amino+acid.">Site-specific epitope tagging of GPCRs by bioorthogonal modification of a genetically-encoded unnatural amino acid.</a> Biochemistry. , (2013).</li><li>Molday, R. S., MacKenzie, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+to+rhodopsin:+characterization,+cross-reactivity,+and+application+as+structural+probes.">Monoclonal antibodies to rhodopsin: characterization, cross-reactivity, and application as structural probes.</a> Biochemistry. 22, 653-660 (1983).</li><li>Gether, 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=Agonists+induce+conformational+changes+in+transmembrane+domains+III+and+VI+of+the+beta2+adrenoceptor.">Agonists induce conformational changes in transmembrane domains III and VI of the beta2 adrenoceptor.</a> The EMBO journal. 16, 6737-6747 (1997).</li><li>Hubbell, W. L., Altenbach, C., Hubbell, C. M., Khorana, H. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzophenone+photophores+in+biochemistry.">Benzophenone photophores in biochemistry.</a> Biochemistry. 33, 5661-5673 (1994).</li><li>Zhang, Z., 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=A+new+strategy+for+the+site-specific+modification+of+proteins+in+vivo.">A new strategy for the site-specific modification of proteins in vivo.</a> Biochemistry. 42, 6735-6746 (2003).</li><li>Dirksen, A., Hackeng, T. M., Dawson, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleophilic+catalysis+of+oxime+ligation.">Nucleophilic catalysis of oxime ligation.</a> Angew. Chem. Int. 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We describe here a robust methodology for site-specific incorporation of a reactive probe, azF, into GPCRs and demonstrate three useful applications of this tool to study the structure and dynamics of GPCRs. Our method to site-specifically incorporate UAAs circumvents a fundamental problem with an alternative strategy based on chemically labeling32, 33 or attaching photocrosslinkers34 to a single accessible cysteine mutant. Although, chemistries that target cysteine thiol groups have been used to at...

Heptahelical G protein-coupled receptors (GPCRs) comprise a superfamily of highly dynamic membrane proteins that mediate important and diverse extracellular signals. In the classical paradigm, receptor activation is coupled with ligand-induced conformational changes.1, 2 Recent advancements in structural biology of GPCRs have provided significant insight on the molecular mechanisms of transmembrane signaling.3-5 However, to understand with greater chemical precision the functional mechanism and structural dynamics of GPCR signaling, a toolkit of approaches is required to incorporate informative molecular and chemical probes to interrogate active signaling complexes.
To this end, we adapted a method to site-specifically introduce non- or minimally perturbing probes into expressed receptors based on unnatural amino acid (UAA) mutagenesis using amber codon suppression technology previously pioneered by Schultz and coworkers.6 We optimized the UAA mutagenesis methodology to achieve a high-yield expression and mutagenesis system for proteins such as GPCRs, which are difficult to express in most heterologous systems other than mammalian cells. Using an orthogonal engineered suppressor tRNA and evolved aminoacyl-tRNA synthetase pair for a specific UAA, we site-specifically introduced UAAs in expressed target GPCRs. The successful incorporation of UAAs, p-acetyl-L-phenylalanine (AcF), p-benzoyl-L-phenylalanine (BzF), and p-azido-L-phenylalanine (azF) has been demonstrated in our model GPCRs - rhodopsin and human C-C chemokine receptor, CCR5.7, 8
In principle, an UAA can be genetically encoded at any position within the protein sequence and this property is an invaluable biochemical tool as it enables single-codon scanning of a target GPCR. We focus here specifically on the versatility of the UAA, azF, which has a reactive azido moiety. In addition to serving as a unique infrared (IR) probe,8, 9 azF can also serve as a photoactivatable cross-linker by reacting with neighboring primary amines or aliphatic hydrogens. Additionally, the biologically inert azido group can participate as a selective chemical handle in bioorthogonal labeling reactions. Here we present examples illustrating the useful applications of site-specific incorporation of azF into GPCRs, such as targeted photocrosslinking to trap a receptor-ligand complex, and modification of GPCRs by bioorthogonal epitope tagging and fluorescent labeling strategies.
Photoactivatable reagents have been used to study biological systems since the 1960s.10 In this period, an abundance of receptor-ligand crosslinking experiments have been reported to study GPCR complexes, most of which involved the use of photoaffinity ligands.11, 12 However, these applications are technically limited, as they require synthesis of ligands bearing a crosslinking group.13-15 Moreover, with the crosslinker moiety in the ligand, it is challenging to identify the location of the crosslink on the GPCR. Site-specifically introducing photocrosslinking groups as UAAs into proteins using the amber codon suppression technology is a valuable advancement. 16, 17 We developed a photocrosslinking technique to identify the binding interface on a receptor that is involved in the formation of a receptor-ligand complex in live cells by introducing photolabile groups into GPCRs.18, 19 Here we describe the experimental protocol and method of data analysis for applying this targeted photocrosslinking technology to identify the binding site of a small-molecule ligand, tritiated maraviroc, on CCR5. This method capitalizes on the precise quantification of the radioactive handle on the ligand, in addition to retaining the native chemical structure of the ligand.
Fluorescence-based techniques support the precise understanding of the structural basis of receptor activation by directly probing the conformational state of the receptor.20, 21 However, techniques possessing the flexibility to introduce fluorescent labels into GPCRs site-specifically are limited. We are interested in employing bioorthogonal chemical modification strategies to facilitate single-molecule detection (SMD) of GPCR signaling complexes.22 The azido group can participate in bioorthogonal chemistries such as Staudinger-Bertozzi ligation,23, 24 copper-free strain-promoted azide-alkyne cycloaddition (SpAAC)25 and copper-catalyzed azide-alkyne cycloaddition (CuAAC).26 We focus here on the Staudinger-Bertozzi ligation reaction that involves the specific reaction between an azide and a phosphine. We demonstrate the use of two different phosphine derivatives, conjugated to a peptide epitope (FLAG peptide) or a fluorescent label (fluorescein) to achieve site-specific modification of GPCRs.
We previously optimized the conditions for site-specific labeling of rhodopsin azF variants using the X-ray crystal structure and dynamic simulations to choose target sites that are solvent exposed. 27, 28 We also illustrated the feasibility to achieve background-free labeling by Staudinger-Bertozzi ligation.29 We demonstrate here the general procedure employed to achieve fluorescent labeling of a detergent-solubilized receptor that is immobilized on an immunoaffinity matrix and subsequently visualized by in-gel fluorescence. Additionally, we demonstrate a useful extension on this labeling strategy to identify positions amenable to labeling on a receptor of unknown structure, CCR5. This is carried out using a targeted peptide-epitope tagging strategy that relies on bioorthogonal modification of azF-GPCR variants in a cell-based semi-high throughput format.30 This method exploits the multi-step detection properties of a cell-surface ELISA to monitor labeling events.
The methodologies we discuss here are general and in principle can be applied to any GPCR incorporated with azF using the amber codon suppression technology. In the protocols presented here we detail the steps involved in mammalian cell expression of receptors incorporated with UAAs such as azF using the unnatural amino acid mutagenesis method and their subsequent applications to facilitate structural and dynamic studies of GPCRs.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmid pSVB. Yam</td>
			<td></td>
			<td></td>
			<td>(Ye et al., 2008)</td>
		</tr>
		<tr>
			<td>Plasmid pcDNA.RS for azF</td>
			<td></td>
			<td></td>
			<td>(Ye et al., 2009)</td>
		</tr>
		<tr>
			<td>Plasmids pMT4.Rho and pcDNA 3.1.CCR5</td>
			<td></td>
			<td></td>
			<td>Optionally contain the amber stop codon (TAG) at a desired position</td>
		</tr>
		<tr>
			<td>HEK 293T cells</td>
			<td></td>
			<td></td>
			<td>Adherent cells</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Gibco</td>
			<td>10566</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco</td>
			<td>14200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gemini Bio-products</td>
			<td>100-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine Plus</td>
			<td>Invitrogen</td>
			<td>Lipofectamine: 18324-012 
			Plus: 11514-015</td>
			<td></td>
		</tr>
		<tr>
			<td>p-azido-L-phenylalanine</td>
			<td>Chem-Impex International</td>
			<td>6162</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. Site-specific genetic incorporation of unnatural amino acids into GPCRs materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>Maxima ML-3500S UV-A lamp</td>
			<td>Spectronics Corporation</td>
			<td></td>
			<td>azF is activated by 365-nm light</td>
		</tr>
		<tr>
			<td>Hank&#39;s Buffered Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14065</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Irvine Scientific</td>
			<td>9319</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Roche</td>
			<td>3117405001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tritium-labeled ligand</td>
			<td></td>
			<td></td>
			<td>From collaborator (Grunbeck et al., 2012)</td>
		</tr>
		<tr>
			<td>1% (w/v) n-dodecyl-&#946;-D-maltoside</td>
			<td>Anatrace</td>
			<td>D310LA</td>
			<td></td>
		</tr>
		<tr>
			<td>1D4-sepharose resin</td>
			<td></td>
			<td></td>
			<td>1D4 mAb immobilized on CNBr-activated sepharose 2B resin</td>
		</tr>
		<tr>
			<td>1% (w/v) sodium dodecyl sulfate (SDS)</td>
			<td>Fisher Scientific</td>
			<td>BP166</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4 - 12% SDS gels</td>
			<td>Invitrogen</td>
			<td>NP0322BOX</td>
			<td>SDS-PAGE performed on NuPAGE apparatus</td>
		</tr>
		<tr>
			<td>Trans-Blot SD apparatus</td>
			<td>Biorad</td>
			<td></td>
			<td>Apparatus for semi-dry transfer</td>
		</tr>
		<tr>
			<td>Immobilon polyvinylidene difluoride (PVDF) membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat powered milk</td>
			<td>Fisher Scientific</td>
			<td>NC9934262</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Aldrich</td>
			<td>274348</td>
			<td></td>
		</tr>
		<tr>
			<td>Ecoscint A</td>
			<td>National Diagnostics</td>
			<td>LS-273</td>
			<td>Scintillation fluid</td>
		</tr>
		<tr>
			<td>Scintillation vials</td>
			<td>Fisher Scientific</td>
			<td>333726</td>
			<td></td>
		</tr>
		<tr>
			<td>LKB Wallac 1209 Rackbeta Liquid Scintillation Counter</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>Beta-Scintillation counter</td>
		</tr>
		<tr>
			<td>Anti-rhodopsin 1D4 mAb</td>
			<td>National Cell Culture Center</td>
			<td>custom</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase (HRP)-conjugated anti-mouse IgG</td>
			<td>KPL, Inc.</td>
			<td>474-1806</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Thermo Scientific</td>
			<td>34080</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyblot CL AR film</td>
			<td>Denville</td>
			<td>E3018</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Targeted photocrosslinking materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>0.25% Trypsin</td>
			<td>Invitrogen</td>
			<td>15050065</td>
			<td></td>
		</tr>
		<tr>
			<td>FLAG-triarylphosphine</td>
			<td>Sigma</td>
			<td>GPHOS1</td>
			<td></td>
		</tr>
		<tr>
			<td>M2 FLAG mAb</td>
			<td>Sigma</td>
			<td>F1804</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CCR5 2D7 mAb</td>
			<td>BD Biosciences</td>
			<td>555990</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Costar</td>
			<td>3601</td>
			<td>Clear bottom, high binding EIA/RIA</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (Calcium, Magnesium)</td>
			<td>Gibco</td>
			<td>14040</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>EMS</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP-conjugated</td>
			<td>KPL, Inc.</td>
			<td>474-1516</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rabbit IgG</td>
			<td>KPL, Inc.</td>
			<td>474-1516</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex Red</td>
			<td>Invitrogen</td>
			<td>A12222</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>DE Healthcare Products</td>
			<td>97-93399</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFluor II fluorescence multi-well plate reader</td>
			<td>Perseptive Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 3. Targeted peptide-epitope tagging and cell surface ELISA materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>Fluorescein-phosphine</td>
			<td></td>
			<td></td>
			<td>(Huber et al., submitted 2012)</td>
		</tr>
		<tr>
			<td>Nonapeptide (C9 peptide)</td>
			<td>AnaSpec</td>
			<td>62190</td>
			<td>Peptide mimicking the 1D4-epitope NH2-TETSQVAPA-COOH</td>
		</tr>
		<tr>
			<td>1% (w/v) n-dodecyl-&#946;-D-maltoside</td>
			<td>Anatrace</td>
			<td>D310LA</td>
			<td></td>
		</tr>
		<tr>
			<td>1D4-sepharose resin</td>
			<td></td>
			<td></td>
			<td>1D4 mAb immobilized on CNBr-activated sepharose 2B resin</td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4 - 12% SDS gels</td>
			<td>Invitrogen</td>
			<td>NP0322BOX</td>
			<td>SDS-PAGE performed on NuPAGE apparatus</td>
		</tr>
		<tr>
			<td>Confocal Typhoon 9400 fluorescence scanner</td>
			<td>GE Healthcare</td>
			<td>discontinued</td>
			<td>Scanner with 488-nm wavelength laser</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 4. Fluorescent labeling materials</td>
		</tr>
	</tbody>
</table>

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 employed the unnatural amino acid mutagenesis methodology to site-specifically introduce molecular probes into a GPCR using the amber codon suppression technology. The scheme in Figure 1 outlines the salient steps of the methodology and the various applications of incorporating the versatile UAA, p-azido-L-phenylalanine (azF), into GPCRs. Expression of azF-GPCRs in mammalian cells enables targeted photocrosslinking to ligands, and targeted peptide-epitope tagging or fluorescent modifications ...

Watch this Scientific Journal Video about Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors at JoVE.com

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

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 ...

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