This work was supported by National Institutes of Health grant DA-000266 and the Medical Scientist Training Program T32 grant (C.V., N.W.Z., and P.C.S.). The authors would also like to acknowledge somersault18:24 (somersault1824.com) for the Library of Science &#38; Medical Illustrations.

1. Expression of Recombinant HA-MOR1 in Cultured Cells
NOTE: Follow all cell culture protocols in a sterile laminar flow hood.
<ol>
	<li>Sterilize the cell culture laminar flow hood with 70% ethanol and maintain sterile technique throughout the cell culture.</li>
	<li>Prepare human embryonic kidney cells 293 (HEK293) cell culture medium, complete Dulbecco&#39;s-modified Eagle Medium (DMEM): DMEM, pH 7.4, supplemented with 2 mM L-glutamine, 1% penicillin/streptomycin, and 10% fetal bovine ser...

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The present protocol describes two separate but complementary methods: a simple approach to fractionate cells and tissues into broad but distinct compartments and a means to investigate GPCR signaling by measuring [35S]GTP&#947;S binding.
Efficient cellular fractionation has a wide range of applications, ranging from the extraction and enrichment of proteins, to the assessment of the subcellular localization of proteins, to the study of receptor pharmacology. Although alternative ap...

G-Protein-Coupled Receptors&#160;(GPCRs)&#160;are a large family of cell-surface receptors responsible for a remarkable array of physiological processes, including analgesia, olfaction, and behavior1. GPCRs act by sensing specific external signals and subsequently stimulating intracellular signaling. They therefore mark a key junction between the external and internal environments of a cell. Due to the critical role GPCRs play in biology, they have become major targets for both basic research and drug discovery2,3.
Unlike other receptor families that bind discrete ligands, GPCRs can bind very different types of molecules. While one GPCR may interact with peptides, another may sense photons, small molecules, or ions1,4. While their ligands are diverse, GPCRs are unified in their overall architecture and function. Individual GPCRs are made up of seven &#945;-helical transmembrane proteins with extracellular amino terminals and intracellular carboxyl terminals5,6. GPCRs are coupled to intracellular G-proteins&#8212;heterotrimeric protein complexes composed of &#945;, &#946;, and &#947; subunits&#8212;which mediate diverse signaling pathways7. The G&#945; subunit is a guanine nucleotide-binding protein that is inactive when bound to guanosine diphosphate (GDP) and active when bound to guanosine triphosphate (GTP)8,9. When GPCRs bind their ligands, they undergo a conformational change that permits G&#945; to dissociate from G&#946;&#947;, thereby allowing G&#945; to exchange GDP for GTP7. The receptor itself is phosphorylated at its carboxyl terminal by various serine/threonine kinases10,11 and internalized to attenuate receptor signaling12,13,14. Meanwhile, the activated G&#945; monomer and G&#946;&#947; dimer proceed to activate distinct signaling pathways7. There are several isoforms of each G-protein subunit, and each isoform targets particular downstream pathways and secondary messenger systems. The major G&#945; isoforms include Gs, Gq, Gi/o, and G12-13. Typically, individual GPCRs associate with a particular G&#945; isoform, thereby linking an external stimulus to a specific cellular response1.
Characterizing a GPCR-ligand interaction is critical to understanding the biology of the receptor. As GDP/GTP exchange is one of the earliest events that follows ligand binding, monitoring GTP binding can measure GPCR activation or inhibition. Assaying more downstream events in GPCR signaling is often not as quantitative or stoichiometric, may not distinguish full agonists from partial ones, and can require expensive reagents. Moreover, increased GTP binding to G&#945; proteins is an almost-universal event following GPCR activation, meaning that measuring GTP binding is a broadly applicable assay for monitoring the activity of most GPCRs. Measuring GTP binding is a simple and rapid approach to monitor GPCR signaling in cells overexpressing the receptor of interest or in native tissue. The present protocol details a functional GTP-binding assay using an archetypal GPCR, the &#181;-opioid receptor (MOR1), to quantitatively determine the activity of an agonist and antagonist on GPCR signaling.
This protocol first outlines how to isolate crude membranes from cells overexpressing MOR1. Note that this protocol is not limited to overexpression systems and can be applied to many sources of membrane, including native tissue or preparations expressing multiple receptors and G proteins15. The protocol then details how to measure the binding of a radioactive GTP analog to these membranes in response to varying concentrations of [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO) or naloxone, a MOR1 agonist and antagonist, respectively. The GTP analog, [35S]guanosine-5&#39;-O-(3-thio) triphosphate ([35S]GTP&#947;S), is non-hydrolyzable. This property is critical because G&#945; subunits exhibit intrinsic GTPase activity7 and would eliminate the labeled gamma phosphate on a hydrolyzable GTP radiochemical. Membranes are then trapped onto glass fiber filters and washed, after which the radiolabeled GTP is quantified by liquid scintillation counting. Multiple pharmacological parameters can be derived to characterize the receptor-ligand interaction, including the half-maximal response (EC50) and Hill coefficient (nH) for agonists and the half-maximal inhibitory concentration (IC50) and equilibrium dissociation constant (Kb) for antagonists16,17,18.

<table border="0" cellpadding="0" cellspacing="0" width="1034">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">DMEM, high glucose, pyruvate, no glutamine</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">10313021</td>
			<td style="width:264px;">Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">L-glutamine</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">25030081</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Penicillin-Streptomycin</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">15140122</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Opti-MEM I Reduced Serum Medium</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">31985070</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Fetal Bovine Serum</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">16000044</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Cell culture 10-cm plate</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">CLS430167</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Lipofectamine 3000 reagent</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">L3000-008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">1.6 mL microcentrifuge tubes</td>
			<td style="width:189px;">USA Scientific</td>
			<td style="width:119px;">1615-5500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Tris(hydroxymethyl)aminomethane (Trizma base)</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">BP152-1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:463px;">ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA)</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">E3889</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:463px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">E9884</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Sucrose</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5016</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:463px;">cOmplete ULTRA Tablets, Mini, EASYpack Protease Inhibitor Cocktail</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">2900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">DL-Dithiothreitol (DTT)</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">DO632</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Sodium chloride (NaCl)</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">BP358-1</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:463px;">Magnesium chloride (MgCl2)</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">M1028-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Pellet pestles motor</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z359971</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Pestles</td>
			<td style="width:189px;">Bel Art</td>
			<td style="width:119px;">F19923-0001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Bovine serum albumin (BSA)</td>
			<td style="width:189px;">Affymetrix</td>
			<td align="right" style="width:119px;">10857</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">[35S]guanosine-5&#8217;-O-(3-thio)triphosphate ([35S]GTP&#947;S)&#160;</td>
			<td style="width:189px;">Perkin Elmer</td>
			<td style="width:119px;">NEG030H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">non-radiolabeled guanosine-5&#8217;-O-(3-thio)triphosphate (GTP&#947;S)&#160;</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">89378</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">guanosine diphosphate (GDP)</td>
			<td style="width:189px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">51060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Bradford reagent</td>
			<td style="width:189px;">Bio-Rad</td>
			<td align="right" style="width:119px;">5000006</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">UV/VIS spectrophotometer</td>
			<td style="width:189px;">Beckman Coulter</td>
			<td style="width:119px;">DU640</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">spectrophotometer cuvettes</td>
			<td style="width:189px;">USA Scientific</td>
			<td style="width:119px;">9090-0460</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">orbital shaker</td>
			<td style="width:189px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:119px;">2314</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">thermomixer</td>
			<td style="width:189px;">Eppendorf</td>
			<td align="right" style="width:119px;">535027903</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">glass fiber filters&#160;</td>
			<td style="width:189px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">1821-021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">vacuum filtration apparatus</td>
			<td style="width:189px;">Millipore Corporation</td>
			<td style="width:119px;">XX2702550</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">desktop microcentrifuge</td>
			<td style="width:189px;">Eppendorf</td>
			<td align="right" style="width:119px;">65717</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Scintillation counter</td>
			<td style="width:189px;">Beckman Coulter</td>
			<td style="width:119px;">LS6500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">scintillation fluid&#160;</td>
			<td style="width:189px;">Ecoscint A</td>
			<td style="width:119px;">LS-273</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">scintillation counter vials</td>
			<td style="width:189px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">592690</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">scintillation vial lids</td>
			<td style="width:189px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">592928</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">Prism 6</td>
			<td style="width:189px;">GraphPad Software</td>
			<td style="width:119px;">PRISM 6</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:463px;">ATP1A1 antibody</td>
			<td style="width:189px;">Developmental Studies Hybridoma</td>
			<td style="width:119px;">a6F</td>
			<td>1:1000 in 3% BSA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">GAPDH antibody</td>
			<td style="width:189px;">EMD Millipore</td>
			<td style="width:119px;">CB1001</td>
			<td>1:5000 in 3% BSA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">H2B antibody</td>
			<td style="width:189px;">Cell Signaling</td>
			<td style="width:119px;">2934S</td>
			<td>1:2500 in 3% BSA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">PDI antibody</td>
			<td style="width:189px;">Cell Signaling</td>
			<td style="width:119px;">3501S</td>
			<td>1:1000 in 3% BSA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:463px;">HA antibody</td>
			<td style="width:189px;">Roche</td>
			<td align="right" style="width:119px;">11867423001</td>
			<td>1:2000 in 3% BSA</td>
		</tr>
	</tbody>
</table>

measuring g-protein-coupled receptor signaling via radio-labeled gtp binding

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a major pharmacological target for multiple indications, including analgesia, blood pressure regulation, and the treatment of psychiatric disease. Upon ligand binding, GPCRs catalyze the activation of intracellular G-proteins by stimulating the incorporation of guanosine triphosphate (GTP). Activated G-proteins then stimulate signaling pathways that elicit cellular responses. GPCR signaling can be monitored by measuring the incorporation of a radiolabeled and non-hydrolyzable form of GTP, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), into G-proteins. Unlike other methods that assess more downstream signaling processes, [35S]GTP&#947;S binding measures a proximal event in GPCR signaling and, importantly, can distinguish agonists, antagonists, and inverse agonists. The present protocol outlines a sensitive and specific method for studying GPCR signaling using crude membrane preparations of an archetypal GPCR, the &#181;-opioid receptor (MOR1). Although alternative approaches to fractionate cells and tissues exist, many are cost-prohibitive, tedious, and/or require non-standard laboratory equipment. The present method provides a simple procedure that enriches functional crude membranes. After isolating MOR1, various pharmacological properties of its agonist, [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO), and antagonist, naloxone, were determined.

Cell fractionation can be used to isolate and enrich membrane-associated proteins from cytosolic and nuclear proteins. Figure 1 is a Western blot demonstrating the contents of the three primary fractions that can be collected during the subcellular fractionation process. Specifically, Figure 1 shows that fractionation cleanly separates membrane proteins (i.e.&#160;Na+/K+ ATPase, protein disulfide isomerase (PDI), and HA-...

Watch this Scientific Journal Video about Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding at JoVE.com

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding

Guanosine triphosphate (GTP) binding is one of the earliest events in G-Protein-Coupled Receptor (GPCR) activation. This protocol describes how to pharmacologically characterize specific GPCR-ligand interactions by monitoring the binding of the radio-labeled GTP analog, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), in response to a ligand of interest.

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a ...

The overall goal of this procedure is to isolate cellular membranes that contain G-protein-coupled receptors or GPCRs and to study GPCR pharmacology using a functional GTP binding essay. This method can help answer key questions in the field of biochemistry regarding the pharmacologic properties of established or orphaned G-protein-coupled receptors. The main advantages are that it is simple, rapid and measures the most proximal event in G-protein-coupled receptors signaling.First, plate human embryonic kidney cells onto a 10 centimeter tissue culture plate in 10 milliliters of complete DMEM. Incubate the cells at 37 degree celsius and 5%carbon dioxide, until 70%to 80%confluency is reached. Following incubation, transfect the cells with HAMOR1 using a suitable transfection reagent per the manufacturer's guidelines.Then, incubate the cells as previously described for 36 to 48 hours. After removing the transfected cells from the incubator, aspirate the medium and rinse the cells with five milliliters of ice cold PBS. Then, aspirate the PBS and excess liquid from the plate.Next, add 600 microliters of buffer 1 to the cells. Using a cell scraper, dislodge the cells from the plate surface and pipette the cell suspension into a 1.6 milliliter micro centrifuge tube. Immediately snap freeze the micro centrifuge tube in liquid nitrogen.Once the sample is frozen, thaw the lysates on ice. Once the lysates have thawed, use a single pulse microtube homogenizer to break open the cells. Pulsing that homogenizer three to five times for 10 seconds.Place the tube on ice for 30 seconds between pulses. Following this, centrifuge the sample for 10 minutes at 1, 000 times g and four degrees celsius. Then, transfer the supernatant to a new 1.6 milliliter micro centrifuge tube on ice.Re-suspend the palate in 100 microliters of buffer 1. Re-homogenize the sample and pulse the homogenizer three to five times for five seconds, placing the tube on ice for 30 seconds between pulses. After centrifuging the sample a second time, combine the supernatants and centrifuge for 20 minutes at 11, 000 times g and four degree celsius.Transfer the supernatant to a new 1.6 milliliter micro centrifuge tube. Re-suspend in 200 microliters of buffer 2. Then, homogenize the palate by triturating it three to five times.After centrifuging the sample and aspirating the supernatant, re-suspend the palate containing the crude membrane faction in 50 microliters of buffer 2. Dilute stock S35GTP gamma S to 50 nanomolar and 10 millimolar trace HCl and 10 millimolar DTT. Make 250 microliter hour quarts for the binding experiments, and flash freeze for storage if desired.Next, prepare complete binding buffer or CBB, by supplementing incomplete binding buffer to include a final concentration of one millimolar DTT, 0.1%BSA, 10 micromolar GTP and 0.1 nanomolar S35GTP gamma S.After thawing the membrane fraction on ice, dilute it to a concentration of 100 micrograms per milliliter and incomplete binding buffer. To essay the effect of the peptide DAMGO on GTP binding via MOR1, prepare a series of DAMGO dilutions and CBB to a final volume of 100 microliters. To evaluate basil GTP binding, prepare a 1.6 milliliter micro centrifuge tube with 100 microliters of CBB.To evaluate non-specific binding, prepare a 1.6 milliliter micro centrifuge tube with 99 microliters of CBB supplemented with one microliter of two millimolar non-radial labeled GTP gamma S.Now, add 100 microliters of the diluted membrane solution to each sample. Incubate the samples for 30 minutes at 25 degrees celsius in a thermo-mixer at 300 rpm. Pre-soak glass fiber filters in distilled water for 10 minutes.After removing the samples from the thermo-mixer, briefly pulse spin them for five seconds to collect each sample at the bottom of the tube. Next, remove the lid of a vacuum filtration apparatus and lay the pre-soaked filters onto the vacuum ports of the apparatus. Re-secure the apparatus lid to form a vacuum seal and turn on the vacuum.Pipette 195 microliters of each sample onto the filters. Wash the filters three times with one milliliter of ice cold wash buffer. Place five milliliters scintillation counting vials in a counting rack.Add five milliliters of scintillation fluid to each vial. Next, turn off the vacuum and remove the lid of the vacuum filtration apparatus. Using tweezers, pick up the filters from the vacuum ports of the filtration apparatus and drop each filter into a scintillation vial.After capping each vial securely, incubate the samples on an orbital shaker at 25 degree celsius for 10 minutes. Following this, turn on the scintillation counter, program the counter to measure sulfur 35 isotope emission for five minutes per sample using the standard associated scintillation program and press start to take account. Self fractionation, cleanly separates membrane proteins from nuclear protein histone h2b and the cytoplasmic protein glycerol to hide three phosphate dehydrogenase.Proteins are enriched in their respective sub-cellular fractions. A representative of Ponceau stain demonstrates equal protein loading in each fraction. Multiple pharmacological parameters can be drive to characterize a GPCR ligand interaction via GTP binding experiments such as the EC50 and hill coefficient.Those responsive GTP binding to MOR1 after DAMGO treatment is demonstrated here. This plot illustrates the agonist activity of DAMGO in the antagonism of Naloxone on MOR1 G-protein signaling. Once mastered, this technique can be done in two to four hours, depending on the number of experimental conditions.While attempting this procedure, it's important to remember to use GPCR ligand concentrations at least five orders of magnitude above and below the expected EC50. Following this procedure, assessing molecules that are structurally similar to your ligand of interest, can help define important structural moieties in the ligand that define a receptor's ligand profile. After its development, this technique has aided in drug discovery and paved the way for researchers to explore detailed aspects of GPCR signaling, such as biased agonism.After watching watching this video, you should have a good understanding of how to isolate cellular membranes and measure pharmacologic properties of GPCRs using radio labeled GTP. Don't forget, that working with radiation can be extremely hazardous. Precaution should be taken, such as wearing appropriate personal protective equipment and working with radiation appropriate lab wear.Institutional guidelines for working with radioactivity should be reviewed prior to performing this procedure.

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16.0K 조회수.  Johns Hopkins University School of Medicine. The overall goal of this procedure is to isolate cellular membranes that contain G-protein-coupled receptors or GPCRs and to study GPCR pharmacology using a functional GTP binding essay. This method can help answer key questions in the field of biochemistry regarding the pharmacologic properties of established or orphaned G-protein-coupled receptors. The main advantages are that it is simple, rapid and measures the most proximal event in G-protein-coupled receptors signaling.First, pla...