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 serum (FBS).</li>
	<li>Plate 2.5 x 106 HEK293 cells onto a 10 cm tissue culture plate in 10 mL of complete DMEM and incubate at 37 &#176;C and 5% CO2 until 70-80% confluency is reached (O/N). 
	NOTE: To collect enough protein for a complete GTP-binding experiment, plate at least 3 x 10 cm plates of cells.</li>
	<li>Transfect the cells with HA-MOR1 using a transfection reagent of choice and by following the manufacturer&#39;s guidelines. Incubate for 36-48 h. 
	NOTE: Human MOR-1 cDNA (NCBI Reference Sequence: NM_000914.4) was a generous gift from Gavril Pasternak and was cloned into pCMV-HA.</li>
</ol>
2. Cell Fractionation and Membrane Collection
<ol>
	<li>Prepare the following lysis buffers:
	<ol>
		<li>Buffer 1: 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4; 1 mM ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA); 10% sucrose; protease inhibitor cocktail; and 1 mM dithiothreitol (DTT). Add the DTT fresh on the day of membrane isolation.</li>
		<li>Buffer 2: 10 mM HEPES, pH 7.4; 1 mM EGTA; 1 mM MgCl2; and 1 mM DTT. Add the DTT fresh on the day of membrane isolation.</li>
	</ol>
	</li>
	<li>Remove the transfected cells from the incubator (step 1.4), aspirate the medium, and rinse the cells with 5 mL of ice-cold Phosphate-buffered Saline (PBS). Aspirate the PBS and excess liquid from the plate.</li>
	<li>Add 600 &#181;L 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 mL microcentrifuge tube.</li>
	<li>Immediately snap-freeze the microcentrifuge tube in liquid nitrogen. Once the samples are frozen, thaw the lysates on ice or place them at -80 &#176;C for long-term storage.</li>
	<li>Repeat steps 2.3 and 2.4 with all plates of cells.</li>
	<li>Once the lysates have thawed, use a single-pulse micro-tube homogenizer to break open the cells. Pulse the homogenizer 3-5 times for 10 s, placing the tube back on ice for 30 s between pulses.
	<ol>
		<li>Collect 20 &#181;L as whole cell fraction.</li>
	</ol>
	</li>
	<li>Centrifuge the remaining sample for 10 min at 1,000 x g and 4&#176;C. Collect the supernatant and place in a new 1.6 mL microcentrifuge tube on ice.</li>
	<li>Resuspend the pellet in 100 &#181;L of Buffer 1 and rehomogenize with a pestle. Pulse the homogenizer 3-5 times for 5 s, placing the tube back on ice for 30 s between pulses.
	<ol>
		<li>Centrifuge the sample a second time for 10 min at 1,000 x g and 4 &#176;C. Combine the supernatant with the supernatant from step 2.7. 
		NOTE: The remaining pellet contains nuclear and membrane proteins.</li>
	</ol>
	</li>
	<li>Centrifuge the combined supernatants for 20 min at 11,000 x g and 4 &#176;C. Separate the supernatant (the cytosolic fraction) into a new 1.6 mL microcentrifuge tube.</li>
	<li>Resuspend the pellet in 200 &#181;L of Buffer 2. Homogenize the pellet by triturating it 3-5 times. Centrifuge the sample for 20 min at 21,100 x g and 4 &#176;C. Aspirate the supernatant. Resuspend the pellet containing the crude membrane fraction in 50 &#181;L of Buffer 2.</li>
	<li>Immediately proceed to the GTP&#947;S binding experiments (section 3) or snap-freeze in liquid nitrogen and store at -80 &#176;C.</li>
</ol>
3. [35S]GTP&#947;S Binding
NOTE: Use standard radiochemical safety protocol when handling [35S]GTP&#947;S and when conducing [35S]GTP&#947;S binding experiments. Wear protective gloves and a lab coat at all times. Check the packaging material for leaks or cracks. Dispose of waste and excess reagents according to institutional protocols.
<ol>
	<li>Prepare Incomplete Binding Buffer (IBB) by mixing 50 mM HEPES, pH 7.4; 5 mM MgCl2; 100 mM NaCl; and 1 mM ethylenediaminetetraacetic acid (EDTA) in distilled water.</li>
	<li>Dilute stock [35S]GTP&#947;S to 50 nM in 10 mM Tris-HCl, pH 7.6, and 10 mM DTT. Make 500 &#181;L aliquots and store them at -80 &#176;C. Use only a freshly thawed aliquot immediately before initiating the experiment. Thaw the aliquots on ice.</li>
	<li>Prepare nonradio-labeled GTP&#947;S by dissolving 1 mg of GTP&#947;S powder in 177.62 &#181;L of pure water for a stock concentration of 10 mM. Make 10-&#181;L aliquots and store them at -20 &#176;C.</li>
	<li>Prepare complete binding buffer (CBB) by supplementing IBB to include a final concentration of 1 mM DTT, 0.1% (wt/vol) bovine serum albumin (BSA), 10 &#181;M GDP, and 0.1 nM [35S]GTP&#947;S. 
	NOTE: The CBB now contains radiolabeled GTP. Take appropriate safety precautions while working with radioactive solutions.</li>
	<li>Thaw the membrane fraction from step 2.11 on ice.</li>
	<li>Quantify the protein concentration of the membrane fraction via a Bradford protein assay using a spectrophotometer at 595 nm.
	<ol>
		<li>Prepare a dilution series of BSA protein standards with Buffer 2, at final concentrations of 0, 250, 500, 750, 1,500, 2,500, and 5,000 &#181;g/mL.</li>
		<li>Add 2 &#181;L of each standard or membrane to 1 mL of Bradford reagent in a cuvette. Mix thoroughly by vortexing.</li>
		<li>Adjust the spectrophotometer to a wavelength of 595 nm and blank using the cuvette with 0 &#181;g/mL protein.</li>
		<li>Wait 5 min and read each of the standards and each of the samples at a 595 nm wavelength.</li>
		<li>Plot the absorbance of the standards versus the concentration. Using the Beer-Lambert Law (A = &#949;cl, where &#949; = extinction coefficient, l = length of cuvette, and c = concentration), calculate the concentration of the membrane samples.</li>
	</ol>
	</li>
	<li>Dilute the membrane fractions to a concentration of 100 &#181;g protein/mL in IBB. 
	NOTE: One 10 cm dish of HEK293 cells typically yields between 1 &#38; 3 mg protein/mL.</li>
	<li>Prepare the following experimental conditions in individual 1.6 mL microcentrifuge tubes: a ligand dilution series, a basal activity condition to measure basal GTP binding, and a nonspecific binding condition to measure nonspecific GTP binding.
	<ol>
		<li>For the ligand dilution series, prepare a dilution series of the ligand of interest in a final volume of 100 &#181;L of CBB. Prepare the ligand series at 2x the desired final concentrations. Space the ligand concentrations to adequately cover a range of responses.
		<ol>
			<li>To assay the effect of DAMGO on GTP binding via MOR1, prepare DAMGO dilutions of 2 mM, 200 &#181;M, 20 &#181;M, 2 &#181;M, 200 nM, 20 nM, 2 nM, 200 pM, 20 pM, and 2 pM in CBB (final volume: 100 &#181;L). 
			NOTE: For example, if the ligand of interest has an expected EC50 value of 10 &#181;M, prepare ligand dilutions of 200 nM, 2 &#181;M, 20 &#181;M, 200 &#181;M, and 2 mM. These five conditions cover a wide range of concentrations and are 2x the concentration desired for the assay.</li>
		</ol>
		</li>
		<li>For basal binding, prepare a tube with 100 &#181;L of CBB only.</li>
		<li>For nonspecific binding, prepare a tube with 99 &#181;L of CBB supplemented with 1 &#181;L of 2 mM nonradio-labeled GTP&#947;S.</li>
	</ol>
	</li>
	<li>Add 100 &#181;L of the diluted membrane solution (step 3.7) to each experimental condition.</li>
	<li>Incubate the membranes with the various experimental conditions in 1.6 mL microcentrifuge tubes for 30 min at 25 &#176;C in a thermomixer or on an orbital shaker.</li>
</ol>
4. Membrane Filtration
<ol>
	<li>Prepare wash buffer by combining 50 mM Tris-HCl, pH 7.4; 5 mM MgCl2; and 50 mM NaCl in distilled water.</li>
	<li>Presoak the glass fiber filters in water for 10 min. 
	NOTE: Filters have a pore size of 1 &#181;m, a diameter of 2.1 cm, and a thickness of 675 &#181;m.</li>
	<li>Remove the samples from the thermomixer or orbital shaker (step 3.10). Briefly pulse-spin the samples for 5 s to collect each sample at the bottom of the tube.</li>
	<li>Remove the lid of the vacuum filtration apparatus. Lay the presoaked filters onto the vacuum ports of the apparatus. Re-secure the apparatus lid to form a vacuum seal. Turn on the vacuum.</li>
	<li>Pipette 195 &#181;L of each 200 &#181;L experimental condition onto filters to minimize error from adsorption to the walls of the tube and/or from pipetting.</li>
	<li>Wash the filters three times with 1 mL of ice-cold wash buffer.</li>
</ol>
5. Liquid Scintillation Counting
<ol>
	<li>Place 5 mL scintillation counting vials in a counting rack. Add 5 mL of scintillation fluid to each vial.</li>
	<li>Turn off the vacuum (step 4.4). 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 an individual 5 mL scintillation vial.</li>
	<li>Prepare one vial with 195 &#181;L of CBB to determine the maximal signal.</li>
	<li>Cap each vial securely. Incubate the vials on an orbital shaker at 25 &#176;C for 10 min.</li>
	<li>Turn on the scintillation counter. Program the scintillation counter to measure 35S isotope emission for 5 min per sample using the standard associated scintillation program.</li>
	<li>Press &#34;Start&#34; to take a count.</li>
	<li>When the counting is done, dispose the counted vials as hazardous waste.</li>
</ol>
6. Data Analysis
<ol>
	<li>Enter the data into statistics and analysis software.
	<ol>
		<li>Subtract the non-specific binding from each of the other measurements to determine the specific binding. 
		NOTE: The degree of nonspecific binding is determined by the sample incubated with non-radiolabeled GTP&#947;S (step 3.8.3).</li>
		<li>Open the statistics and analysis software and select an XY dataset entry.</li>
		<li>Enter the specific binding data from the [35S]GTP&#947;S-binding experiments into a data table in the statistics and analysis software. Enter the agonist concentrations, as molar concentrations, in the &#34;X&#34; column. Enter the associated 35S counts as &#34;Y&#34; values.</li>
	</ol>
	</li>
	<li>Transform the data.
	<ol>
		<li>Convert the X values to their respective logarithm by clicking &#34;Analyze.&#34; Select &#34;Transform&#34; from the built-in analyses.</li>
		<li>Within the &#34;Transform&#34; dialog box, select &#34;Transform X values using&#34; and select the function &#34;X=log(X).&#34; Choose to create a new graph of the results.</li>
		<li>Click &#34;OK.&#34;</li>
	</ol>
	</li>
	<li>Normalize the Y-values.
	<ol>
		<li>With the transformed results displayed, click &#34;Analyze.&#34; Select &#34;Normalize&#34; from the built-in analyses.</li>
		<li>Within the &#34;Normalize&#34; dialog box, select the radio button to define 0% as the &#34;smallest value in each data set&#34; and 100% as the &#34;largest value in each data set.&#34; Select the box to present the results as percentages. Select the box to create a new graph of the results.</li>
		<li>Click &#34;OK.&#34;</li>
	</ol>
	</li>
	<li>Fit a nonlinear regression curves to the plotted data. Perform a nonlinear regression analysis.
	<ol>
		<li>With the normalized results displayed, click &#34;Analyze.&#34; From the built-in analyses, select &#34;Nonlinear regression (curve fit).&#34;</li>
		<li>If investigating an agonist, select &#34;log(agonist) vs. normalized response - variable slope&#34; from the dialog box.</li>
		<li>If investigating an antagonist, &#34;select log(antagonist) vs. normalized response - variable slope&#34; from the dialog box.</li>
		<li>Click &#34;OK.&#34;</li>
	</ol>
	</li>
	<li>Review the graph and results to ensure that the fit and data are in reasonable agreement. Ensure that the regression matches the general pattern of the plotted data and that the residuals are not so large that they render the dose-response curve flat. 
	NOTE: The software will summarize the results of the nonlinear regression and details the concentration that elicits the half-maximal response (EC50), Hill coefficient (nH), and half-maximal inhibitory concentration (IC50).</li>
	<li>Derive agonist/antagonist parameter potency.
	<ol>
		<li>Derive the agonist equilibrium dissociation constants (Kb) from either of the following experiments16,17,18
		<ol>
			<li>Assess the shift in the dose-response of an agonist in competition with a fixed concentration of antagonist. Here, EC50&#39; = EC50 (1 + [antagonist]/antagonistKb), where EC50&#39; is the right-shifted EC50.</li>
			<li>Assess [35S]GTP&#947;S binding with varying concentrations of antagonist in competition with a fixed concentration of agonist. Here, Kb = IC50/ (2 +([agonist]/agonist EC50)n)1/n - 1, where n is the Hill coefficient of the agonist.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Depending upon the variability of the data, repeat the experiment (steps 3.8-5.6) for each ligand approximately 3-5 times and average the results using a statistics and analysis software.</li>
</ol>

<ol>
	<li>Kobilka, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+protein+coupled+receptor+structure+and+activation.">G protein coupled receptor structure and activation.</a> Biochim. Biophys. Acta. 1768 (4), 794-807 (2007).</li><li>Neubig, R. R., Siderovski, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulators+of+G-protein+signalling+as+new+central+nervous+system+drug+targets.">Regulators of G-protein signalling as new central nervous system drug targets.</a> Nat. Rev. Drug Discov 1. (3), 187-197 (2002).</li><li>Overington, J. P., Al-Lazikani, B., Hopkins, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+many+drug+targets+are+there?.">How many drug targets are there?.</a> Nat. Rev. Drug Discov. 5 (12), 993-996 (2006).</li><li>Buck, L., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multigene+family+may+encode+odorant+receptors:+a+molecular+basis+for+odor+recognition.">A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.</a> Cell. 65 (1), 175-187 (1991).</li><li>Kobilka, B. K., 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,+sequencing,+and+expression+of+the+gene+coding+for+the+human+platelet+alpha+2-adrenergic+receptor.">Cloning, sequencing, and expression of the gene coding for the human platelet alpha 2-adrenergic receptor.</a> Science. 238 (4827), 650-656 (1987).</li><li>Palczewski, K., 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+rhodopsin:+A+G+protein-coupled+receptor.">Crystal structure of rhodopsin: A G protein-coupled receptor.</a> Science. 289 (5480), 739-745 (2000).</li><li>Neer, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotrimeric+c+proteins:+organizers+of+transmembrane+signals.">Heterotrimeric c proteins: organizers of transmembrane signals.</a> Cell. 80 (2), 249-257 (1995).</li><li>Londos, C., Salomon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5'-Guanylylimidodiphosphate,+a+Potent+Activator+of+Adenylate+Cyclase+Systems+in+Eukaryotic+Cells.">5'-Guanylylimidodiphosphate, a Potent Activator of Adenylate Cyclase Systems in Eukaryotic Cells.</a> Proc. Natl. Acad. Sci. USA. 71 (8), 3087-3090 (1974).</li><li>Ross, E. M., Gilman, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+Some+Components+of+Adenylate-Cyclase+Necessary+for+Catalytic+Activity.">Resolution of Some Components of Adenylate-Cyclase Necessary for Catalytic Activity.</a> J. Biol. Chem. 252 (20), 6966-6969 (1977).</li><li>Stadel, J. M., Nambi, P., Shorr, R., Sawyer, D. F., Caron, M. G., 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=Catecholamine-Induced+Desensitization+of+Turkey+Erythrocyte+Adenylate-Cyclase+Is+Associated+with+Phosphorylation+of+the+Beta-Adrenergic-Receptor.">Catecholamine-Induced Desensitization of Turkey Erythrocyte Adenylate-Cyclase Is Associated with Phosphorylation of the Beta-Adrenergic-Receptor.</a> Proc. Natl. Acad. Sci. USA. 80 (11), 3173-3177 (1983).</li><li>Wilden, U., Kuhn, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-Dependent+Phosphorylation+of+Rhodopsin+-+Number+of+Phosphorylation+Sites.">Light-Dependent Phosphorylation of Rhodopsin - Number of Phosphorylation Sites.</a> Biochemistry. 21 (12), 3014-3022 (1982).</li><li>Lohse, M., Benovic, J., Codina, J., Caron, M., Lefkowitz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Arrestin:+a+protein+that+regulates+beta-adrenergic+receptor+function.">beta-Arrestin: a protein that regulates beta-adrenergic receptor function.</a> Science. 248 (4962), 1547-1550 (1990).</li><li>Leftowitz, R. J., Shenoy, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+of+receptor+signals+by+beta-arrestins.">Transduction of receptor signals by beta-arrestins.</a> Science. 308 (5721), 512-517 (2005).</li><li>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=Historical+review:+a+brief+history+and+personal+retrospective+of+seven-transmembrane+receptors.">Historical review: a brief history and personal retrospective of seven-transmembrane receptors.</a> Trends Pharmacol. Sci. 25 (8), 413-422 (2004).</li><li>Salah-Uddin, H., Thomas, D. 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=Pharmacological+assessment+of+m1+muscarinic+acetylcholine+receptor-gq/11+protein+coupling+in+membranes+prepared+from+postmortem+human+brain+tissue.">Pharmacological assessment of m1 muscarinic acetylcholine receptor-gq/11 protein coupling in membranes prepared from postmortem human brain tissue.</a> J. Pharm. Exp. Ther. 325 (3), 869-874 (2008).</li><li>Yung-Chi, C., Prusoff, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+the+inhibition+constant+(K1)+and+the+concentration+of+inhibitor+which+causes+50+per+cent+inhibition+(I50)+of+an+enzymatic+reaction.">Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction.</a> Biochem. Pharmacol. 22 (23), 3099-3108 (1973).</li><li>Lazareno, S., Birdsall, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+competitive+antagonist+affinity+from+functional+inhibition+curves+using+the+Gaddum,+Schild+and+Cheng-Pruso&#237;f+equations.">Estimation of competitive antagonist affinity from functional inhibition curves using the Gaddum, Schild and Cheng-Pruso&#237;f equations.</a> Br. J. Pharmacol. 109 (4), 1110-1119 (1993).</li><li>Mar&#233;chal, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+5,+Measuring+Bioactivity:+KI,+IC50+and+EC50.">Chapter 5, Measuring Bioactivity: KI, IC50 and EC50.</a> Chemogenomics and Chemical Genetics. , 55-65 (2011).</li><li>Salah-Uddin, H., Thomas, D. 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=Pharmacological+Assessment+of+M1+Muscarinic+Acetylcholine+Receptor-Gq/11+Protein+Coupling+in+Membranes+Prepared+from+Postmortem+Human+Brain+Tissue.">Pharmacological Assessment of M1 Muscarinic Acetylcholine Receptor-Gq/11 Protein Coupling in Membranes Prepared from Postmortem Human Brain Tissue.</a> J. Pharm. Exp. Ther. 325 (3), 869-874 (2008).</li><li>Milligan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles:+extending+the+utility+of+[35S]GTP+gamma+S+binding+assays.">Principles: extending the utility of [35S]GTP gamma S binding assays.</a> Trends. Pharmacol. Sci. 24 (2), 87-90 (2003).</li><li>Harrison, C., Traynor, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+[35S]GTPgammaS+binding+assay:+approaches+and+applications+in+pharmacology.">The [35S]GTPgammaS binding assay: approaches and applications in pharmacology.</a> Life Sci. 74 (4), 489-508 (2003).</li><li>Traynor, J. R., Nahorski, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+by+mu-opioid+agonists+of+guanosine-5'-O-(3-[35S]thio)triphosphate+binding+to+membranes+from+human+neuroblastoma+SH-SY5Y+cells.">Modulation by mu-opioid agonists of guanosine-5'-O-(3-[35S]thio)triphosphate binding to membranes from human neuroblastoma SH-SY5Y cells.</a> Mol. Pharmacol. 47 (4), 848-854 (1995).</li><li>Sittampalam, G. 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=GTP&#947;S+Binding+Assays.">GTP&#947;S Binding Assays.</a> Assay Guidance Manual. , (2004).</li><li>Strange, P. G. <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+the+GTP&#947;S+([35S]GTP&#947;S+and+Eu-GTP&#947;S)+binding+assay+for+analysis+of+ligand+potency+and+efficacy+at+G+protein-coupled+receptors.">Use of the GTP&#947;S ([35S]GTP&#947;S and Eu-GTP&#947;S) binding assay for analysis of ligand potency and efficacy at G protein-coupled receptors.</a> Br. J. Pharmacol. 161 (6), 1238-1249 (2010).</li></ol>

The authors declare no competing interests.

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><tbody><tr><td>DMEM, high glucose, pyruvate, no glutamine</td><td>Thermo Fisher Scientific</td><td>10313021</td><td>Warm in a 37&amp;deg;C water bath before use</td></tr><tr><td>L-glutamine</td><td>Thermo Fisher Scientific</td><td>25030081</td><td>Warm in a 37&amp;deg;C water bath before use</td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>Warm in a 37&amp;deg;C water bath before use</td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Thermo Fisher Scientific</td><td>31985070</td><td>Warm in a 37&amp;deg;C water bath before use</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Thermo Fisher Scientific</td><td>16000044</td><td>Warm in a 37&amp;deg;C water bath before use</td></tr><tr><td>Cell culture 10 cm plate</td><td>Sigma-Aldrich</td><td>CLS430167</td><td></td></tr><tr><td>Lipofectamine 3000 reagent</td><td>Thermo Fisher Scientific</td><td>L3000-008</td><td></td></tr><tr><td>1.6 mL microcentrifuge tubes</td><td>USA Scientific</td><td>1615-5500</td><td></td></tr><tr><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane (Trizma base)</td><td>Thermo Fisher Scientific</td><td>BP152-1</td><td></td></tr><tr><td>ethylene glycol-bis(&amp;beta;-aminoethyl ether)-N,N,N&#039;,N&#039;-tetraacetic acid (EGTA)</td><td>Sigma-Aldrich</td><td>E3889</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>E9884</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S5016</td><td></td></tr><tr><td>cOmplete ULTRA Tablets, Mini, EASYpack Protease Inhibitor Cocktail</td><td>Sigma-Aldrich</td><td>2900</td><td></td></tr><tr><td>DL-Dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>DO632</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Thermo Fisher Scientific</td><td>BP358-1</td><td></td></tr><tr><td>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>M1028-1</td><td></td></tr><tr><td>Pellet pestles motor</td><td>Sigma-Aldrich</td><td>Z359971</td><td></td></tr><tr><td>Pestles</td><td>Bel Art</td><td>F19923-0001</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Affymetrix</td><td>10857</td><td></td></tr><tr><td>[&lt;sup&gt;35&lt;/sup&gt;S]guanosine-5&amp;rsquo;-O-(3-thio)triphosphate ([&lt;sup&gt;35&lt;/sup&gt;S]GTP&amp;gamma;S)&amp;nbsp;</td><td>Perkin Elmer</td><td>NEG030H</td><td></td></tr><tr><td>nonradiolabeled guanosine-5&amp;rsquo;-O-(3-thio)triphosphate (GTP&amp;gamma;S)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>89378</td><td></td></tr><tr><td>guanosine diphosphate (GDP)</td><td>Sigma-Aldrich</td><td>51060</td><td></td></tr><tr><td>Bradford reagent</td><td>Bio-Rad</td><td>5000006</td><td></td></tr><tr><td>UV/VIS spectrophotometer</td><td>Beckman Coulter</td><td>DU640</td><td></td></tr><tr><td>spectrophotometer cuvettes</td><td>USA Scientific</td><td>9090-0460</td><td></td></tr><tr><td>orbital shaker</td><td>Thermo Fisher Scientific</td><td>2314</td><td></td></tr><tr><td>thermomixer</td><td>Eppendorf</td><td>535027903</td><td></td></tr><tr><td>glass fiber filters&amp;nbsp;</td><td>GE Healthcare Life Sciences</td><td>1821-021</td><td></td></tr><tr><td>vacuum filtration apparatus</td><td>Millipore Corporation</td><td>XX2702550</td><td></td></tr><tr><td>desktop microcentrifuge</td><td>Eppendorf</td><td>65717</td><td></td></tr><tr><td>Scintillation counter</td><td>Beckman Coulter</td><td>LS6500</td><td></td></tr><tr><td>scintillation fluid&amp;nbsp;</td><td>Ecoscint A</td><td>LS-273</td><td></td></tr><tr><td>scintillation counter vials</td><td>Beckman Coulter</td><td>592690</td><td></td></tr><tr><td>scintillation vial lids</td><td>Beckman Coulter</td><td>592928</td><td></td></tr><tr><td>Prism 6</td><td>GraphPad Software</td><td>PRISM 6</td><td></td></tr><tr><td>ATP1A1 antibody</td><td>Developmental Studies Hybridoma</td><td>a6F</td><td>1:1000 in 3% BSA</td></tr><tr><td>GAPDH antibody</td><td>EMD Millipore</td><td>CB1001</td><td>1:5000 in 3% BSA</td></tr><tr><td>H2B antibody</td><td>Cell Signaling</td><td>2934S</td><td>1:2500 in 3% BSA</td></tr><tr><td>PDI antibody</td><td>Cell Signaling</td><td>3501S</td><td>1:1000 in 3% BSA</td></tr><tr><td>HA antibody</td><td>Roche</td><td>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 ...

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16.3K Views.  Johns Hopkins University School of Medicine.  Guanosine trifosfaat (GTP) binding is een van de vroegste gebeurtenissen bij G-proteïne-koppelde receptor (GPCR) activatie. Dit protocol beschrijft hoe u specifieke GPCR-ligandinteracties farmacologisch kunt karakteriseren door de binding van het radioactief gemerkte GTP-analoog, [35S] guanosine-5'-O- (3-thio) trifosfaat ([35S] GTPγS) te monitoren in Reactie op een ligand van interesse. 

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

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent &#946;-arrestin2 recruitment assay, the universality of &#946;-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango&#39;s sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient &#946;-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

A Pipeline to Investigate the Structures and Signaling Pathways of Sphingosine 1-Phosphate Receptors

Lysophospholipids (LPLs) are bioactive lipids that include sphingosine 1-phosphate (S1P), lysophosphatidic acid, etc. S1P, a metabolic product of sphingolipids in the cell membrane, is one of the best-characterized LPLs that regulates a variety of cellular physiological responses via signaling pathways mediated by sphingosine 1-phosphate receptors (S1PRs). This implicated that the S1P-S1PRs signaling system is a remarkable potential therapeutic target for disorders, including multiple sclerosis (MS), autoimmune disorders, cancer, inflammation, and even COVID-19. S1PRs, a small subset of the class A G-protein coupled receptor (GPCR) family, are composed of five subtypes: S1PR1, S1PR2, S1PR3, S1PR4, and S1PR5. The lack of detailed structural information, however, impedes the drug discovery targeting S1PRs. Here, we applied the cryo-electron microscopy method to solve the structure of the S1P-S1PRs complex, and elucidated the mechanism of activation, selective drug recognition, and G-protein coupling by using cell-based functional assays. Other lysophospholipid receptors (LPLRs) and GPCRs can also be studied using this strategy.

S1P exerts its diverse physiological effects through the S1P receptors (S1PRs) subfamily. Here, a pipeline is described to expound on the structures and function of S1PRs.

Lysophospholipids (LPLs) are bioactive lipids that include sphingosine 1-phosphate (S1P), lysophosphatidic acid, etc. S1P, a metabolic product of ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Biology

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include well-validated targets for therapeutic intervention. Discovery of lead compounds, including small synthetic molecules, that specifically inhibit the receptor&#39;s function, is an important initial step in drug development and relies on sensitive, specific, and robust cell-based assays. Here, we describe a kinetic cellular assay with a fluorescent readout primarily designed to identify receptor-specific antagonists that inhibit the intracellular Ca2+ release evoked upon the activation of the CXC chemokine receptor 4 (CXCR4) by its endogenous ligand, the CXC chemokine ligand 12 (CXCL12). A key advantage of this method is that it also enables screening of compounds endowed with intrinsic agonistic properties (i.e., compounds eliciting an increase in intracellular Ca2+ concentration in the absence of CXCL12) or compounds modulating the receptor&#39;s function via interaction with allosteric binding sites (i.e., positive and negative allosteric modulators (PAMs and NAMs, respectively)). On the down side, autofluorescent compounds might interfere with the assay&#39;s readout, thereby hampering reliable data interpretation. Most likely this assay can be implemented, with minimal adaptations, as a generic drug discovery assay for many other GPCRs of which the activation leads to a release of intracellular Ca2+.

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

The described cellular assay is designed for the identification of CXC chemokine receptor 4 (CXCR4)-interacting agents that inhibit or stimulate, either competitively or allosterically, the intracellular Ca2+ release initiated by CXCR4 activation.

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include ...

Medicine

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 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 ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

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.

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 ...

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

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