This work was supported by a National Institutes of Health Grant GM 69829.

1. Measurement of agonist concentration-response curves: no constitutive activity
<ol>
 <li>For estimation of relative agonist Kb values (RAi), a series of at least two agonist concentration-response curves is required. Any in vitro assay for the functional response of a GPCR can be measured provided that the concentration of agonist can be controlled and a single type of receptor mediates the response. Suitable cell-based assays include measurement of cAMP 4,7 and inositolphosphate accumulation 8,9 in a cell line expressing a recombinant receptor. Examples of whole tissue assays include measurement of contraction of smooth muscle 10 or M2 muscarinic receptor- and &beta;1-adrenoceptor-mediated changes in contraction of the field-stimulated rat left atrium 11.</li>
 <li>Choose a series of agonists for analysis including a highly efficacious agonist (e.g., endogenous ligand). In a given experiment, measure complete concentration-response curves for each agonist. If the number of agonists is too large to complete the assay in a single experiment, divide the agonists into subgroups, each consisting of a manageable number of agonists for a single experiment. For each subgroup, measure the concentration-response curve of the highly efficacious agonist together with those of the other agonists in the subgroup (see Figure 3). Repeat the experiment for each subgroup 3 - 6 times, approximately, depending upon the variability of the data.</li>
 <li>For each concentration-response curve, measure the response in the absence of agonist (basal response), and in the presence of increasing concentrations of agonist. Space the agonist concentrations uniformly on a log scale approximately every 0.3 - 0.7 log10 units, covering the range of responses and defining the maximal response (Emax) (see Figure 3). For experiments on cell lines, each measurement is done in triplicate.</li>
 <li>Subtract the basal response from that measured in the presence of each concentration of agonist. The responses plotted in Figure 3 were calculated in this manner.</li>
</ol>
2. Preliminary analysis of agonist concentration-response curves: no constitutive activity
<ol>
 <li>Enter the data from the concentration-response experiments (e.g., Figure 5) into a Data Table in Prism. All of the log agonist concentrations are entered under the column labeled X. The response measurements for the agonist that appears to have the largest Emax value are entered into column A, and those for the other agonist are entered into adjacent lettered columns. Replicate measurements of a concentration-response curves are entered into sub-columns of a given lettered column.</li>
 <li>Select the graph sheet on which the data are plotted, and analyze the data by nonlinear regression analysis using the equation entitled, log(agonist) vs. response - Variable slope (four parameters). Constrain the "Bottom" parameter to zero, and perform the regression analysis. Copy the "Top" (Emax) and log EC50 parameters into an Excel spreadsheet for use in the calculation of initial parameter estimates. The agonist with the largest Emax estimate is designated as the "standard agonist", whereas the other agonists are designated as "test agonists".</li>
 <li>
 Calculate the initial estimate of log &tau;Kobs (LOGR1) as the negative log EC50 value of the standard agonist (-log EC50'). 
 Calculate the initial estimate of the log RAi value of each test agonist (LOGRA) as log {(Top*EC50')/(Top'*EC50)}, in which the parameters of the standard agonist are denoted with an apostrophe. 
Calculate the log affinity constants of the test agonists (LOGK2 - LOGK5) as the negative log of their respective EC50 values.</li>
</ol>
3. Estimation of agonist RAi values using nonlinear regression analysis: no constitutive activity
<ol>
 <li>Enter the data into a Data Table in Prism as described above under 2.1. Make sure that the data for the standard agonist are entered in column A.</li>
 <li>Enter a user-defined equation, "log RAi", into Prism on several lines as shown for the case involving a total of five agonists:
<ul>
<li>&lt;A&gt;P=LOGK1</li>
<li>&lt;A&gt;Q=LOGR</li>
<li>&lt;~A&gt;Q=LOGR+LOGRA</li>
<li>&lt;B&gt;P=LOGK2</li>
<li>&lt;C&gt;P=LOGK3</li>
<li>&lt;D&gt;P=LOGK4</li>
<li>&lt;E&gt;P=LOGK5</li>
<li>Y=Msys/(1+{[(1+10^(X+P))/(10^(X+Q))]^M})</li></ul>
</li>
<li>Enter the initial parameter estimates as follows: 
Enter an arbitrarily low value of 0 for the log affinity constant (LOGK1) of the standard agonist. 
Enter the initial estimate of LOGR and the affinity constants (LOGK2 - LOGK5) and log RAi values (LOGRA) of the test agonists. 
Assign the initial estimate of the transducer slope factor (M) a value of 1.0. 
 Assign the initial estimate for the maximal response of the system (Msys) a value equivalent to the Emax of the standard agonist (Top&prime;).</li>
 <li>Apply the following parameter constraints: LOGK1 constrained to the constant, 0; LOGR1, Msys and M, shared value for all data sets; LOGK2 - LOGK5 and LOGRA, no constraints.</li>
 <li>Initiate nonlinear regression analysis. The results yield the log Kobs and log RAi values of the test agonists. If the standard agonist is a full agonist, then the both the log Kobs and log RAi values are accurate. If the most efficacious agonist (standard agonist) is actually a partial agonist, then the Kobs values of the more efficacious agonists may be overestimated. Nonetheless, the estimates of log RAi are still accurate in this situation.</li>
 <li>If the regression does not converge, the experimenter may wish to plot the theoretical curve defined by the initial parameter estimates. If there are large deviations between the data points and the theoretical curves, check the calculation of the initial parameter estimates. If one or more agonists have Emax values equivalent to that of the standard agonist, it may be necessary to constrain their log Kobs values to 0 for the regression to converge on a solution.</li>
</ol>
4. Measurement of agonist concentration-response curves in cell-based assays exhibiting constitutive receptor activity
<ol>
 <li>For estimation of agonist Kb values in absolute units of M-1, cell-based in vitro assays are used that exhibit constitutive receptor activity. Constitutive receptor activity is defined as the increase in response above the basal level caused by expression of the receptor of interest.</li>
 <li>Choose agonists and design the experiment as described above in section 1.2.</li>
 <li>For each concentration-response curve, measure the response in the absence of agonist in non-transfected cells (basal response) and in cells transfected with the receptor of interest (basal response + constitutive response). Measure the response in the presence of different concentrations of the different agonists as described above in Section 1.3.</li>
 <li>Calculate constitutive receptor activity and the response to each concentration of agonist as the measured response minus the basal response in cells not transfected with the receptor. Figure 6 shows responses to various agonists calculated in this manner for a signaling pathway exhibiting very low constitutive activity.</li>
</ol>

5. Preliminary analysis of agonist concentration-response curves exhibiting constitutive activity
<ol>
 <li>Enter the data (e.g., Figure 6) into a Data Table in Prism as described in Section 3.1, but also enter the response caused by constitutive receptor activity in the appropriate lettered columns corresponding to a log agonist concentration of -20. A large negative logarithm is entered to approximate an agonist concentration of zero.</li>
 <li>Analyze the data as described above in Section 2.2, but with the "Bottom" parameter constrained so that it is "shared for all data sets." Copy the "Top" (Emax), shared "Bottom" and log EC50 parameters into an Excel spreadsheet for use in the calculation of initial parameter estimates.</li>
 <li>Calculate the initial parameter estimates as follows: 
 The log affinity constant of the standard agonist (LOGK1) or any full, test agonist must be determined in separate experiments as described previously 3. 
 Calculate the initial estimate of the log Kobs value of each partial test agonist (i.e., only LOGK2 in this case) as log {(Top'-Top)/(EC50(Top&prime;- Bottom))}, in which "Bottom" denotes the response caused by constitutive receptor activation. 
 Calculate the initial estimate of log Kb (LOGKb) for each agonist as log {Top/(EC50Bottom)}. 
 Calculate the initial estimate of log &tau;sys (LOGTsys) as log {Bottom/(Top&prime; - Bottom)}.</li>
</ol>
6. Estimation of agonist Kb values for responses exhibiting constitutive receptor activity using nonlinear regression analysis
<ol>
<li>Enter the data into a Data Table in Prism as described above under 3.1</li>
<li>Enter a user-defined equation, "Log Kb", into Prism on several lines as shown for the condition of two agonists:
<ul>
<li>&lt;A&gt; LOGKOBS = LOGK1</li>
<li>&lt;B&gt; LOGKOBS = LOGK2</li>
<li>A=(10^(X+LOGKOBS))+1</li>
<li>B=10^(X+LOGTSYS+LOGKB)</li>
<li>C=(10^(LOGTSYS))</li>
<li>Y=Msys/{1+[(A/(B+C))^M]}</li>
</ul>
</li>
<li>Enter the initial parameter estimates as follows: 
 Enter the affinity constants of each agonist (LOGK1 - enter the value determined under step 5.3). 
 Enter the Kobs estimates of each test agonist. 
 Enter the Kb estimates of the agonists. 
 Enter log &tau;sys (LOGTsys) estimate. 
 Assign the transducer slope factor (M) and the maximal response of the system (Msys) values of 1.0 and Top' (Emax of the standard agonist), respectively.</li>
<li>Apply the following parameter constraints: LOGTsys, Msys and M, shared value for all data sets; LOGK2 and LOGKb, no constraints.</li>
<li>Initiate nonlinear regression analysis. The results yield the log Kb of the standard agonist, the log Kobs and log Kb values of partial agonists, and the maximal response of the system (Msys), the &tau;sys value for the free receptor complex (LOGTsys), and the transducer slope factor in the operational model (M).</li>
<li>If the regression does not converge, follow the steps described above under point 3.6.</li>
</ol>
7. Representative Results
Figure 5 shows some of our previously published data on muscarinic agonist-induced phosphoinositide hydrolysis in Chinese hamster ovary cells stably expressing the M3 muscarinic receptor 12. The concentration-response curves of selected muscarinic agonists were measured in this assay. The data were analyzed as described above under Section 3 to estimate the Kb value of each agonist, expressed relative to that of oxotremorine-M. These log RAi estimates are, carbachol, -0.56 &plusmn; 0.063; arecoline, -0.60 &plusmn; 0.074; pilocarpine, -1.20 &plusmn; 0.15; and McN-A-343, -1.92 &plusmn; 0.31. The theoretical curves represent the least-squares fit of the regression equation to the data (Section 3.2). The corresponding estimates of the maximum response of the system (Msys) and the transducer slope factor (M) were 53.9 &plusmn; 1.3 and 1.15 &plusmn; 0.09, respectively. The log Kobs values of the agonists, except that of oxotremorine-M were, carbachol, 5.19 &plusmn; 0.14; arecoline, 5.49 &plusmn; 0.12; pilocarpine, 5.58 &plusmn; 0.16 and McN-A-343, 5.27 &plusmn; 0.33.
Figure 6 shows the results of experiments on muscarinic agonist-stimulated phosphoinositide hydrolysis in HEK 293 cells stably expressing G&alpha;15 3. Transient expression of the M3 muscarinic receptor caused an increase in basal phosphoinositide hydrolysis, which was attributed to constitutive receptor activity. The data were analyzed to estimate the log Kb values of oxotremorine-M and McN-A-343 as described under Section 6. This analysis yielded log Kb values of 8.30 &plusmn; 0.59 and 6.59 &plusmn; 0.77 for oxotremorine-M and McN-A-343, respectively. The theoretical curves represent the least-squares fit of the regression equation to the data (see Section 6.2). The estimates of log &tau;sys, Msys and M were -2.29 &plusmn; 0.59, 95.8 &plusmn; 2.8 and 0.72 &plusmn; 0.08, respectively. The estimate of the Kobs value of McN-A-343 was 5.35 &plusmn; 0.46.
<img alt="Figure 1" src="/files/ftp_upload/3179/3179fig1.jpg" /> 
Figure 1. Relationship between single receptor activity and the average level of activation of the receptor population. Single receptor activity, the theoretical behavior of a single receptor undergoing transitions between active (On) and inactive (Off) states in the presence of an agonist is shown. The affinity constants of the agonist for the active and inactive states are denoted by Kb and Ka, respectively. Population behavior, the theoretical behavior of several receptors undergoing random transitions between active and inactive states in the presence of an agonist is shown. Population average, the average level of receptor activation in a population of receptors in the presence of a specific concentration of agonist is shown. The activation level of the receptor population is known as the stimulus.
<img alt="Figure 2" src="/files/ftp_upload/3179/3179fig2.jpg" /> 
Figure 2. Relationship between the receptor activation function and the efficacy (&#949;) and observed affinity constant (Kobs) of the agonist. Population average, the average level of receptor activation elicited by increasing concentrations of agonist is shown. Receptor activation, the average level of receptor activation is plotted agonist the log concentration of agonist. The negative log concentration of agonist required for half-maximal receptor activation is equivalent to the log observed affinity constant of the agonist (log Kobs). The maximum of the receptor activation function is denoted as efficacy (&#949;).
<img alt="Figure 3" src="/files/ftp_upload/3179/3179fig3.jpg" /> 
Figure 3. Examples of concentration-response curves for the stimulation of [3H]inositolphosphate accumulation by various muscarinic agonists in Chinese hamster ovary cells expressing the human M3 muscarinic receptor. A total of nine agonist concentration-response curves were measured. The experiment can be divided into two parts. For each part, the standard agonist (oxotremorine-M) is always tested together with additional agonists. Thus, two experiments of the type shown in a and b are required to measure the concentration-response curves of nine agonists if only five agonists can be assayed at once. The data are from Ehlert et al. 12.
<img alt="Figure 4" src="/files/ftp_upload/3179/3179fig4.jpg" /> 
Figure 4. Examples of concentration-response curves for stimulation of [3H]inositolphosphate accumulation by various muscarinic agonists in HEK 293 cells expressing the human M2 muscarinic receptor and G&alpha;15. Expression of the M2 receptor caused a 30 - 35% [3H]inositolphosphate response, which was attributed to constitutive receptor activity. The data are from Ehlert et al. 12.
<img alt="Figure 5" src="/files/ftp_upload/3179/3179fig5.jpg" /> 
Figure 5. Muscarinic agonist-mediated phosphoinositide hydrolysis in Chinese hamster ovary cells stably expressing the human M3 muscarinic receptor. The concentration-response curves of selected muscarinic agonists for stimulating phosphoinositide hydrolysis are shown. Mean values from three experiments &plusmn; SEM are shown. The data are from Ehlert et al. 12.
<img alt="Figure 6" src="/files/ftp_upload/3179/3179fig6.jpg" /> 
Figure 6. Oxotremorine-M- and McN-A-343-mediated phosphoinositide hydrolysis in HEK 293 cells expressing G&alpha;15 and the M3 muscarinic receptor. Phosphoinositide hydrolysis is expressed relative to the Emax of oxotremorine-M. In the absence of agonist, phosphoinositide hydrolysis attributed to expression of the M3 receptor accounted for approximately 2% of the response. The means of three experiments &plusmn; SEM are shown. The data are from Ehlert et al. 3.

<ol>
	<li>Stephenson, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modification+of+receptor+theory.">A modification of receptor theory.</a> British Journal of Pharmacology. 11, 379-393 (1956).</li><li>Furchgott, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+b-haloalkylamines+in+the+differentiation+of+receptors+and+in+the+determination+of+dissociation+constants+of+receptor-agonist+complexes.">The use of b-haloalkylamines in the differentiation of receptors and in the determination of dissociation constants of receptor-agonist complexes.</a> Advances in Drug Research. 3, 21-55 (1966).</li><li>Ehlert, F. J., Suga, H., Griffin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+agonism+and+inverse+agonism+in+functional+assays+with+constitutive+activity:+Estimation+of+orthosteric+ligand+affinity+constants+for+active+and+inactive+receptor+states.">Analysis of agonism and inverse agonism in functional assays with constitutive activity: Estimation of orthosteric ligand affinity constants for active and inactive receptor states.</a> J. Pharmacol. Exp. Ther. 33, 671-686 (2011).</li><li>Griffin, M. T., Figueroa, K. W., Liller, S., Ehlert, F. J. <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+Agonist+Activity+at+G+Protein-Coupled+Receptors:+Analysis+of+M2+Muscarinic+Receptor+Signaling+through+Gi/o,Gs,+and+G15.">Estimation of Agonist Activity at G Protein-Coupled Receptors: Analysis of M2 Muscarinic Receptor Signaling through Gi/o,Gs, and G15.</a> J. Pharmacol. Exp. Ther. 321, 1193-1207 (2007).</li><li>Black, J. W., Leff, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operational+models+of+pharmacological+agonism.">Operational models of pharmacological agonism.</a> Proceedings of the Royal Society of LondonSeries B: Biological Sciences. 220, 141-162 (1983).</li><li>Tran, J. A., Chang, A., Matsui, M., Ehlert, F. J. <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+relative+microscopic+affinity+constants+of+agonists+for+the+active+state+of+the+receptor+in+functional+studies+on+M2+and+M3+muscarinic+receptors.">Estimation of relative microscopic affinity constants of agonists for the active state of the receptor in functional studies on M2 and M3 muscarinic receptors.</a> Mol. Pharmacol. 75, 381-396 (2009).</li><li>Schultz, J., Hamprecht, B., Daly, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+adenosine+3':5'-cyclic+monophosphate+in+clonal+glial+cells:+labeling+of+intracellular+adenine+nucleotides+with+radioactive+adenine.">Accumulation of adenosine 3':5'-cyclic monophosphate in clonal glial cells: labeling of intracellular adenine nucleotides with radioactive adenine.</a> Proceedings of the National Academy of Sciences of the United States of America. 69, 1266-1270 (1972).</li><li>Berridge, M. J., Downes, C. P., Hanley, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lithium+amplifies+agonist-dependent+phosphatidylinositol+responses+in+brain+and+salivary+glands.">Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands.</a> Biochemical Journal. 206, 587-595 (1982).</li><li>Kendall, D. A., Hill, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Neurotransmitter Receptor Analysis. , 68-87 (1990).</li><li>Pulido-Rios, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitro+Isolated+Tissue+Functional+Muscarinic+Receptor+Assays.">Vitro Isolated Tissue Functional Muscarinic Receptor Assays.</a> Current Protocols in Pharmacology. 48, 4-15 (2010).</li><li>Kenakin, T., Enna, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Pharmacology. , (2001).</li><li>Ehlert, F. J., Griffin, M. T., Sawyer, G. W., Bailon, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=simple+method+for+estimation+of+agonist+activity+at+receptor+subtypes:+comparison+of+native+and+cloned+M3+muscarinic+receptors+in+guinea+pig+ileum+and+transfected+cells.">simple method for estimation of agonist activity at receptor subtypes: comparison of native and cloned M3 muscarinic receptors in guinea pig ileum and transfected cells.</a> Journal of Pharmacology and Experimental Therapeutics. 289, 981-992 (1999).</li><li>Burstein, E. S., Spalding, T. A., Brann, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology+of+muscarinic+receptor+subtypes+constitutively+activated+by+G+proteins.">Pharmacology of muscarinic receptor subtypes constitutively activated by G proteins.</a> Mol. Pharmacol. 51, 312-319 (1997).</li></ol>

No conflicts of interest declared.

Because our method for estimating RAi (relative Kb value) only requires the measurement of agonist concentration-response curves, our analysis can be done anytime these curves are measured.
If day-to-day variation in the response of the experimental preparation (e.g., cells or tissue) is large, the response measurements of each concentration-response curve can be normalized relative to the "Top" estimate of the standard agonist for each day's experiment....

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.

Watch this Scientific Journal Video about Quantifying Agonist Activity at G Protein-coupled Receptors at JoVE.com

Quantifying Agonist Activity at G Protein-coupled Receptors

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 ...

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18.2K Views.  University of California, Irvine. 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.

Video: Quantifying Agonist Activity at G Protein-coupled Receptors

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

Hui Zhang and Niko G. Gubernator contributed equally to this work.

A new means to measure neurotransmission optically using fluorescent dopamine analogs.

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

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 ...

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 ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...