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</sup...

<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.

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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 조회수.  University of California, Irvine. 활성 상태 작용제의 친화력이 상수 계산하는 방법 ( K B