Quantifying Agonist Activity at G Protein-Coupled Receptors

Abstract

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 (ε) (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 (τ). The estimate of τKobs 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 (τsys). In this case, the Kb value of an agonist is equivalent to τKobs/τsys 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. Video LinkThe , 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 β1-adrenoceptor-mediated...

Biased Type 1 Cannabinoid Receptor Signaling Influences Neuronal Viability in a Cell Culture Model of Huntington Disease

“…Concentration-response curves for ERK, BRET 2 (CB 1 /b-arrestin1), CREB, phospholipase C (PLC)b3, and Akt are presented as % of WIN E max in STHdh Q7/Q7 cells (Griffin et al, 2007). Concentration-response curves were fit to nonlinear regression with variable slope (four-parameter) model to determine pEC 50 and E max (Table 1), or global nonlinear regression using the operational model (Black and Leff, 1983;Ehlert et al, 2011;Kenakin et al, 2012) (eq. 1) to estimate the transduction coefficient [logR (t/K A )], change in transducer coefficient relative to the reference ligand (DlogR), and bias factor (DDlogR) (Prism v. 5.0, GraphPad Software Inc., San Diego, CA), as indicated.…”

Estimation of ligand affinity constants for receptor states in functional studies involving the allosteric modulation of G protein-coupled receptors: Implications for ligand bias

“…RA i is also equivalent to the corresponding ratio of K b values. The theoretical basis for this estimate was first described by Ehlert et al (1999) using a response clamp analysis (null method) as well as in subsequent publications where explicit methods on how to calculate this parameter are given (Ehlert, 2008; Ehlert, et al, 2011c; Griffin, et al, 2007). A similar method for estimating the same parameter has been recently described by Kenakin and coworkers (2012).…”

“…Equations 31 and 32 can also be used to estimate the RA i values of both orthosteric and allosteric ligands when the latter are tested in the absence of other ligands (Ehlert, 2008; Ehlert, et al, 2011c; Figueroa, et al, 2008; Griffin, et al, 2007). They are also valid for the case of reduced receptor expression with no constitutive activity.…”

Intrinsic relative activities of κ opioid agonists in activating Gα proteins and internalizing receptor: Differences between human and mouse receptors

“…Intrinsic reactive activity ( RAi ) of each agonist was estimated by global nonlinear regression analysis using the following two equations (Griffin et al, 2007; Ehlert, 2008; Ehlert et al, 2011b): $$y=\frac{M_{\text{italicsys}}}{1+{true(\frac{1+{10}^{\text{log}X\prime +\text{log}K\prime }}{{10}^{\text{log}X\prime +\text{log}R\prime }}true)}^m}$$ $$y=\frac{M_{\text{italicsys}}}{1+{true(\frac{1+{10}^{\text{log}X\prime +\text{log}K}}{{10}^{\text{log}X+\text{log}R\prime +\log R{A}_i}}true)}^m}$$ The parameters of the standard agonist [dynorphin A (1–17)] are denoted with an apostrophe. In these equations, y represents the measured response, M sys , the maximal response of the signaling pathway, m , the transducer slope factor, X , the concentration of agonist, and K , the observed affinity constant of the agonist (inverse concentration units, e.g., M −1 ).…”

“…We used N2a cells transfected with the hKOP receptor or mKOP receptor and performed [ 35 S]GTPγS binding as a measure of G protein activation and the on-cell western (OCW) assay as a measure of β-arrestin-mediated receptor internalization. We then used an approach, originally developed and refined by Ehlert and colleagues (Griffin et al, 2007; Ehlert, 2008; Ehlert et al, 2011b), to estimate the relative affinity constant of an agonist for the active receptor state that elicits the response [intrinsic relative activity ( RA i )]. We used this analysis to detect differences in ligand affinity for the active receptor states that engage G proteins and β-arrestins following receptor activation.…”