Overview of Receptor Allosterism
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Abstract
In addition to the orthosteric site, which recognizes endogenous ligands, most G protein?coupled receptors (GPCRs) possess topographically distinct allosteric sites that can be recognized by small molecules and accessory cellular proteins. Ligand binding to allosteric sites promotes a conformational change in the GPCR that can alter orthosteric ligand affinity and/or efficacy. Moreover, there has been an increase in recent years in the identification of allosteric agonists that can directly activate the receptor in the absence of orthosteric ligand. Allosteric sites are attractive therapeutic targets because they can be exploited to achieve modes of selectivity and signaling that are not attainable by orthosteric means. However, an important challenge in this field remains the quantification of the myriad of possible allosteric effects on binding and signaling events. This unit provides an overview on GPCR allosterism and the different pharmacological approaches to understanding allosteric behaviors. Curr. Protoc. Pharmacol. 51:1.21.1?1.21.34. © 2010 by John Wiley & Sons, Inc.
Keywords: allosteric ternary complex model; bitopic; G protein?coupled receptor; modulator; orthosteric; radioligand binding
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PDF or HTML at Wiley Online LibraryTable of Contents
- Historical Perspective
- The Many Shades of Receptor Allosterism
- An Allosteric Ternary Complex Model (ATCM) and Its Variants
- Detecting Allosteric Interactions
- Classes of Allosteric Ligands
- Utility of Allosteric Ligands
- Summary
- Literature Cited
- Figures
- Tables
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Figure 1.21.1 Allosterism between a receptor and its G protein: the effects of GTP on the competition between [3 H] N ‐methylscopolamine and the agonist, carbachol, at the muscarinic acetylcholine M2 receptor in rat myocardial membranes. Competition is measured in the absence (▪) or presence of GTP at concentrations of 0.1 µM (□), 0.3 µM (▴), 1 µM (Δ), 10 µM (•), or 100 µM (○). The data was taken from Ehlert and Rathbun (). View Image -
Figure 1.21.2 Effect of: (A ) negative allosteric modulator, (B ) positive allosteric modulator, or (C ) a competitive antagonist on orthosteric ligand‐receptor occupancy (ρA ). For all the simulations, p K A = 6 and p K B = 9. The modulator, B, modifies orthosteric ligand affinity to a limit determined by the cooperativity factor (α) that characterizes the interaction between allosteric and orthosteric sites. In these examples, ligand affinity is either diminished (A) or enhanced (B) by a factor of 10. In contrast, simple competitive interactions (C) are characterized by mutually exclusive binding of the two ligands for the same site and thus they allow for a theoretically limitless dextral shift of orthosteric ligand occupancy. View Image -
Figure 1.21.3 The effects of increasing concentrations of an allosteric modulator on the binding of a fixed concentration of radiolabeled orthosteric ligand ([undefined]). For all the simulations, p K A = 6 and p K B = 9. Panel A shows the saturation‐binding profile of the ligand [undefined] in the absence or presence of increasing concentrations of positive or negative modulator. The solid circles on the control saturation‐binding isotherm of [undefined] indicate three arbitrarily chosen levels of occupancy (denoted L, I, and H) by the radioligand. The lower two panels (B, C ) illustrate the effect of a negative (B) or positive (C) allosteric modulator on the binding of [undefined] at each of these three initial occupancy levels. The insets to each panel show the same data normalized as a percentage of the control level of binding. View Image -
Figure 1.21.4 Effect of a negative (A ) or positive (B ) allosteric modulator that does not alter receptor signaling capacity on the hyperbolic concentration‐response curve of an orthosteric ligand, A. Curves were simulated according to Equation with p K A = 6, p K B = 7, τA = 10, E m = 100, n = 1. Cooperativity factors (α) are indicated in each panel. Under these conditions, the effect of allosteric modulation on orthosteric ligand responses is identical to the effect on orthosteric ligand occupancy. View Image -
Figure 1.21.5 Effect of an allosteric modulator that completely suppresses orthosteric ligand efficacy (β = 0). Concentration‐response curves were simulated using Equation . The following parameters were used: p K A = 6, p K B = 7, E m = 100, n = 1. The simulations show the effect of increasing agonist efficacy (τA ) on the ability of a modulator to depress the response to agonist, in panel A τA = 10, in panel B τA = 100. View Image -
Figure 1.21.6 Effect of an allosteric ligand that has the capacity to modulate both affinity and efficacy. For all simulations Equation was used where p K A = 6, p K B = 7, n = 1, and E m = 100. The upper panels simulate the effect of a negative modulator of affinity (α = 0.1) that is either a positive (A ; β = 3) or negative (B and C ; β = 0.3) modulator of orthosteric ligand efficacy. The lower panels simulate the effect of a positive allosteric modulator of affinity (α = 10), which is either a positive (D and F ; β = 3) or negative (E ; β = 0.3) modulator of orthosteric ligand efficacy. Panels C and F illustrate the influence of changing agonist coupling efficiency (logτA = 2 compared to 0.1 for panels A, B, D, E). View Image -
Figure 1.21.7 Effect of an allosteric agonist that has the ability to modulate orthosteric agonist affinity and efficacy. For all simulations Equation was used where p K A = 6, p K B = 7, n = 1, and E m = 100. Panels A and B simulate the behavior of an allosteric agonist that is also a negative modulator of orthosteric affinity and efficacy (α = 0.1, β = 0.3). Panels C and D simulate the behavior of an allosteric agonist that is also a positive allosteric modulator of both affinity and efficacy (α = 10, β = 3). In panels B and D the coupling efficiency of both orthosteric and allosteric agonists is ten times higher (τA = 100, τB = 3) than in panels A and C (τA = 10, τB = 0.3). It is apparent that the coupling efficiency of the system under investigation can have a profound effect on the window to visualize allosteric modulation between two agonists. View Image -
Figure 1.21.8 Allosteric modulation by oleamide of the saturation binding of [3 H]5‐HT in HeLa cell membranes transiently transfected with the 5‐HT7 receptor. (A ) Radioligand saturation‐binding curves obtained in presence of the following concentrations of oleamide: 0 (▪), 0.1 nM (□), 10 nM (▴), 30 nM (▵), 100 nM (•), 300 nM (○), and 1 µM (♦). (B ) Effect of oleamide on the ratio of [3 H]5‐HT K D values (“affinity‐shift”) determined in the presence or absence of the modulator. The dashed line shows the predicted behavior of a competitive antagonist. The data was taken from Hedlund et al. (). View Image -
Figure 1.21.9 Effect of the allosteric modulators gallamine and alcuronium on the binding of the orthosteric antagonist, [3 H] N ‐methylscopolamine, at M2 muscarinic acetylcholine receptors in guinea pig atrial membranes. (A ) Negative cooperativity between gallamine and two different concentrations of the radioligand: 0.2 nM (•; K A ) and 1 nM (○; 5 × K A ). (B ) Positive cooperativity between alcuronium and 0.1 nM radioligand. Data were fitted to Equation to derive the parameter estimates shown in the figure. View Image -
Figure 1.21.10 Mixed modes of allosteric/competitive interactions. (A ) Inhibition of [3 H]spiperone binding by methylisobutylamiloride (MIA) at dopamine D2 receptors in membranes from Ltk59 cells. The dotted line corresponds to a curve fit based on the simple allosteric ternary complex model (Eq. ) or to a simple model of competitive interaction. The solid line denotes the fit of the data to a model allowing for the allosteric modulator to recognize both orthosteric and allosteric sites. Data taken from Hoare and Strange (). (B ) Enhancement and inhibition of the binding of the agonist, [3 H] N6 ‐cyclohexyladenosine ([3 H]CHA) by the modulator PD 81,723 at the A1 adenosine receptor in rat brain membranes. Data taken from Bruns and Fergus (). (C ) Simulations based on a model of concomitant orthosteric and allosteric binding by an allosteric modulator (Eqs. and ). The following parameters were used p K A = 4.6, p K B1 = p K B2 = 4.7, α = 0.25, β = 27, Log[A] = –5. (D ) Simulations based on the same model, but with the following parameters p K A = 9, p K B1 = p K B2 = 5, α = 7, β = 1, Log[A] = −9. View Image -
Figure 1.21.11 Effects of allosteric modulators on orthosteric ligand dissociation kinetics. (A ) Enhancement of the dissociation rate of [3 H]yohimbine from the human α2A receptor expressed in CHO cell membranes by the modulator 5‐( N ‐ethyl‐ N ‐isopropyl)‐amiloride (EPA). Data taken from Leppik et al. (). (B ) Slowing of the dissociation rate of [3 H]CHA from the adenosine A1 receptor in rat brain membranes by the modulator PD 117,975. Data taken from Bruns and Fergus (). View Image -
Figure 1.21.12 Neutral cooperativity between [3 H] N ‐methylscopolamine and the modulator, Tetra‐W84 at the M2 muscarinic acetylcholine receptor. Increasing concentrations of modulator are able to decrease the rate of radioligand association (▪) and dissociation (□), thus revealing the allosteric nature of the interaction. However, because the kinetics of the radioligand are influenced over similar concentration ranges and to similar extents, equilibrium‐binding studies show minimal effects on levels of radioligand binding (○). Data taken from Kostenis and Mohr (). View Image -
Figure 1.21.13 Allosteric modulation under non‐equilibrium conditions. Orthosteric radioligand binding was simulated for a positive allosteric modulator (A and C ; α = 10) or a negative allosteric modulator (B and D ; α = 0.1) using Equations ‐ and the following parameters: p K A = 6, p K B = 7, k offA = 0.5 min−1 , k offAB = 0 min−1 , Log[A] = −7 (A and B only). The curves represent the concentration‐occupancy relationship for the interaction at the different times (hours) shown in the figure. It is evident that allosteric modulators may slow the kinetics of the system to such an extent that equilibrium is unachievable during the time course of the experiment, thus yielding complex binding curves. The simulations in panels C and D illustrate the influence of radioligand concentration ([A] = K A , 10 × K A or 100 × K A ) on the attainment of equilibrium within 1 hr. View Image -
Figure 1.21.14 Effect of non‐equilibrium conditions on allosteric modulation of [3 H] N ‐methylscopolamine binding by gallamine and alcuronium at M2 muscarinic acetylcholine receptors. Incubations were for 60 min (gallamine) or 90 min (alcuronium). Note the complex curve profiles due to lack of equilibration of radioligand in the presence of high concentrations of each modulator. Data taken from Avlani et al. (). View Image -
Figure 1.21.15 Effect of the allosteric modulator, gallamine, on the ability of acetylcholine to inhibit the electrically evoked contractions of the guinea pig left atrium. (A ) Effects of acetylcholine (ACh) in the absence (▪) or presence of gallamine at the following concentrations: 10 µM (▴), 30 µM (▾), 100 µM (♦), 300 µM (□), and 500 µM (•). All experiments were conducted in the presence of the cholinesterase inhibitor, diisopropylfluorophosphate. (B ) Schild plot of the data shown in A. The solid line (slope = 1) denotes the behavior expected for a competitive antagonist, while the dashed line shows the best‐fit linear regression (and associated slope factor) through the points. The curve through the points and associated parameter estimates represent the fit of the allosteric model (Eq. ) to the data. View Image -
Figure 1.21.16 Potential error in the pharmacological estimation of the cooperativity factor from a functional receptor assay. The figure shows the Schild plots based on simulated data using Equation (p K A = 6, LogτA = 0.1) and the parameter values shown in the figure. It can be seen that the correct estimate of α (0.1) is only obtained when the allosteric modulator does not change signaling capacity (β = 1). Points were fitted to the allosteric model (Eq. ). The dashed line denotes the behavior expected for a competitive antagonist. View Image -
Figure 1.21.17 Application of an operational model of allosterism to the interaction between ACh and LY2033298 at M4 muscarinic ACh receptors. Data represent receptor‐mediated [35 S]GTPγS binding to activated G proteins in membranes stably expressing the human M4 muscarinic receptor. Data were fitted to Equation to obtain the parameters shown in the figure. View Image
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