Receptor complexes ternary complex models

A classic example of where definitive experimental data necessitated refinement and extension of a model of drug-receptor interaction involved the discovery of constitutive receptor activity in GPCR systems. The state of the art model before this finding was the ternary complex model for GPCRs, a model that cannot accommodate ligand-independent (constitutive) receptor activity. 

The resulting modification is called the extended ternary complex model [3], which describes the spontaneous formation of active state receptor ([Ra]) from an inactive state receptor ([RJ) according to an allosteric constant (L = [Ra]/[RJ). The active state receptor can form a complex with G-protein ([G]) spontaneously to form RaG, or agonist activation can induce formation of a ternary complex ARaG ... 

The extended ternary complex model can take into account the phenomenon of constitutive receptor activity. In genetically engineered systems where receptors can be expressed in high density, Costa and Herz [2] noted that high levels of receptor expression uncovered the existence of a population of spontaneously active receptors and that these receptors produce an elevated basal response in the system. The relevant factor is the ratio of receptors and G-proteins (i.e., elevated levels of receptor cannot yield constitutive activity in the absence of adequate amounts of G-protein, and vice versa). Constitutive activity (due to the [RaG] species) in the absence of ligand ([A] = 0) is expressed as... 

While the extended ternary complex model accounts for the presence of constitutive receptor activity in the absence of ligands, it is thermodynamically incomplete from the standpoint of the interaction of receptor and G-protein species. Specifically, it must be possible from a thermodynamic point of view for the inactive state receptor (ligand bound and unbound) to interact with G-proteins. The cubic ternary complex model accommodates this possibility [23-25]. From a practical point of view, it allows for the potential of receptors (whether unbound or bound by inverse agonists) to sequester G-proteins into a nonsignaling state. 

A schematic representation of receptor systems in terms of the cubic ternary complex model is shown in Figure 3.13. The amount of signaling species (as a fraction of total receptor) as defined by the cubic ternary complex model see Section 3.13.8 is expressed as... 

There are some specific differences between the cubic and extended ternary complex models in terms of predictions of system and drug behavior. The first is that the receptor, either ligand bound or not bound, can form a complex with the G-protein and that this complex need not signal (i.e., [ARiG] and [RjG]). Under these circumstances an inverse agonist (one that stabilizes the inactive state of the receptor) theoretically can form inactive ternary complexes and thus sequester G-proteins away from signaling pathways. There is evidence that this can occur with cannabi-noid receptor [26]. The cubic ternary complex model also... 

FIGURE 3.13 Major components of the cubic ternary complex model [25-27]. The major difference between this model and the extended ternary complex model is the potential for formation of the [ARjG] complex and the [RiG] complex, both receptor/ G-protein complexes that do not induce dissociation of G-protein subunits and subsequent response. Efficacy terms in this model are a, y, and 5. 

The ternary complex model followed by the extended ternary complex model were devised to describe the action of drugs on G-protein-coupled receptors. 

The cubic ternary complex model considers receptors and G-proteins as a synoptic system with some interactions that do not lead to visible activation. 

The extended ternary complex model [23] was conceived after it was clear that receptors could spontaneously activate G-proteins in the absence of agonist. It is an amalgam of the ternary complex model [12] and two-state theory that allows proteins to spontaneously exist in two conformations, each having different properties with respect to other proteins and to ligands. Thus, two receptor species are described [Ra] (active state receptor able to activate G-proteins) and [RJ (inactive state receptors). These coexist according to an allosteric constant (L = [Ra]/[Ri]) ... 

Samama, P., Cotecchia, S., Costa, T., and Lefkowitz, R. J. (1993). A mutation-induced activated state of the p2-adrener-gic receptor Extending the ternary complex model. J. Biol. Chem. 268 4625-4636. 

Cubic ternary complex model, a molecular model (J. Their. Biol 178, 151-167, 1996a 178, 169-182, 1996b 181, 381-397, 1996c) describing the coexistence of two receptor states that can interact with both G-proteins and ligands. The receptor/G-protein complexes may or may not produce a physiological response see Chapter 3.11. 

Extended ternary complex model, a modification of the original ternary complex model for GPCRs (J. Biol. Chem. 268, 4625-4636, 1993) in which the receptor is allowed to spontaneously form an active state that can then couple to G-proteins and produce a physiological response due to constitutive activity. 

Ternary complex (model), this model describes the formation of a complex among a ligand (usually an agonist), a receptor, and a G-protein. Originally described by De Lean and colleagues (J. Biol. Chem. 255, 7108-7117, 1980), it has been modified to include other receptor behaviors (see Chapters 3.8 to 3.11), such as constitutive receptor activity. 

One current model of G-protein receptor activation is the allosteric ternary complex model of Lefkowitz and Costa. The agonist, receptor and G-protein must combine to... 

However, based on the concept that GPCRs are able to adopt a variety of conformations, an extended model can also be described, as shown in Figure 2. In this extended cubic ternary complex model of receptor activation and modulation, the receptor can interconvert between an active (R) and an inactive conformation (R), each with a different... 

Figure 2 Representation of a "cubic ternary complex" model of allosteric interaction R, the inactive state of the receptor R, the active state of the receptor A, ligand X, allosteric agent. (From Ref. 14.)...

An extended (seven-sided) ternary complex model (Fig. 2B) was proposed by Samama et al. (1993) to accommodate mutant receptors that exhibited constitutive activity and to link receptor affinity with efficacy. This model includes the isomerization of the receptor between two conformational states, inactive (R) and active (R ), and only allows for the active R conformational state to interact with the G protein. Conceptually, the model allows for the receptor to toggle between on and off states where ligand or G protein manipulates the population size of these two conformational states, rather than affecting the activation strength of a particular conformational state. The different types of ligands influence... 

A measurement system that is able to quantitatively determine the interactions of receptor and G protein has the potential for more detailed testing of ternary complex models. The soluble receptor systems, ([l AR and FPR) described in Section II, allow for the direct and quantitative evaluation of receptor and G protein interactions (Simons et al, 2003, 2004). Soluble receptors allow access to both the extracellular ligandbinding site and the intracellular G protein-binding site of the receptor. As the site densities on the particles are typically lower than those that support rebinding (Goldstein et al, 1989), simple three-dimensional concentrations are appropriate for the components. Thus, by applying molar units for all the reaction components in the definitions listed in Fig. 2A, the units for the equilibrium dissociation constants are molar, not moles per square meter as for membrane-bound receptor interactions. These assemblies are also suitable for kinetic analysis of ternary complex disassembly. 

Fig. 3. Experimental dose-response data on G-beads from previous work (Simons et al, 2003, 2004) fitted to simulations of the ternary complex model including soluble G protein (Fig. 1C). The inclusion of soluble G protein in the model (Fig. 1C) is required due to the presence of extra G protein from the solubilized receptors and without which resulted in simulations that overestimated bead-bound receptors. Note that the same equilibrium dissociation constant values were used for the interactions with G protein on bead as with soluble G protein (Gtotbead and Gtots0l). Although the individual kinetic reaction rate constants for the interactions with soluble G protein might be faster than those for the bead-bound G protein, their ratios (the equilibrium dissociation constants) are expected to remain the same. The calibrated GFP per bead as...

The ternary complex model with both bead-bound as well as soluble G proteins (Fig. 2C) reproduces the experimental data from the G-beads (Fig. 3). Because the amount of precoupled receptors on beads at even the highest concentrations of receptors (up to 100 nM) is insignificant, a low... 

The ternary complex model allows for cooperative interactions among ligand, receptor, and G protein as defined by the cooperativity factor a. With a lower limit of Kg established from the levels of RG precoupling, the comparisons of K,.A with K l indicate for both / 2AR and FPR that precoupled receptors (RG) would exhibit higher affinity for an agonist... 

Simulations of ternary complex model were performed with total receptor concentration of 30 nM, soluble G protein concentration of 100 nM for /LAR or 200 nM for FPR, and bead-bound G protein concentration of 0.6 nM per experimental conditions. Values for Aa were fixed from previously experimental values see receptor affinity in Table I (Simons et al, 2003, 2004). Best fits for all partial and full agonists were achieved with the value of Kg fixed at 5 x 10-6 M (log Kg = —5.3), which resulted in insignificant amount of precoupled receptor (RG) in the absence of ligand. 

De Lean, A., Stadel, J. M., and Lefkowitz, R. J. (1980). A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled beta-adrenergic receptor./. Biol. Chem. 255, 7108-7117. 

Receptors, like all proteins, are known to spontaneously adopt a variety of conformations, and current concepts of receptor function (e.g., the extended and cubic ternary complex models) are based on the allosteric transitions between multiple conformational states. Some of these conformations are called active because they are able to interact with cellular signal transduction mechanisms (and are the basis of constitutive receptor activity ). If one assumes that different receptor conformations might present different intracellular portions of a... 

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