The general allosteric ternary complex model

The term 'allosteric' (from the Greek meaning 'other site') was introduced by Monod et al. (1965) to define binding of ligands to sites on enzymes that were topographically distinct from the substrate-binding site. These accessory binding sites were called 'allosteric sites', in contrast to the substrate-binding (active) site, which was defined as the 'isosteric site'. For GPCRs, the orthosteric site refers to the agonist binding

Figure 213 Allosteric models for the binding of two ligands (A and B) to one receptor (R, R', R", R* and R** represent different conformations). Left: the simplest 'allosteric terenary complex model' assumes that the conformation of the ternary complex is the same regardless of which ligand bound first (Christopoulos and Kenakin, 2002). Right: the more complete sequential 'KNF' model (Koshland et at., 1966) allows the conformation of the ternary complex to depend on whether A or B bound first (reprinted from Fundamental and Clinical Pharmacotogy, 19, Vauquelin, G. and Van Liefde, I., G protein-coupled receptors: a count of 1001 conformations, 45-56, Copyright (2005) Blackwell Publishing).

Figure 213 Allosteric models for the binding of two ligands (A and B) to one receptor (R, R', R", R* and R** represent different conformations). Left: the simplest 'allosteric terenary complex model' assumes that the conformation of the ternary complex is the same regardless of which ligand bound first (Christopoulos and Kenakin, 2002). Right: the more complete sequential 'KNF' model (Koshland et at., 1966) allows the conformation of the ternary complex to depend on whether A or B bound first (reprinted from Fundamental and Clinical Pharmacotogy, 19, Vauquelin, G. and Van Liefde, I., G protein-coupled receptors: a count of 1001 conformations, 45-56, Copyright (2005) Blackwell Publishing).

site on a receptor. Allosteric sites are additional binding sites on a receptor that are topographically distinct (i.e. not overlapping) from the orthosteric site, but that can modulate receptor activity. Allosteric interactions are interactions between two distinct binding sites on the same receptor complex. These interactions involve the transmission of a conformational change across the GPCR from one binding site to another. Hence allosteric interactions are reciprocal in nature: for GPCRs the binding of an allosteric ligand modulates the binding/function of the orthosteric ligand and vice versa. As discussed below, this allosteric modulation can be either negative or positive.

The general allosteric ternary complex model (Figure 213) is the simplest model to describe the binding of two ligands A and B to one receptor to form a ternary ARB complex. A binds to the orthosteric site whereas B, the allosteric modulator, binds to the allosteric site and whatever ligand A does to B, B does to A (Christopoulos and Kenakin, 2002). K-values are equilibrium dissociation constants for binding and a represents a co-operativity factor. It represents the magnitude by which the affinity of one ligand is changed by the other ligand when both are bound to the receptor (Figure 214).

a < 1 reflects positive co-operativity (i.e. the affinity of a ligand increases when the receptor is occupied by the other ligand). Saturation binding curves for A (where receptor occupation is defined as ([AR] + [ARB])/[R])) will shift to the left in the presence of B and a maximal shift will be reached when all receptors are occupied by B. In other words, as B increases, the apparent KD of A will gradually decrease from Ka to aKa.

a = 1 reflects no co-operativity (i.e. the affinity of a ligand is the same for the free receptor and when the receptor is already occupied by the other ligand).

a > 1 reflects negative co-operativity (i.e. the affinity of a ligand decreases when the receptor is occupied by the other ligand). Negative co-operativity between A and B will be manifested as a rightward shift of the binding curve for A.

Figure 214 Saturation binding curves for an orthosteric ligand. The arrow represents the effect of increasing concentrations of a negative allosteric modulator (a = 10), positive allosteric modulator (a = 0.1), or a competitive ligand. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Drug Discovery, 1, Christopoulos, A., Allosteric binding sites on cell-surface receptors: novel targets for drug discovery, 198-210, © (2002).

Figure 214 Saturation binding curves for an orthosteric ligand. The arrow represents the effect of increasing concentrations of a negative allosteric modulator (a = 10), positive allosteric modulator (a = 0.1), or a competitive ligand. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Drug Discovery, 1, Christopoulos, A., Allosteric binding sites on cell-surface receptors: novel targets for drug discovery, 198-210, © (2002).

Whereas the allosteric terenary complex model implies a strict reciprocality between the mandatory effects of orthosteric or allosteric ligands (to reach the same end state R*), the more complete sequential 'KNF' model (Figure 213) allows non-reciprocal modulations. The end state of the receptor will then depend on which ligand bound first.

Exogenous and endogenous allosteric modulators

An increasing number of rather small molecules have been identified as behaving as allosteric modulators at GPCRs (Table 25). However, they do not constitute the only ones.

The formation of a bond between two proteins also causes their conformational change and hence alters their properties. In this vein, G proteins may be regarded as behaving like allosteric GPCR 'ligands' since they affect the agonist-receptor interaction, but do not couple to the same site of the receptor as the agonist. The interaction between agonist binding and G protein coupling is positively co-operative in nature. Agonist binding to the receptor increases its affinity

Table 25 Small molecule allosteric modulators at GPCRs.

GPCR

Allosteric modulators

Adenosine A1

Thieno[2,3-c]pyridine derivatives, 2-amino-3-

heteroaroylthiophenes, 2-aminothiophene-3-carboxylates,

amilorides

Adenosine A3

1H-imidazo[4,5-c]quinolin-4-amine and 3-

(2-pyridinyl)isoquinoline derivatives, amilorides

a1-adrenergic

Conopeptide rho-TIA

a2-adrenergic

SCH-202676, amilorides

P2-adrenergic

Zinc

D1 dopamine

Methylisobutylamiloride, zinc

D2 dopamine

Homocysteine, L-prolyl-L-leucyl-glycinamide and analogues,

methylisobutylamiloride, zinc

5-HT2C serotonin

L-threo-alpha-D-galacto-octopyranoside, PNU-69176E

5-HT3 serotonin

Verapamil, ifenprodi, GYKI-46903, 5-hydroxyindole

and analogues

5-HT7 serotonin

Oleamide

Ml muscarinic

MT7 toxin, KT5720, AC-42

M2 muscarinic

Alkane-bisammonio-type and bispyridinium-type compounds,

NGD-3366, W-84, gallamine

M3 muscarinic

Rapacuronium

M4 muscarinic

WIN 62,577, alcuronium, brucine

GABAB

CGP7930, GS39783

Calcium-sensing

Calindol, NPS R-568

M5 metabotropic glutamate

CDPPB, VU-29, DFB, CPPHA

for the G protein and, reciprocally, agonists display increased affinity for the G protein-coupled receptor.

From the perspective of the GPCR, the orthosteric site is the agonist binding site, whereas for the G protein, the orthosteric site may be defined as the guanine nucleotide binding site on the G subunit. The binding interface of the two proteins constitutes their allosteric site. The binding of GTP to the orthosteric site of the G protein will weaken the affinity of its allosteric site for the receptor. Following the dissociation of the complex, the receptor no longer senses the positive allosteric interaction of the G protein so that its orthosteric site again displays low agonist affinity. This explains the GTP-mediated rightward shift of agonist/labelled antagonist competition binding curves (Figure 44 and 157).

As addressed in previous sections, the classic picture of isolated monomeric GPCRs has given way to models in which they can form dimers and even combine with 'accessory proteins' that may act as partners in signalling events. In all of these instances, the possibility exists for allosterism as a consequence of protein-protein interactions. It is even possible that accessory proteins are required to unmask the pharmacology of specific orphan receptors (i.e. receptors for which the gene product has been identified but not yet the endogenous ligand):

• GPCR homo- and heterodimers have been shown to generate receptor subtypes with a pharmacological profile that is distinct from that of either monomer alone. For the GABAb receptor, heterodimerization between its constituents GABAbR1 and GABABR2 has even been shown to be essential for its ligand recognition and signalling functions. In other instances, homo- or heterodimerization may merely be the consequence of receptor overexpression in recombinant systems and, hence, have no or little physiological significance.

• A particularly well characterized case of GPCR interaction with accessory proteins is the association of the calcitonin receptor-like receptor (CRLR) with single TM-spanning receptor activity modifying proteins (RAMPs). These accessory proteins clearly change the phenotype of the receptor: association with RAMP1 produces a high-affinity CGRP receptor while association with RAMP2 or RAMP3 produces a receptor for adrenomedullin.

• Calcyon, another single TM-spanning protein, has been shown to physically associate with D1 dopamine receptors in neurones and to potentiate their ability to increase the cytosolic Ca2+ concentration, a typical Gq/11-mediated response. Whereas calcyon does not seem to affect the affinity of agonists, it significantly enhances the proportion of the high-affinity state. This finding suggests a complex allosteric interaction involving calcyon, the receptor and the G protein.

Other endogenous allosteric modulators include cations. By interacting with a highly conserved aspartic acid located in TM2, sodium ions can exert an allosteric effect on the binding properties of both agonists and G proteins. Indeed, mutagenesis studies have shown that altering the charge of this single amino acid exerts a global alteration in GPCR conformational states. Other cations have been suggested to allosteri-cally modulate GPCR binding properties by interacting with extracellular amino acid contact points.

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