With synoptic GPCR systems, G protein binding can greatly modify the interaction of ligands with receptors, that is, the observed affinities may be modified by the influence of the G protein. For example, if the ligand alters the affinity of the receptor for G proteins, then the observed overall affinity will be an amalgam of ligand affinity for the receptor and the degree of modification of G protein affinity (referred to as receptor isomerization). Thus the binding of ligand [A] to receptor [R] and subsequent isomerization to a receptor state R* is shown in the scheme below:
where Ka is the equilibrium dissociation constant of the ligand-receptor complex (1/affinity) and ç is a term quantifying the ability of the ligand to induce receptor isomerization (conversion from R to R*). In such a system, the observed affinity of A for the receptor (denoted Kobs) is augmented by the ability of the ligand to isomerize the receptor to a different species (Colquhoun 1985):
Thus, if the ligand isomerizes the receptor, the observed affinity of the ligand for the receptor will be greater than the affinity of [A] for R (the observed equilibrium dissociation constant ofthe agonist-receptor complex will be <Ka). This system modification is affected by the relative stoichiometry between receptor and G protein. For example, the observed affinity of a ligand for a receptor, in terms of the ETC model, is given as:
As can be seen from equation (8), there are efficacy terms (namely a and y) which dictate the observed potency of the ligand, that is, the change of affinity of the receptor upon ligand binding and the propensity of the ligand to cause formation of the active state receptor Ra.
There are two practical ramifications of these effects. The first is that, observed affinity maybe a result of ligand affinity and efficacy and thus may change in different systems due to differences in receptor/G protein relative stoichiometry (Kenakin 1997a). This is a problem when system independent measures of affinity are required for prediction of therapeutic effect. There is also the possibility, especially in engineered recombinant receptor systems, that system relative stoichiometry (receptor/G protein ratios) can confound observation of characteristic binding patterns and lead to complex binding curves (Kenakin 1997a). For example, Fig. 8.4a shows competitive displacement curves of an antagonist radioligand by a non-radioactive agonist ligand systems with varying but limiting amounts of G protein.
Fig. 8.4 Complex binding kinetics for stoichiometrically deficient (a) or oligomeric binding (b) systems. a. Displacement of an antagonist radioligand by an agonist radioligand in four different systems of varying amount of G protein available for high affinity binding. Numbers next to the curves correspond to the amount of G protein expressed as a percentage of the amount of receptor. (b) Displacement of a submaximal amount of tracer radioligand by concentrations of the same non-radioactive ligand in a system where the receptor forms a dimer and the binding of the ligand favours dimerization and thus enhances the affinity of the receptor for the ligand. Numbers refer to various concentrations of radioligand (ordinate values normalized).
Finally, GPCRs are known to form homodimers (Mukhopadhyay et al. 2000; Cvejic and Devi 1997, 2000; Jordan et al. 2000; Hebert et al. 1996; Angers 2000; Zeng et al. 2000), heterodimers (Pauwells et al. 1998; Benkirane et al. 1997; White et al. 1998; Jones et al. 1998; Marshall et al. 1999; Rocheville et al. 2000) and higher order oligomers (Kuhman et al. 2000) (Chapter 1) and this also can lead to complex binding phenomena. Figure 8.4b shows displacement of a radioligand which interacts with a GPCR dimer by ligands with differing propensity to affect the dimerization process (Kenakin 2001b). This figure shows a system whereby the receptor forms a dimer and the binding of the ligand to that dimer is enhanced. An interesting effect is created whereby the addition of non-radioligand actually increases radioligand binding before it decreases it within a certain concentration range (see Fig. 8.4b).
The measurement of the ability of ligands to isomerize the receptor to a form that binds to G proteins can be a measure of ligand efficacy. Thus, the more a ligand facilitates the conversion from R to R* (see scheme 6) then the greater is the ligand efficacy; in essence the magnitude of the term ç (equation (7)) corresponds to the magnitude of the ligand efficacy. Experimentally, in binding assays, the affinity of the ligand is measured in the binding assay as a displacement curve under conditions where G protein coupling can occur (observed affinity according to equation (7)). To measure efficacy, the affinity then is measured again under conditions whereby G protein binding is cancelled. This latter condition can be achieved with an excess amount of unhydolyzable analogue of GTP in the assay to prevent accumulation of the ARaG complex (see Fig. 8.1). Under these conditions, a measure of Ka (affinity with no efficacy modification) is obtained. The ratio of these affinities (referred to as the 'GTP-shift') represents the ability of the ligand to induce G protein coupling (i.e. efficacy). Within the ETC
and CTC model, the relative GTP-shift for two agonists designated A and B is represented by:
rTn [1 + «AL + ya[G]/Kg(1 + 8a«apL)](1 + «bL + yb[G]/Kg)
[1 + «bL + yb[G]/Kg(1 + 8b«bpL)](1 + «aL + ya[G]/Kg)
Note that under the conditions of non-limiting G protein, the magnitude of the GTP-shift depends upon efficacy terms a, y, and 8. It should be pointed out that GTP-shift experiments are kinetic in nature and depend upon the rate of GDP-GTP exchange. For some systems this can be quite slow and thus incomplete cancellation of G protein coupling can occur.
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