K2 il

(inactive)

(active)

Figure 173 In the 'conformational selection model' ligands may have different affinities for the inactive (R) and the active (R*) receptor states.

higher affinity for R*. The equilibrium dissociation constant for the transition between the two forms of the receptor (M) is very high since the great majority of receptors are inactive in the absence of ligand. Nevertheless, this model allows unoccupied receptors to produce a small stimulus. Ligands are able to bind both to R and R* with the 'microscopic' equilibrium dissociation constants K and K2, respectively:

In this model, agonists can be discriminated from antagonists on the basis of differences between their binding affinities for the active and non-active receptors. This model also provides an explanation for the existence of so-called 'inverse agonists':

• Antagonists are supposed to bind with equal affinity to both receptor conformations (i.e. Kj = K2); the [R*]/[R] ratio remains the same as in the basal situation.

• Agonists bind with higher affinity to R* as compared to R (i.e. K > K2) so that the whole equilibrium will be pulled to the right, resulting in an increase in the [R*]/[R] ratio. The Kj/K2 ratio is higher for full agonists than for partial agonists.

Figure 174 Conformational selection model for noradrenaline-induced p2-adrenergic receptor activation (Gether and Kobilka, 1998, reproduced by permission of the American Society for Biochemistry and Molecular Biology).

Conformational Selection

Figure 174 Conformational selection model for noradrenaline-induced p2-adrenergic receptor activation (Gether and Kobilka, 1998, reproduced by permission of the American Society for Biochemistry and Molecular Biology).

Figure 175 A strict receptor two-state model that integrates 'induced fit' and 'conformational fit'. Reproduced from Krumins, A. M. and Barber, R. (1997) Molecular Pharmacology, 52, 144-154, with permission from the American Society for Pharmacology and Experimental Therapeutics.

• Inverse agonists bind with higher affinity to R compared to R* (i.e. K < K2) so that the whole equilibrium will be pulled to the left, resulting in a decrease in the [R*]/[R] ratio.

In general, it is useful to think of conformational induction and conformational selection, but it is unclear which is the predominant one for agonism. The terenary complex model (De Lean et al., 1980) integrates 'induced fit' and 'conformational selection'. The slightly more complex 'strict two-state model' (Krumins et al., 1997) (Figure 175) also refers to the receptor being in an active conformation (R*) in order to couple to the G protein. In fact, conformational selection and confor-mational induction represent two extremes of this model. This model makes it possible for the receptor to:

• Be in the active conformation even when not coupled to a G protein.

• Couple to a G protein even in the absence of agonist.

Figure 176 The 'extended ternary complex model' as initially proposed by Samama et al. (1993) implies the existence of three or more receptor states (unless a and p = 1) (Samama et al., 1993, with permission from the American Society for Biochemistry and Molecular Biology).

By increasing the number of receptors present in the system, the number of spontaneously active receptors can be increased until a threshold is attained where the resulting response from the spontaneously formed R*G species can be observed. This explains why constitutive receptor activity is often observed in recombinant systems in where receptors are overexpressed.

The currently most widely accepted model for GPCR activation is the 'extended ternary complex model' (Samama et al., 1993) (Figure 176). This model has often been referred to as the 'two-state model'. However, it must be emphasised that in a strict two-state model (Figure 175) R and R* represent uniquely defined conformational states. This means that the affinity of G for R is identical to that for HR (K2) and its affinity for R* is identical to that for HR* (K3) (Krumins and Barber, 1997). In the 'extended ternary complex model' different affinities are used for the binding of H to the R* and the R*G states. This means that a different conformation is assumed for R* when bound and when not bound to a G protein. Since there are at least three states, the 'extended ternary complex model' is actually a multistate model.

The 'extended ternary complex model' model only allows for the active receptor to interact with a G protein. However, some sparse experimental data also suggest that the inactive receptor is also capable of coupling to G proteins, giving rise to inactive RG and ARG complexes. This has culminated in the development of the even more complex, cubic ternary complex model (Weiss et al., 1996) (Figure 177). Obviously, this model carries so many parameters that it is no longer possible to estimate them based on experimental observations.

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