Constitutively active GPCR systems

It can be seen intuitively from both the ETC and CTC models that active state receptors can form spontaneously according to the magnitude of the allosteric constant L. When this occurs to the extent that a response is observed, the system is referred to as being constitutively active. Thus, the system itself emanates an elevated basal activity resulting from spontaneous receptor activation. Experimentally, constitutive GPCR systems can be engineered through over-expression of receptors (Whaley et al. 1994; Barker et al. 1994; Van Sande et al. 1995; Chen etal. 2000), G proteins (Senogles et al. 1990), point mutation (Kjelsberg et al. 1992; Scheer et al. 1996, 1997; Porter et al. 1996) and changes in biochemical milieu (i.e. removal of Na+, Costa and Herz 1989; Tian et al. 1994). Such systems then can detect positive agonism (providing the system maximum is not attained) and, in fact, are more sensitive to positive agonists than are quiescent (non-constitutively active) systems. The effects of a positive agonist on systems of increasing amounts of constitutive activity are shown in Fig. 8.6a. Constitutively active GPCR systems can also be used to detect inverse agonism caused by ligands that preferentially stabilize the inactive state of the receptor. Figure 8.6b shows the effects of an inverse agonist on GPCR systems of various levels of constitutive activity. Providing that the inverse agonist does not completely eliminate constitutive activity (i.e. reduce the observed basal response to zero), the EC5o is a reasonable estimate of the reciprocal of affinity (KB). As with partial agonists, the antagonism of agonist responses by an inverse agonist can be used to measure the affinity of the inverse agonist. Equiactive concentrations of the agonist can be used for analysis in Schild regressions (see Fig. 8.6c). However, the effects of the inverse agonist on basal response cause a slight over-estimation of the inverse agonist affinity with this method.

Theoretically, constitutive receptor activity can be studied either in binding or functional studies. However, in practice, functional studies are much more practical. This is because, the amount of constitutively active receptor species producing the signal, namely RaG, is usually relatively low due to the low magnitude of most receptor allosteric constants. Changes in

Log[A]/KA

Fig. 8.6 Effects of agonists (a) and inverse agonists (b and c) in constitutively active GPCR systems. (a) Under conditions of increasing constitutive activity, baseline response increases as well as sensitivity to agonists. (b) Inverse agonists depress constitutive activity in a concentration-dependent manner. (c) Antagonism of agonist-induced response by an inverse agonist. Dextral displacement of the agonist dose--response curve is observed concomitantly with depressed basal constitutive activity.

Fig. 8.6 Effects of agonists (a) and inverse agonists (b and c) in constitutively active GPCR systems. (a) Under conditions of increasing constitutive activity, baseline response increases as well as sensitivity to agonists. (b) Inverse agonists depress constitutive activity in a concentration-dependent manner. (c) Antagonism of agonist-induced response by an inverse agonist. Dextral displacement of the agonist dose--response curve is observed concomitantly with depressed basal constitutive activity.

very low levels of RaG are difficult to quantify with binding. In contrast, the amplification of receptor signalling produced by cellular stimulus response mechanisms allow very small levels of RaG to produce measurable and stable levels of tissue response.

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