Constitutive activity

With the experimental observation of constitutive activity for GPCRs by Costa and Herz [2], a modification was needed. Subsequently, Samama and colleagues [3] presented the extended ternary complex model to fill the void. This chapter discusses relevant mathematical models and generally offers a linkage between empirical measures of activity and molecular mechanisms. 

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... 

Constitutive activity can be produced in a recombinant system by increasing the level of receptors expressed on the cell membrane. The formation of the constitutively active... 

FIGURE 3.10 Constitutive activity due to receptor overexpression visualization through binding and function, (a) Constitutive activity observed as receptor species ([RaG]/[RL0J) and cellular function ([RaG]/ ([RaG] + 3), where P = 0.03. Stimulus-response function ([RaG]/([RaG] + p)) shown in inset. The output of the [RaG] function becomes the input for the response function. Dotted line shows relative amounts of elevated receptor species and functional response at [R]/KG= 1. (b) Effects of an inverse agonist in a system with [R]/ Kq= 1 (see panel a) as observed through receptor binding and cellular function. 

FIGURE 3.11 Constitutive activity in melanophores expressing hCTR2 receptor, (a) Basal melanophore activity, (b) Effect of transfection with human cDNA for human calcitonin receptors (16 j-ig/ml). (c) Concentration response curve for cDNA for human calcitonin receptors (abscissae as log scale) and constitutive activity. Data redrawn from [27]. 

It can be seen from this equation that maximal constitutive activity need not reach a maximal asymptote of unity. Submaximal constitutive activity has been observed with some receptors with maximal receptor expression [28]. While there is scattered evidence that the cubic ternary complex is operative in some receptor systems, and while it is thermodynamically more complete, it also is heuristic... 

FIGURE 5.10 Effects of co-expressed G-protein (G ) on neuropeptide NPY4 receptor responses (NPY-4). (a) Dose-response curves for NPY-4. Ordinates Xenopus laevis melanophore responses (increases light transmission). Ordinates logarithms of molar concentrations of neuropeptide Y peptide agonist PYY. Curves obtained after no co-transfection (labeled 0 jig) and co-transfection with cDNA for Gai6. Numbers next to the curves indicate jig of cDNA of Ga]g used for co-transfection, (b) Maximal response to neuropeptide Y (filled circles) and constitutive activity (open circles) as a function of pg cDNA of co-transfected G g. 

In constitutively active receptor systems (where the baseline is elevated due to spontaneous formation of receptor active states, see Chapter 3 for full discussion), unless the antagonist has identical affinities for the inactive receptor state, the spontaneously formed active state, and the spontaneously G-protein coupled state (three different receptor conformations, see discussion in Chapter 1 on receptor conformation) it will alter the relative concentrations of these species—and in so doing alter the baseline response. If the antagonist has higher affinity for the... 

The effects of high values of constitutive activity can be determined for functional systems where function is defined by the operational model. Thus, it can be assumed in a simplified system that the receptor exists in an active (R ) and inactive (R) form and that agonists stabilize... 

FIGURE 6.12 Schild analysis for constitutively active receptor systems, (a) Competitive antagonism by the inverse agonist in a constitutively active receptor system with DR values calculated at the EC80. 

Equation 6.19 predicts an increasing IC50 with either increases in L or 1. In systems with low-efficacy inverse agonists or in systems with low levels of constitutive activity, the observed location parameter is still a close estimate of the KB (equilibrium dissociation constant of the ligand-receptor complex, a molecular quantity that transcends test system type). In general, the observed potency of inverse agonists only defines the lower limit of affinity. 

Used to estimate system independent potency of an inverse agonist in a constitutively active receptor system. 

X = transducer function for response to the full agonist and constitutively active receptor state. 

FIGURE 11.2 Paired experimental data. Values of constitutive calcitonin receptor activity [1 -(Tr/Tj) units] in transiently transfected melanophores. Five separate experiments are shown. Points to the left indicate the basal level of constitutive activity before (filled circles) and after (open circles) addition of 100 nM AC512 (calcitonin receptor inverse agonist). Lines join values for each individual experiment. Points to the right are the mean values for constitutive activity in control (filled circles) and after AC512 (open circles) for all five experiments (bars represent standard errors of the mean). Data shown in Table 11.3. 

Values are levels of constitutive activity [1 — (Tr/Tj)] for four individual transfection experiments (denoted X ) x2 are the constitutive receptor activity values after exposure to AC512 in the same experiment. 

As can be seen from the analysis in Table 11.3, the paired t-test indicates that the effect of AC512 on the constitutive activity is significant at the 99% level of confidence (p<0.01 and AC512 is an inverse agonist and does decrease the constitutive receptor activity of calcitonin receptors). 

FIGURE 11.3 One-way ANOVA (analysis of variance). One-way analysis of variance of basal rates of metabolism in melanophores (as measured by spontaneous dispersion of pigment due to G,.-protein activation) for four experiments. Cells were transiently transfected with cDNA for human calcitonin receptor (8 j-ig/ml) on four separate occasions to induce constitutive receptor activity. The means of the four basal readings for the cells for each experiment (see Table 11.4) are shown in the histogram (with standard errors). The one-way analysis of variance is used to determine whether there is a significant effect of test occasion (any one of the four experiments is different with respect to level of constitutive activity). 

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