Quantifying Allosteric Modulation of G Protein-Coupled Receptors
In this chapter, methods for the analysis of allosterism in equilibrium binding and functional assays are described. The functional response to activation of a G protein-coupled receptor is usually measured at a point downstream from receptor activation in the signaling pathway, and the effects of allosteric modulation on the response can be attributed to scalar changes in the observed affinity (α) and intrinsic efficacy (β) of the agonist-receptor complex. If the concentration-response curve of the agonist is measured in the absence and presence of a range of concentrations of the allosteric modulator, then it is always possible to estimate the affinity constant of the modulator (K 2) and the product (γ) of the modulatory changes in the observed affinity and efficacy of the agonist-receptor complex (γ = a β). In many instances, it is impossible through analysis of receptor function only to determine the α and β components of the modulation. Regardless, the parameter γ is unique in that it represents the ratio of the microscopic affinity constants of the modulator for the active and inactive states of the receptor in situations where there is one orthosteric activation site directly linked to the allosteric site. Radioligand binding assays can be used to estimate affinity modulation between the allosteric modulator and the orthosteric radioligand or another nonlabeled orthosteric ligand. By measuring allosterism in both functional and binding assays, it is possible to measure the affinity and efficacy components of allosteric modulation.
Key wordsAllosterism Affinity Efficacy Modulation Pathway selectivity Ligand-directed signaling
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