Quantifying Allosteric Modulation of G Protein-Coupled Receptors
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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.
