Activation of G protein-coupled receptors entails cysteine modulation of agonist binding

Abstract:   - The increase of the affinity of agonists with an increase in pH and experiments using thiol-specific reagents indicate that G protein-coupled receptors contain an ionizable cysteine residue at the ligand binding site. Since treatment of receptors with reducing agents produces functional activation and potentiates agonist stimulation, it is likely that this free sulfhydryl modulates receptor activation. We have derived a two-state acid-base model for cysteine modulation of ligand binding which leads to a description of ligand efficacy. We have shown that pH-dependent binding of agonists is closely correlated with measurements of ligand efficacy at the 5-HT2A receptor. In general, efficacy is determined by the preference of a ligand for the base of the receptor. Efficacy may also be described in thermodynamic terms as the coupling free energy involving a ligand and the acid and base states of the receptor. Molecular modeling of the third transmembrane domain of the 5-HT2A receptor, which contains a conserved cysteine, shows that efficacy is determined by the difference between the electrostatic interaction energies of a ligand with the acid and base forms of the receptor model. The difference in interaction energy between the two forms of cysteine makes the largest contribution to this electrostatic interaction energy difference. Therefore, the cysteine makes the largest contribution to ligand efficacy. Using this approach, we can distinquish between the efficacies of agonists with varying molecular structures and account for the differences between the properties of agonists and antagonists.