RT Journal Article
SR Electronic
T1 Catechol-Binding Serines of β<sub>2</sub>-Adrenergic Receptors Control the Equilibrium between Active and Inactive Receptor States
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 198
OP 210
VO 57
IS 1
A1 Ambrosio, Caterina
A1 Molinari, Paola
A1 Cotecchia, Susanna
A1 Costa, Tommaso
YR 2000
UL http://molpharm.aspetjournals.org/content/57/1/198.abstract
AB The binding free energy for the interaction between serines 204 and 207 of the fifth transmembrane helix of the β2-adrenergic receptor (β2-AR) and catecholic hydroxyl (OH) groups of adrenergic agonists was analyzed using double mutant cycles. Binding affinities for catecholic and noncatecholic agonists were measured in wild-type and mutant receptors, carrying alanine replacement of the two serines (S204A, S207A β2-AR), a constitutive activating mutation, or both. The free energy coupling between the losses of binding energy attributable to OH deletion from the ligand and from the receptor indicates a strong interaction (nonadditivity) as expected for a direct binding between the two sets of groups. However, we also measured a significant interaction between the deletion of OH groups from the receptor and the constitutive activating mutation. This suggests that a fraction of the decrease in agonist affinity caused by serine mutagenesis may involve a shift in the conformational equilibrium of the receptor toward the inactive state. Direct measurements using a transient transfection assay confirm this prediction. The constitutive activity of the (S204A, S207A) β2-AR mutant is 50 to 60% lower than that of the wild-type β2-AR. We conclude that S204 and S207 do not only provide a docking site for the agonist, but also control the equilibrium of the receptor between active (R*) and inactive (R) forms. The American Society for Pharmacology and Experimental Therapeutics