title: Catechol-binding serines of beta(2)-adrenergic receptors control the equilibrium between active and inactive receptor states.
abstract: The binding free energy for the interaction between serines 204 and 207 of the fifth transmembrane helix of the beta(2)-adrenergic receptor (beta(2)-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 beta(2)-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) beta(2)-AR mutant is 50 to 60% lower than that of the wild-type beta(2)-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.
authors.affiliation: Department of Pharmacology, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Ambrosio
authors.forename: P
authors.initials: P
authors.lastname: Molinari
authors.forename: S
authors.initials: S
authors.lastname: Cotecchia
authors.forename: T
authors.initials: T
authors.lastname: Costa
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002396:Catechols / Q000378:metabolism*; D003001:Cloning, Molecular; D017353:Gene Deletion; D006900:Hydroxylation; D008024:Ligands; D016296:Mutagenesis; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013816:Thermodynamics
chemical_list: D002396:Catechols; D008024:Ligands; D018343:Receptors, Adrenergic, beta-2; D012694:Serine; C034221:catechol