Abstract
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.
Footnotes
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Send reprint requests to: Dr. Tommaso Costa, Laboratorio di Farmacologia, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. E-mail:tomcosta{at}iss.it
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We acknowledge support from the EU BIOMED 2 program “Inverse Agonism. Implications for Drug Design” (to T.C.) and from the Fonds National Suisse de la Recherche Scientifique, Grant 31-51043.97 (to S.C.).
- Abbreviations:
- 7TM
- 7 transmembrane domains
- β2-AR
- β2-adrenergic receptor
- AA
- S204A, S207A mutation
- CHO
- Chinese hamster ovary
- cam
- constitutive active mutant
- mape
- (±)2-(methylamino)-1-phenyl-1-ethanol
- Received May 24, 1999.
- Accepted October 1, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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