Mutations inducing divergent shifts of constitutive activity reveal different modes of binding among catecholamine analogues to the β2‐adrenergic receptor

Department of Neuroscience, University of Rome, 'Tor Vergata', Rome, Italy.
British Journal of Pharmacology (Impact Factor: 4.84). 05/2002; 135(7):1715-22. DOI: 10.1038/sj.bjp.0704622
Source: PubMed


We compared the changes in binding energy generated by two mutations that shift in divergent directions the constitutive activity of the human β2 adrenergic receptor (β2AR).
A constitutively activating mutant (CAM) and the double alanine replacement (AA mutant) of catechol-binding serines (S204A, S207A) in helix 5 were stably expressed in CHO cell lines, and used to measure the binding affinities of more than 40 adrenergic ligands. Moreover, the efficacy of the same group of compounds was determined as intrinsic activity for maximal adenylyl cyclase stimulation in wild-type β2AR.
Although the two mutations had opposite effects on ligand affinity, the extents of change were in both cases largely correlated with the degree of ligand efficacy. This was particularly evident if the extra loss of binding energy due to hydrogen bond deletion in the AA mutant was taken into account. Thus the data demonstrate that there is an overall linkage between the configuration of the binding pocket and the intrinsic equilibrium between active and inactive receptor forms.
We also found that AA mutation-induced affinity changes for catecholamine congeners gradually lacking ethanolamine substituents were linearly correlated to the loss of affinity that such modifications of the ligand cause for wild-type receptor. This indicates that the strength of bonds between catechol ring and helix 5 is critically dependent on the rest of interactions of the β-ethanolamine tail with other residues of the β2-AR binding pocket.
British Journal of Pharmacology (2002) 135, 1715–1722; doi:10.1038/sj.bjp.0704622

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    • "All fitting using the equiactive approach and operational model was performed using Prism. For the ␤2AR, reported dissociation constants for ligands from Del Carmine et al. (2002) were used. "
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    • "Membranes were prepared as described previously (Vachon et al., 1987) and stored at 80°C until assay. The binding of [ 125 I]()-pindolol was performed, as reported previously (Del Carmine et al., 2002), for membrane preparations expressing wild-type 2 -AR and mutants: S204A; S207A; S204,S207A; S204T; S207T; S204T,S207T; S204C; S207C; S204C,S207C. In each binding assay, the six ligands were tested simultaneously, using competition curves consisting of 8 log-spaced concentrations. "
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    • "). However, detailed studies with such human receptor models typically focused on the characterization of single receptor subtypes (Liggett 1992; Lattion et al. 1999; Candelore et al. 1999; Del Carmine et al. 2002). "
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