Abstract

The structural basis of ligand affinity can be approached by studying the interactions between a drug and receptor residues; the basis for efficacy is more complex and must involve activation-associated conformational changes. We have used wild-type (WT), a constitutively active mutant (CAM), and a “constitutively inactive” mutant β2 adrenergic receptor (β₂AR) to investigate changes in the binding site that accompany binding and activation. The active state (R^*) probably involves repositioning of at least some of the agonist-contact residues, thereby optimizing their interactions with agonist and resulting in a higher affinity for agonist. A comparison of the binding affinities of a series of phenethylamine derivatives for WT revealed a remarkable synergism between the various functional groups present in epinephrine. Binding affinity was essentially unchanged with addition of β-OH, N-CH₃, or catechol OHs to phenethylamine. In contrast, when each of these same groups was added to the appropriate compound, already containing the other two groups, to make epinephrine, the increase in affinity was quite large (60- to 120-fold). An initial interaction between two or more contacts may stabilize an intermediate conformation of β₂AR, R′, either by altering amino acid side chain rotamer conformations or by a more global conformational change involving the repositioning of transmembrane segments. The pattern of these effects was different in the CAM in that fewer interactions were required to observe the synergistic effect, consistent with the hypothesis that the CAM mutation enriches the proportion of receptors in R^* or in R′ from which R^* is more readily assumed.