Sustained activation of a G protein-coupled receptor via "anchored" agonist binding. Molecular localization of the salmeterol exosite within the 2-adrenergic receptor.

An inherent therapeutic limitation of many G protein-coupled receptor agonists is a short duration of action due to rapid dissociation from receptors. Salmeterol is a modified beta-adrenergic receptor (betaAR) agonist that has a long duration of action at the beta2AR (but not the beta1AR) both in vitro and in vivo and that is persistent despite extensive washout of the agonist. It has been proposed that salmeterol binds not only to the active site of the beta2AR (localized to receptor transmembrane spanning domains (TMDs) 3 and 5) but also to another site (termed the "exosite") that anchors it to the receptor and provides for repetitive active-site binding events. To identify the location of this exosite, we used site-directed mutagenesis to replace beta2AR amino acids 149-173 (within TMD4) with beta1AR sequence. The resulting constructs were then expressed in COS-7 cells for radioligand binding studies. Using this approach, when this domain was replaced with the analogous beta1AR sequence, the ability of salmeterol to persist at the receptor under washout conditions was reduced by 67%. The results from more selective mutants (S-(149-166), S-(164-173), and S-(149-158)) indicated that a limited 10-amino acid region (beta2AR residues 149-158), localized at the interface of the cytoplasm and the transmembrane domain, contains a critical determinant for exosite binding. Whereas CHW cells stably expressing wild-type beta2AR displayed persistent salmeterol-promoted cAMP accumulation despite agonist washout, substitution of beta2AR residues 149-158 with beta1AR sequence resulted in a 56% attenuation of salmeterol-promoted cAMP accumulation under identical washout conditions. A reverse chimera was also studied, which consisted of a substitution of beta2AR residues 152-156 into the beta1AR. This substitution was found to confer exosite binding to the beta1AR. None of these mutations decreased the affinity of salmeterol for the receptor at the active site as assessed in competition binding studies. Anchored binding to this motif thus represents a novel mechanism by which agonists like salmeterol can repetitively activate receptors. Conceivably, with other G protein-coupled receptors that have similar motifs, anchored ligands can be designed to provide for long durations of action by this mechanism.