Tyr199 in transmembrane domain 5 of the beta2-adrenergic receptor interacts directly with the pharmacophore of a unique fluorenone-based antagonist.

Mutagenesis of the beta2-adrenergic receptor (beta2AR) has suggested that amino acids in transmembrane domain 5 (TMD 5) play an important role in the interaction of the receptor with the catechol end of adrenergic agonists. However, little direct biochemical evidence for the interaction of any beta2AR agonist or antagonist with TMD 5 has been reported. To identify receptor amino acids that contribute to the beta2AR antagonist binding site, we identified the precise amino acid photoinsertion site of a novel carazolol-like fluorenone antagonist photoaffinity label, [125I]iodoaminoflisopolol ([125I]IAmF). A unique property of this photolabel is that the photoreactive centre is also the binding pharmacophore, which corresponds to the catechol end of related beta2AR agonists. [125I]IAmF specifically photolabels membrane-bound and purified beta2AR from a baculovirus/Spodoptera frugiperda (fall armyworm) ('Sf9') expression system. When the photolabelled beta2AR was cleaved by trypsin or Factor Xa, 30 kDa labelled peptides were generated. On the basis of concanavalin A binding and amino acid sequencing, these contain the N-terminus of the beta2AR, including TMDs 1-5. Further cleavage of the 30 kDa peptides with endoproteinase Lys-C generated a 4 kDa labelled peptide with an N-terminal amino acid sequence between TMDs 4 and 5. Radiosequencing of this peptide demonstrated that the precise [125I]IAmF photoinsertion site was Tyr(199) in TMD 5. Since the photoreactive centre and the binding pharmacophore of IAmF are the same, these data demonstrate that Tyr(199) interacts with the planar fluorenone moiety of a carazolol-like beta2AR antagonist, and contributes significant new information regarding the binding site for beta2AR antagonists.