Tyr199 in transmembrane domain 5 of the β2-adrenergic receptor interacts directly with the pharmacophore of a unique fluorenone-based antagonist

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Abstract
Mutagenesis of the β2-adrenergic receptor (β2AR) 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 β2AR agonist or antagonist with TMD 5 has been reported. To identify receptor amino acids that contribute to the β2AR 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 β2AR agonists. [125I]IAmF specifically photolabels membrane-bound and purified β2AR from a baculovirus/Spodoptera frugiperda (fall armyworm) (‘Sf9’) expression system. When the photolabelled β2AR was cleaved by trypsin or Factor Xa, 30kDa labelled peptides were generated. On the basis of concanavalin A binding and amino acid sequencing, these contain the N-terminus of the β2AR, including TMDs 1–5. Further cleavage of the 30kDa peptides with endoproteinase Lys-C generated a 4kDa 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 Tyr199 in TMD 5. Since the photoreactive centre and the binding pharmacophore of IAmF are the same, these data demonstrate that Tyr199 interacts with the planar fluorenone moiety of a carazolol-like β2AR antagonist, and contributes significant new information regarding the binding site for β2AR antagonists.