Tyr(199) in transmembrane domain 5 of the beta(2)-adrenergic receptor interacts directly with the pharmacophore of a unique fluorenone-based antagonist

Mutagenesis of the beta (2)-adrenergic receptor (beta (2)AR) has suggested that amino acids in transmembrane domain 5 (TMD 5) play an important role i n the interaction of the receptor with the catechol end of adrenergic agoni sts. However, little direct biochemical evidence for the interaction of any beta (2)AR agonist or antagonist with TMD 5 has been reported. To identify receptor amino acids that contribute to the beta (2)AR antagonist binding site, we identified the precise amino acid photoinsertion site of a novel c arazolol-like fluorenone antagonist photoaffinity label, [I-125]iodoaminofl isopolol ([I-125]IAmF). A unique property of this photolabel is that the ph otoreactive centre is also the binding pharmacophore, which corresponds to the catechol end of related beta (2)AR agonists, [I-125]IAmF specifically p hotolabels membrane-bound and purified beta (2)AR from a baculovirus/Spodop tera frugiperda (fall armyworm) ('Sf9') expression system. When the photola belled beta (2)AR was cleaved by trypsin or Factor Xa, 30 kDa labelled pept ides were generated. On the basis of concanavalin A binding and amino acid sequencing, these contain the N-terminus of the beta (2)AR, including TMDs 1-5. Further cleavage of the 30 kDa peptides with endoproteinase Lys-C gene rated a 4 kDa labelled peptide with an N-terminal amino acid sequence betwe en TMDs 4 and 5. Radiosequencing of this peptide demonstrated that the prec ise [I-125]IAmF photoinsertion site was Tyr(199) in TMD 5. Since the photor eactive centre and the binding pharmacophore of IAmF are the same, these da ta demonstrate that Tyr(199) interacts with the planar fluorenone moiety of a carazolol-like beta (2)AR antagonist, and contributes significant new in formation regarding the binding site for beta (2)AR antagonists.