Probing the salmeterol binding site on the beta(2)-adrenergic receptor using a novel photoaffinity ligand, [I-125]iodoazidosalmeterol

Salmeterol is a long-acting beta(2)-adrenergic receptor (beta(2)AR) agonist used clinically to treat asthma. In addition to binding at the active agon ist site, it has been proposed that salmeterol also binds with very high af finity at a second site, termed the "exosite", and that this exosite contri butes to the long duration of action of salmeterol. To determine the positi on of the phenyl ring of the aralkyloxyalkyl side chain of salmeterol in th e beta(2)AR binding site, we designed and synthesized the agonist photoaffi nity label [I-125]iodoazidosalmeterol ([I-125]IAS). In direct adenylyl cycl ase activation, in effects on adenylyl cyclase after pretreatment of intact cells, and in guinea pig tracheal relaxation assays, IAS and the parent dr ug salmeterol behave essentially the same. Significantly, the photoreactive azide of IAS is positioned on the phenyl ring at the end of the molecule w hich is thought to be involved in exosite binding. Carrier-free radioiodina ted [I-125]IAS was used to photolabel epitope-tagged human beta(2)AR in mem branes prepared from stably transfected HEK 293 cells. Labeling with [I-125 ]IAS was blocked by 10 mu M (-)-alprenolol and inhibited by addition of GTP gamma S, and [I-125]IAS migrated at the same position on an SDS-PAGE gel a s the beta(2)AR labeled by the antagonist photoaffinity label [I-125]iodoaz idobenzyIpindolol ([I-125]IABP). The labeled receptor was purified on a nic kel affinity column and cleaved with factor Xa protease at a specific seque nce in the large loop between transmembrane segments 5 and 6, yielding two peptides. While the control antagonist photoaffinity label [I-125]IABP labe led both the large N-terminal fragment [containing transmembranes (TMs) 1-5 ] and the smaller C-terminal fragment (containing TMs 6 and 7), essentially all of the [I-125]IAS labeling was on the smaller C-terminal peptide conta ining TMs 6 and 7, This direct biochemical evidence demonstrates that when salmeterol binds to the receptor, its hydrophobic aryloxyalkyl tail is posi tioned near TM 6 and/or TM 7. A model of IAS binding to the beta(2)AR is pr oposed.