IDENTIFICATION OF CRITICAL EXTRACELLULAR LOOP RESIDUES INVOLVED IN ALPHA(1)-ADRENERGIC RECEPTOR SUBTYPE-SELECTIVE ANTAGONIST BINDING

alpha(1)-Adrenergic receptor (AR) subtypes mediate many effects of the sympathetic nervous system. Although structurally similar, the three cloned subtypes (alpha(1a)-AR, alpha(1b)-AR, and alpha(1d)-AR) bind a series of ligands with different relative potencies. This is particula rly true for the alpha(1a)-AR, which recognizes a number of ligands wi th 10-100-fold higher affinity than the alpha(1b) or alpha(1d) subtype s. Because ligands are hypothesized to bind to receptor residues that are located in the transmembrane (TM) spanning domains, subtype differ ences in ligand recognition are likely the result of differences in th e binding properties of non-conserved TM residues, We previously repor ted on the identification of two TM residues in the alpha(1b)-AR that converted the agonist binding profile entirely to that of the alpha(1a )-AR when mutated to corresponding alpha(1a)-AR residues. We now repor t on the determinants of antagonist selectivity between these two alph a(1)-AR subtypes. Construction of a chimera in which the entire fifth TM and a portion of the putative second extracellular loop of the hams ter alpha(1b)-AR was replaced with the corresponding region of the rat alpha(1a)-AR revealed that the chimera accounted for all of the highe r binding affinity (8-29-fold) seen in the alpha(1a)-AR for two antago nists, phentolamine and WB4101, Using site-directed mutagenesis, we fu rther analyzed individual point mutations making up this chimera. We f ound that three adjacent residues, which were located on the extracell ular loop of the fifth TM, are fully responsible for this higher antag onist binding affinity in the alpha(1a)-AR, These three point mutation s (G196Q, V197I, T198N) in the alpha(1b)-AR were additive and sufficie nt in their effects on changing antagonist-binding profiles to that of the alpha(1a)-AR. Reversal of these three residues in the alpha(1a)-A R to their corresponding residues in the alpha(1b)-AR completely rever sed the antagonist affinity to wild-type alpha(1b)-AR values. To aid i n molecular modeling, the use of organic chemicals that mimic key stru ctures of the antagonists were used in competitive ligand-binding stud ies with the mutated receptors. These results indicated the orientatio n of both phentolamine and WB4101 in the alpha(1)-AR binding pocket. T ogether, the data indicate that alpha(1)-antagonists may bind near the surface of the receptor, much like the peptide hormone receptors, and not deep within the TM regions, where the ligand-binding pocket was f irst proposed and identified for alpha(1) agonists.