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 particularly true for the alpha(1a)-AR, which recognizes a number of ligands with 10-100-fold higher affinity than the alpha(1b) or alpha(1d) subtypes. Because ligands are hypothesized to bind to receptor residues that are located in the transmembrane (TM) spanning domains, subtype differences in ligand recognition are likely the result of differences in the binding properties of non-conserved TM residues, We previously reported 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 report on the determinants of antagonist selectivity between these two alpha(1)-AR subtypes. Construction of a chimera in which the entire fifth TM and a portion of the putative second extracellular loop of the hamster alpha(1b)-AR was replaced with the corresponding region of the rat alpha(1a)-AR revealed that the chimera accounted for all of the higher binding affinity (8-29-fold) seen in the alpha(1a)-AR for two antagonists, phentolamine and WB4101, Using site-directed mutagenesis, we further analyzed individual point mutations making up this chimera. We found that three adjacent residues, which were located on the extracellular loop of the fifth TM, are fully responsible for this higher antagonist binding affinity in the alpha(1a)-AR, These three point mutations (G196Q, V197I, T198N) in the alpha(1b)-AR were additive and sufficient in their effects on changing antagonist-binding profiles to that of the alpha(1a)-AR. Reversal of these three residues in the alpha(1a)-AR to their corresponding residues in the alpha(1b)-AR completely reversed the antagonist affinity to wild-type alpha(1b)-AR values. To aid in molecular modeling, the use of organic chemicals that mimic key structures of the antagonists were used in competitive ligand-binding studies with the mutated receptors. These results indicated the orientation of both phentolamine and WB4101 in the alpha(1)-AR binding pocket. Together, 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 first proposed and identified for alpha(1) agonists.