Mutational Analysis of the α_1a-Adrenergic Receptor Binding Pocket of Antagonists by Radioligand Binding Assay

Abstract

Computer simulations of the human α_1a-adrenergic receptor (α_1a-AR) based on the crystal structure of rhodopsin have been combined with experimental site-directed mutagenesis to investigate the role of residues in the transmembrane domains in antagonist binding. Previous molecular dynamics studies from our laboratory indicated that the amino acids Asp106 in the third transmembrane domain (TMD), Gln167 in TMD IV of α_1a-AR were directly involved in prazosin, tamsulosin and KMD-3213 binding. The Asp106Ala mutant did not exhibit any affinity for [³H]prazosin. On the other hand, the Gln167Phe mutant α_1a-AR showed reduced binding affinity for [³H]prazosin. In competition binding experiment the binding affinities of prazosin and tamsulosin were increased 11-fold and 33-fold respectively to Gln167Phe mutant in comparison with wild type receptor. It seems that mutation of this residue by phenylalanine has offered more interaction for the ligands with its aromatic ring. The results provide direct evidence that these amino acid residues are responsible for the interactions between α_1a-AR and radioligand [³H]prazosin as well as tamsulosin and KMD-3213.