Abstract

We present a mechanism for agonist-promoted α_2A-adrenergic receptor (α_2A-AR) activation based on structural, pharmacological, and theoretical evidence of the interactions between phenethylamine ligands and α_2A-AR. In this study, we have: 1) isolated enantiomerically pure phenethylamines that differ both in their chirality about the β-carbon, and in the presence/absence of one or more hydroxyl groups: the β-OH and the catecholic meta- andpara-OH groups; 2) used [³H]UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; agonist] and [³H]RX821002 [2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline; antagonist] competition binding assays to determine binding affinities of these ligands to the high- and low-affinity forms of α_2A-AR; 3) tested the ability of the ligands to promote receptor activation by measuring agonist-induced stimulation of [³⁵S]GTPγS binding in isolated cell membranes; and 4) used automated docking methods and our α_2A-AR model to predict the binding modes of the ligands inside the α_2A-AR binding site. The ligand molecules are sequentially missing different functional groups, and we have correlated the structural features of the ligands and ligand-receptor interactions with experimental ligand binding and receptor activation data. Based on the analysis, we show that structural rearrangements in transmembrane helix (TM) 5 could take place upon binding and subsequent activation of α_2A-AR by phenethylamine agonists. We suggest that the following residues are important in phenethylamine interactions with α_2A-AR: Asp113 (D_3.32), Val114 (V_3.33), and Thr118 (T_3.37) in TM3; Ser200 (S_5.42), Cys201 (C_5.43), and Ser204 (S_5.46) in TM5; Phe391 (F_6.52) and Tyr394 (Y_6.55) in TM6; and Phe411 (F_7.38) and Phe412 (F_7.39) in TM7.