Molecular mechanism for agonist-promoted alpha(2A)-adrenoceptor activationby norepinephrine and epinephrine

We present a mechanism for agonist-promoted alpha (2A)-adrenergic receptor (alpha (2A)-AR) activation based on structural, pharmacological, and theore tical evidence of the interactions between phenethylamine ligands and alpha (2A)-AR. In this study, we have: 1) isolated enantiomerically pure pheneth ylamines that differ both in their chirality about the beta -carbon, and in the presence/absence of one or more hydroxyl groups: the beta -OH and the catecholic meta- and para-OH groups; 2) used [H-3]UK-14,304 [5-bromo-N-(4,5 -dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; agonist] and [H-3]RX821002 [2 -(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline; antagonist] competition bi nding assays to determine binding affinities of these ligands to the high- and low-affinity forms of alpha (2A)-AR; 3) tested the ability of the ligan ds to promote receptor activation by measuring agonist-induced stimulation of [S-35]GTP gammaS binding in isolated cell membranes; and 4) used automat ed docking methods and our alpha (2A)-AR model to predict the binding modes of the ligands inside the alpha (2A)-AR binding site. The ligand molecules are sequentially missing different functional groups, and we have correlat ed 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 (T M) 5 could take place upon binding and subsequent activation of alpha (2A)- AR by phenethylamine agonists. We suggest that the following residues are i mportant in phenethylamine interactions with alpha (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 TM 6; and Phe411 (F-7.38) and Phe412 (F-7.39)in TM7.