Molecular mechanisms of ligand-receptor interactions in transmembrane domain V of the alpha2A-adrenoceptor

Abstract

1. The structural determinants of catechol hydroxyl interactions with adrenergic receptors were examined using 12 alpha2-adrenergic agonists and a panel of mutated human alpha2A-adrenoceptors. The alpha2ASer201 mutant had a Cys --> Ser201 (position 5.43) amino-acid substitution, and alpha2ASer201Cys200 and alpha2ASer201Cys204 had Ser --> Cys200 (5.42) and Ser --> Cys204 (5.46) substitutions, respectively, in addition to the Cys --> Ser201 substitution. 2. Automated docking methods were used to predict the receptor interactions of the ligands. Radioligand-binding assays and functional [35S]GTPgammaS-binding assays were performed using transfected Chinese hamster ovary cells to experimentally corroborate the predicted binding modes. 3. The hydroxyl groups of phenethylamines were found to have different effects on ligand affinity towards the activated and resting forms of the wild-type alpha2A-adrenoceptor. Substitution of Ser200 or Ser204 with cysteine caused a deterioration in the capability of catecholamines to activate the alpha2A-adrenoceptor. The findings indicate that (i) Cys201 plays a significant role in the binding of catecholamine ligands and UK14,304 (for the latter, by a hydrophobic interaction), but Cys201 is not essential for receptor activation; (ii) Ser200 interacts with the meta-hydroxyl group of phenethylamine ligands, affecting both catecholamine binding and receptor activation; while (iii) substituting Ser204 with a cysteine interferes both with the binding of catecholamine ligands and with receptor activation, due to an interaction between Ser204 and the para-hydroxyl group of the catecholic ring.

Figure 1

Alignment of amino-acid sequences from TM5 of the human AR subtypes.

Figure 2

Chemical structures of the α_2A-AR ligands examined in this study.

Figure 3

Stereo view of the superposition of the α_2A-wt, α_2ASer201, α_2ASer201Cys200 and α_2ASer201Cys204 binding sites after flexible docking of R-noradrenaline. Amino-acid residues that form the binding cavity in our models are shown. Atom colour codes: grey=carbon, red=oxygen, blue=nitrogen, yellow=sulphur.

Figure 4

Binding mode models (see Experimental procedures for details) of superposed R-isomers of adrenaline, noradrenaline, octopamine, norphenephrine and 2-amino-1-phenylethanol (a), and UK14,304 (b). Some key amino acids that line the ligand-binding cavity model are shown. The yellow surface depicts the solvent-accessible surface of these residues, and the blue surface depicts the solvent-accessible surface of the ligand. Hydrogens are not shown for clarity, but were included in the calculation of the solvent-accessible surface. Atom colour codes: grey=carbon, red=oxygen, blue=nitrogen, yellow=sulphur, brown=bromine (only in UK14,304).

Figure 5

The correlation between the low-/high-affinity competition-binding K_i-ratio and the efficacy for seven agonists in the wild-type α_2A-AR (a) and the α_2ASer201 mutant (b). The K_i (high affinity) values are presented in Table 2 and the K_i (low affinity) values are presented in Table 3. The agonists are numbered: 1 – R-2-amino-1-phenylethanol, 2 – R-norphenephrine, 3 – R-octopamine, 4 – UK14,304, 5 – dopamine, 6 – R-adrenaline, 7 – R-noradrenaline.