Molecular mechanisms of ligand–receptor interactions in transmembrane domain V of the α_2A‐adrenoceptor

Abstract

1 The structural determinants of catechol hydroxyl interactions with adrenergic receptors were examined using 12 a 2 -adrenergic agonists and a panel of mutated human a 2A -adrenoceptors. The a 2A Ser201 mutant had a Cys-Ser201 (position 5.43) amino-acid substitution, and a 2A Ser201Cys200 and a 2A Ser201Cys204 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 [35 S]GTPgS-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 a 2A -adrenoceptor. Substitution of Ser200 or Ser204 with cysteine caused a deterioration in the capability of catecholamines to activate the a 2A -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.

Fluorescence Resonance Energy Transfer Analysis of α2a-Adrenergic Receptor Activation Reveals Distinct Agonist-Specific Conformational Changes

“…Radioligand binding experiments in cell membranes (in the presence of GTP to uncouple the receptors from G-proteins and thereby induce a low-affinity state) showed that all three constructs were virtually indistinguishable from wild-type ␣ 2A -adrenergic receptors regarding saturation binding with the antagonist [ 3 H]RX821002 and competition with the various full and partial agonists used in this study (Table 1). The obtained K D and K i values were similar to published data (Peltonen et al, 2003;Nikolaev et al, 2006). These data indicate that the tetracysteine motifs did not affect ligand binding by the receptors.…”

“…This has been proposed for the M 1 muscarinic receptor (Allman et al, 2000), the herpesvirus 8-encoded CXC-chemokine receptor ORF74-HHV8 (Rosenkilde et al, 2006), and specifically for the ␣ 2A -adrenergic receptor (Marjamaki et al, 1999;Nyronen et al, 2001). TMV binds to the catechol OH groups via several serine residues in the ␣ 2A -and the ␤ 2 -adrenergic receptor (Peltonen et al, 2003;Xhaard et al, 2006). This is compatible with our observation that agonists lacking one of the catechol groups fail to induce FRET signals close to TMV (I3-C construct).…”

“…Figure 2 shows that all constructs induced [ 35 S]GTP␥S binding well above the level of nontransfected HEK cells. These assays were done with the full agonist norepinephrine, the strong partial agonists dopamine and clonidine (a structurally independent compound), and two weak partial agonists, norphenephrine and octopamine (Audinot et al, 2002;Peltonen et al, 2003). With all five agonists, wild-type and mutant receptors were indistinguishable in the rates and in the amplitudes of stimulated [ 35 S]GTP␥S binding.…”

Homology Modelling and Docking Studies of Human α2-Adrenergic Receptor Subtypes

“…Grid was generated at the centroid of the active site residues consisting of residues D3.32 (α2a: Asp113, α2b: Asp92, α2c: Asp131), S5.42 (α2a: Ser200, α2b: Ser176, α2c: Ser214) S5.46 (α2a: Ser204, α2b: Ser180; α2c: Ser218) [18,20,53]. The grid box generated is of the dimensions-outer/ grid box: 30, 30, 30 and ligand centre box/inner box: 10, 10, 10.…”

Transmembrane Segment Five Serines of the D4 Dopamine Receptor Uniquely Influence the Interactions of Dopamine, Norepinephrine, and Ro10-4548

“…That TM5 serines in the D4 receptor have not been studied was surprising given the interest in D4 receptor agonists as potential therapeutics, evidence that conserved TM5 serines in biogenic amine G protein-coupled receptors (GPCRs) are sites of interaction for their amine neurotransmitters, and the conserved TM5 serines in the D1, D2, and D3 subtypes of dopamine receptor are key sites of catecholamine agonist interaction (for review see Floresca and Schetz, 2004). In addition, the high affinity of norepinephrine for only the D4 subtype of dopamine receptor, when coupled with the knowledge that the endogenous function of noradrenergic receptors relies on key interactions between conserved TM5 serines and norepinephrine (Peltonen et al, 2003), suggests that norepinephrine's affinity and efficacy at the D4 receptors may also involve the conserved serines of TM5. From the viewpoint of drug design and the tailoring of ligand selectivity and function, it would also be very informative to identify whether the interactions of TM5 serines, which are presumed to be important for the interaction of dopamine by analogy to the D1, D2, and D3 dopamine receptors, are necessary for D4 receptor activation by selective agonists.…”