Conserved structural, pharmacological and functional properties among the three human and five zebrafish α₂‐adrenoceptors

Abstract

1 Zebrafish has five distinct a 2 -adrenoceptors. Two of these, a 2Da and a 2Db , represent a duplicated, fourth a 2 -adrenoceptor subtype, while the others are orthologue of the human a 2A -, a 2B -and a 2C -adrenoceptors. Here, we have compared the pharmacological properties of these receptors to infer structural determinants of ligand interactions. 2 The zebrafish a 2 -adrenoceptors were expressed in Chinese hamster ovary cells and tested in competitive ligand binding assays and in a functional assay (agonist-stimulated [ 35 S]GTPgS binding). The affinity results were used to cluster the receptors and, separately, the ligands using both principal component analysis and binary trees. 3 The overall ligand binding characteristics, the order of potency and efficacy of the tested agonists and the G-protein coupling of the zebrafish and human a 2 -adrenoceptors, separated by B350 million years of evolution, were found to be highly conserved. The binding affinities of the 20 tested ligands towards the zebrafish a 2 -adrenoceptors are generally comparable to those of their human counterparts, with a few compounds showing up to 40-fold affinity differences. 4 The a 2A orthologues and the zebrafish a 2D duplicates clustered as close pairs, but the relationships between the orthologues of a 2B and a 2C were not clearly defined. Applied to the ligands, our clustering methods segregated the ligands based on their chemical structures and functional properties. As the ligand binding pockets formed by the transmembrane helices show only minor differences among the a 2 -adrenoceptors, we suggest that the second extracellular loop -where significant sequence variability is located -might contribute significantly to the observed affinity differences.

A PET Tracer for Brain α2C Adrenoceptors, 11C-ORM-13070: Radiosynthesis and Preclinical Evaluation in Rats and Knockout Mice

“…Several a 2 adrenoceptor PET tracer candidates have been evaluated previously, but they have either lacked receptor subtype selectivity or shown poor penetration into the brain (6-11). Receptor binding assays have been performed with ORM-13070 and recombinant human a 2 adrenoceptor subtypes (17) and more than 100 other potential receptor targets (Cerep Discovery Profile) to determine the receptor profile of the tracer. The calculated binding affinities were as follows (binding affinity values [nM], with corresponding 95% confidence intervals in parentheses): a 2C adrenoceptor, 3.8 (2.0-7.3); a 2B adrenoceptor, 23 (14-38); and a 2A adrenoceptor, 109 (86-138).…”

Dopamine binds to α2-adrenergic receptors in the song control system of zebra finches (Taeniopygia guttata)

“…Previous pharmacological studies have established [ 3 H]RX821002 as a suitable radioligand to label the total population of α 2 -adrenergic receptors in various tissues and species (DiezAlarcia, et al, 2006, Halme, et al, 1995, O'Rourke, et al, 1994, Ruuskanen, et al, 2005. This ligand does not select between different α 2 -receptor subtypes and lacks binding at I 1 and I 2 imidazoline receptors but displays a relatively high affinity for 5HT 1A receptors (Clarke and Harris, 2002).…”

“…Most in vitro studies published so far have investigated the existence of an interaction between dopamine and noradrenergic receptors in cell lines transfected with cloned receptors. Only a few of these reports examined the actual binding of dopamine to adrenergic receptors in vitro (Nyronen, et al, 2001, Ruuskanen, et al, 2005, Zhang, et al, 1999. Therefore, the occurrence and the distribution of this phenomenon in vivo remains unknown.…”

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

“…Twenty four ligands (agonist (Figure 1) and antagonist ( Figure 2)), were selected from the literature [22,46,52,53]. These ligands have been reported by the research studies to bind to and interact with the α2-AR.…”

“…A set of eight agonists including endogenous ligands such as Dopamine, Adrenaline, known α2 agonists such as Clonidine, Dexmedetomidine and subtype selective agonist Guanfacine were selected from literature for docking studies [23,39,45]. Sixteen antagonists including subtype specific ligands such as BRL-44408, JP-1302, OPC-2836 and others such as ARC239, Clozapine, WB4101 that have been reported to bind to α2 adrenergic receptors were chosen for the docking studies to analyse their interactions with the α2-AR models based on Dopamine receptor (PDB ID:3PBL) [22,46,52]. The 3D structures were obtained from small molecule database Pubchem, present in NCBI server (http://pubchem.ncbi.nlm.nih.gov/).…”