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

1 Zebrafish has five distinct α2‐adrenoceptors. Two of these, α2Da and α2Db, represent a duplicated, fourth α2‐adrenoceptor subtype, while the others are orthologue of the human α2A‐, α2B‐ and α2C‐adrenoceptors. Here, we have compared the pharmacological properties of these receptors to infer structural determinants of ligand interactions. 2 The zebrafish α2‐adrenoceptors were expressed in Chinese hamster ovary cells and tested in competitive ligand binding assays and in a functional assay (agonist‐stimulated [35S]GTPγS 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 α2‐adrenoceptors, separated by ∼350 million years of evolution, were found to be highly conserved. The binding affinities of the 20 tested ligands towards the zebrafish α2‐adrenoceptors are generally comparable to those of their human counterparts, with a few compounds showing up to 40‐fold affinity differences. 4 The α2A orthologues and the zebrafish α2D duplicates clustered as close pairs, but the relationships between the orthologues of α2B and α2C 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 α2‐adrenoceptors, we suggest that the second extracellular loop – where significant sequence variability is located – might contribute significantly to the observed affinity differences. British Journal of Pharmacology (2005) 144, 165–177. doi:10.1038/sj.bjp.0706057