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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.

G protein-coupled octopamine receptors of insects and other invertebrates represent counterparts of adrenoceptors in vertebrate animals. The ␣ 2 -adrenoceptor agonist medetomidine, which is in clinical use as a veterinary sedative agent, was discovered to inhibit the settling process of barnacles, an important step in the ontogeny of this crustacean species. Settling of barnacles onto ship hulls leads to biofouling that has many harmful practical consequences, and medetomidine is currently under development as a novel type of antifouling agent. We now report that medetomidine induces hyperactivity in the barnacle larvae to disrupt the settling process. To identify the molecular targets of medetomidine, we cloned five octopamine receptors from the barnacle Balanus improvisus. We show by phylogenetic analyses that one receptor (BiOct␣) belongs to the ␣-adrenoceptor-like subfamily, and the other four (BiOct␤-R1, BiOct␤-R2, BiOct␤-R3, and BiOct␤-R4) belong to the ␤-adrenoceptor-like octopamine receptor subfamily. Phylogenetic analyses also indicated that B. improvisus has a different repertoire of ␤-adrenoceptor-like octopamine receptors than insects. When expressed in CHO cells, the cloned receptors were activated by both octopamine and medetomidine, resulting in increased intracellular cAMP or calcium levels. Tyramine activated the receptors but with much lesser potency than octopamine. A hypothesis for receptor discrimination between tyramine and octopamine was generated from a homology three-dimensional model. The characterization of B. improvisus octopamine receptors is important for a better functional understanding of these receptors in crustaceans as well as for practical applications in development of environmentally sustainable antifouling agents.

The goal of the present study was to modulate the receptor interaction properties of known alpha 2-adrenoreceptor (AR) antagonists to obtain novel alpha 2-AR agonists with desirable subtype selectivity. Therefore, a phenyl group or one of its bioisosteres or aliphatic moieties with similar steric hindrance were introduced into the aromatic ring of the antagonist lead basic structure. The functional properties of the novel compounds allowed our previous observations to be confirmed. The high efficacy of 7, 12, and 13 as alpha 2-AR agonists and the significant alpha 2C-AR subtype selective activation displayed by 11 and 15 demonstrated that favorable interactions to induce alpha 2-AR activation were formed between the pendant groups of the ligands and the aromatic cluster present in transmembrane domain 6 of the binding site cavity of the receptors.

BACKGROUND AND PURPOSESome large antagonist ligands (ARC239, chlorpromazine, prazosin, spiperone, spiroxatrine) bind to the human a2A-adrenoceptor with 10-to 100-fold lower affinity than to the a2B-and a2C-adrenoceptor subtypes. Previous mutagenesis studies have not explained this subtype selectivity. EXPERIMENTAL APPROACHThe possible involvement of the extracellular amino terminus and transmembrane domain 1 (TM1) in subtype selectivity was elucidated with eight chimaeric receptors: six where TM1 and the N-terminus were exchanged between the a2-adrenoceptor subtypes and two where only TM1 was exchanged. Receptors were expressed in CHO cells and tested for ligand binding with nine chemically diverse antagonist ligands. For purposes of interpretation, molecular models of the three human a2-adrenoceptors were constructed based on the b2-adrenoceptor crystal structure. KEY RESULTSThe affinities of three antagonists (spiperone, spiroxatrine and chlorpromazine) were significantly improved by TM1 substitutions of the a2A-adrenoceptor, but reciprocal effects were not seen for chimaeric receptors based on a2B-and a2C-adrenoceptors. Molecular docking of these ligands suggested that binding occurs in the orthosteric ligand binding pocket. CONCLUSIONS AND IMPLICATIONSTM1 is involved in determining the low affinity of some antagonist ligands at the human a2A-adrenoceptor. The exact mechanism is not known, but the position of TM1 at a large distance from the binding pocket indicates that TM1 does not participate in specific side-chain interactions with amino acids within the binding pocket of the receptor or with ligands bound therein. Instead, molecular models suggest that TM1 has indirect conformational effects related to the charge distribution or overall shape of the binding pocket. AbbreviationsBmax, receptor density; TM, transmembrane (domain); XL2, second extracellular loop BJP British Journal of Pharmacology

Background and purpose: Rodent a 2A -adrenoceptors bind the classical a 2 -antagonists yohimbine and rauwolscine with lower affinity than the human a 2A -adrenoceptor. A serine-cysteine difference in the fifth transmembrane helix (TM; position 5.43) partially explains this, but all determinants of the interspecies binding selectivity are not known. Molecular models of a 2A -adrenoceptors suggest that the second extracellular loop (XL2) folds above the binding cavity and may participate in antagonist binding. Experimental approach: Amino acids facing the binding cavity were identified using molecular models: side chains of residues 5.43 in TM5 and xl2.49 and xl2.51 in XL2 differ between the mouse and human receptors. Reciprocal mutations were made in mouse and human a 2A -adrenoceptors at positions 5.43, xl2.49 and xl2.51, and tested with a set of thirteen chemically diverse ligands in competition binding assays.

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