Identification of Duplicated Fourth α2-Adrenergic Receptor Subtype by Cloning and Mapping of Five Receptor Genes in Zebrafish

Abstract

The alpha(2)-adrenergic receptors (alpha(2)-ARs) belong to the large family of rhodopsinlike G-protein-coupled receptors that share a common structure of seven transmembrane (TM) alpha-helices. The aims of this study were (1) to determine the number of alpha(2)-AR genes in a teleost fish, the zebrafish (Danio rerio), (2) to study the gene duplication events that generated the alpha(2)-AR subtypes, and (3) to study changes in receptor structure that have occurred since the divergence of the mammalian and fish lineages. Here, we report the cloning and chromosomal mapping of fish orthologs for all three mammalian alpha(2)-ARs. In addition, we identified a fourth alpha(2)-AR subtype with two duplicates in zebrafish. Chromosomal mapping showed that the zebrafish alpha(2)-AR genes are located within conserved chromosomal segments, consistent with the origin of the four alpha(2)-AR subtypes by two rounds of chromosome or block duplication before the divergence of the ray fin fish and tetrapod lineages. Thus, the fourth subtype has apparently been present in the common ancestor of vertebrates but has been deleted or is yet to be identified in mammals. The overall percentage identity between the fish and mammalian orthologs is 53% to 67%, and in the TM regions 80% to 87%. These values are clearly lower than what is observed between mammalian orthologs. Still, all of the residues thought to be important for alpha(2)-adrenergic ligand binding are conserved across species and subtypes, and even the most divergent regions of the fish receptors show clear "molecular fingerprints" typical for orthologs of a given subtype.

The zebrafish genome in context: ohnologs gone missing

“…But the ray-finned lineage retained all four of the original ohnologs of the Adra2 ohnology group, and this ohnolog was retained in duplicate copies after the teleost genome duplication (adra2da and adra2db, Fig. 5D) even though ADRA2D has been eliminated from mammalian genomes (Ruuskanen et al, 2003). The location of ACSL orthologs in zebrafish compared to human as reflected by the zebrafish genome assembly verifies these analyses.…”

β-Adrenergic Signaling Regulates Evolutionarily Derived Sleep Loss in the Mexican Cavefish

“…3 ). While the Danio genome encodes for four ␤ -adrenergic receptors [Ruuskanen et al, 2004], the number of ␤ -adrenergic receptor genes in the A. mexicanus genome remains unknown. To gain insight into the subclass of ␤ -adrenergic receptors modulating sleep, we assayed the effects of subtype-specific ␤ -adrenergic antagonists on sleep.…”

Neuronal and glial localization of α2A‐adrenoceptors in the adult zebrafish (Danio rerio) brain

“…ARs of ␣ 2 and ␤ types have been identified in the nervous system and skin of some teleost species (Holmgren and Nilsson, 1982;Svensson et al, 1993;Katayama et al, 1999; Zikopoulos and Dermon, 2005), including zebrafish (Ruuskanen et al, 2004(Ruuskanen et al, , 2005a. The ␣ 2A -ARs are located both postsynaptically and presynaptically, the latter limiting the release of NA and other neurotransmitters, such as serotonin and glutamate.…”

“…The LC projection pattern (Ekström et al, 1986;Ma, 1994b) and recently the localization of the monoamine oxidase (MAO), the NA metabolic pathway enzyme (Anichtchik et al, 2006) have been reported for the zebrafish brain. Furthermore, ␣ 2 -ARs have been identified in zebrafish, by cloning and mapping of five distinct receptor genes (Ruuskanen et al, 2004), each coding for a functional receptor coupled to G-mediated second messenger signaling system, similar to the mammalian ␣ 2 -ARs (Ruuskanen et al, 2005a). Specifically, three of the zebrafish adra2 genes code for the orthologues of the human ␣ 2A -AR, ␣ 2B -AR, and ␣ 2C -AR, whereas the two genes code for ␣ 2Da -and ␣ 2Db -AR subtypes, representing a duplicated fourth ␣ 2 -AR subtype (Ruuskanen et al, 2004(Ruuskanen et al, , 2005a.…”

“…Furthermore, ␣ 2 -ARs have been identified in zebrafish, by cloning and mapping of five distinct receptor genes (Ruuskanen et al, 2004), each coding for a functional receptor coupled to G-mediated second messenger signaling system, similar to the mammalian ␣ 2 -ARs (Ruuskanen et al, 2005a). Specifically, three of the zebrafish adra2 genes code for the orthologues of the human ␣ 2A -AR, ␣ 2B -AR, and ␣ 2C -AR, whereas the two genes code for ␣ 2Da -and ␣ 2Db -AR subtypes, representing a duplicated fourth ␣ 2 -AR subtype (Ruuskanen et al, 2004(Ruuskanen et al, , 2005a. Although it is difficult to establish homologies among vertebrate functional neural systems, the distribution pattern of ␣ 2 -AR subtypes in all brain regions would provide a better understanding of their possible role in the zebrafish brain and illuminate comparative aspects of noradrenergic systems in vertebrates.…”