Identification of Duplicated Fourth a2-Adrenergic Receptor Subtype by Cloning and Mapping of Five Receptor Genes in Zebrafish (original) (raw)
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Molecular Biology and Evolution, 2003
The a 2 -adrenergic receptors (a 2 -ARs) belong to the large family of rhodopsinlike G-protein-coupled receptors that share a common structure of seven transmembrane (TM) a-helices. The aims of this study were (1) to determine the number of a 2 -AR genes in a teleost fish, the zebrafish (Danio rerio), (2) to study the gene duplication events that generated the a 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 a 2 -ARs. In addition, we identified a fourth a 2 -AR subtype with two duplicates in zebrafish. Chromosomal mapping showed that the zebrafish a 2 -AR genes are located within conserved chromosomal segments, consistent with the origin of the four a 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 a 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.
Molecular Biology and Evolution, 2004
The a 2-adrenergic receptors (a 2-ARs) belong to the large family of rhodopsinlike G-protein-coupled receptors that share a common structure of seven transmembrane (TM) a-helices. The aims of this study were (1) to determine the number of a 2-AR genes in a teleost fish, the zebrafish (Danio rerio), (2) to study the gene duplication events that generated the a 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 a 2-ARs. In addition, we identified a fourth a 2-AR subtype with two duplicates in zebrafish. Chromosomal mapping showed that the zebrafish a 2-AR genes are located within conserved chromosomal segments, consistent with the origin of the four a 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 a 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.
Mapping of Five Receptor Genes in Zebrafish
2003
The 2 -adrenergic receptors ( 2 -ARs) belong to the large family of rhodopsin-like G-protein coupled receptors that share a common structure of 7 transmembrane (TM) -helices. The aims of this study were (1) to determine the number of 2 -AR genes in a teleost fish, the zebrafish (Danio rerio); (2) to study the gene duplication events that generated the 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 2 -ARs. In addition, we identified a fourth 2 -AR subtype with two duplicates in zebrafish. Chromosomal mapping showed that the zebrafish 2 -AR genes are located within conserved chromosomal segments, consistent with the origin of the four 2 -AR subtypes by two rounds of chromosome or block duplication prior to 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 53-67 %, and in the TM regions 80-87 %. These values are clearly lower than what is observed between mammalian orthologs. Still, all of the residues thought to be important for 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. by guest on August 10, 2015 http://mbe.oxfordjournals.org/ Downloaded from by guest on August 10, 2015 http://mbe.oxfordjournals.org/ Downloaded from Origin of 2 -adrenergic receptor subtypes 4 AR. There are two duplicates of this receptor subtype in zebrafish, encoded by the genes adra2da and adra2db.
British Journal of Pharmacology, 2009
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 2Cadrenoceptors. 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.
In silico analysis of Human and Zebrafish ?-2 Adrenergic Receptors
Journal of Biology Agriculture and Healthcare, 2012
α-2 adrenoceptors, belong to class of Rhodopsin-like G-protein coupled receptors. Proteins of the G-protein coupled receptor (GPCR) family are involved in many pathophysiological conditions and hence are targets for various drug discovery methods. The current information on the structure of GPCRs is limited to few structures like Rhodopsin, β adrenergic receptors, adenosine A2A receptors, Human Dopamine D3 and Chemokine receptor. In our study α-2 adrenergic receptors of Human and Zebrafish were modeled using MODELLER with Human Dopamine D3 receptor (PDB ID: 3PBL) as template. Through our modeling studies we have identified the critical role played by Proline residues (2.38, 2.59, 4.39, 4.59, 4.60, 7.50) of transmembrane helices and extracellular loop in stabilizing structural deviations in the transmembrane. Novel ligand binding residues S/T (6.56) and F (7.35) along with the positional significance of Y (3.28), Y (6.55) in regulating function were identified. Our models have shown that the Phenylalanine at 7.39 in TM7 can favourably interact with positively charged N-methyl group of the catecholamine ligands via hydrophobic contacts rather than 7.38 as reported previously. Furthermore, we are able to correctly show the orientation of Serine at 5.42 and 5.46 and discuss the relevance of residues at position 3.37 and 5.43 in the receptor regulation. We also demonstrate and propose that the orientation of V (2.61)/S should be taken into account in drug/ pharmacophore design specific for α-2 adrenergic receptors. We believe that these findings will open new lead for ligand/ pharmacophore design, in silico leading further to experimental validation using Zebrafish as experimental model.
Journal of Neurochemistry, 2005
The a 2 -adrenoceptors are G-protein-coupled receptors that mediate many of the physiological effects of norepinephrine and epinephrine. Mammals have three subtypes of a 2 -adrenoceptors, a 2A , a 2B and a 2C . Zebrafish, a teleost fish used widely as a model organism, has five distinct a 2 -adrenoceptor genes. The zebrafish has emerged as a powerful tool to study development and genetics, with many mutations causing diseases reminiscent of human diseases. Three of the zebrafish adra2 genes code for orthologues of the mammalian a 2 -adrenoceptors, while two genes code for a 2Da -and a 2Db -adrenoceptors, representing a duplicated, fourth a 2 -adrenoceptor subtype. The three different mammalian a 2 -adrenoceptor subtypes have distinct expression patterns in different organs and tissues, and mediate different physiological functions. The zebrafish a 2 -adrenergic system, with five different a 2 -adrenoceptors, appears more complicated. In order to deduce the physiological functions of the
In silico analysis of Human and Zebrafish α-2 Adrenergic Receptors
2012
α-2 adrenoceptors, belong to class of Rhodopsin-like G-protein coupled receptors. Proteins of the G-protein coupled receptor (GPCR) family are involved in many pathophysiological conditions and hence are targets for various drug discovery methods. The current information on the structure of GPCRs is limited to few structures like Rhodopsin, β adrenergic receptors, adenosine A2A receptors, Human Dopamine D3 and Chemokine receptor. In our study α-2 adrenergic receptors of Human and Zebrafish were modeled using MODELLER with Human Dopamine D3 receptor (PDB ID: 3PBL) as template. Through our modeling studies we have identified the critical role played by Proline residues (2.38, 2.59, 4.39, 4.59, 4.60, 7.50) of transmembrane helices and extracellular loop in stabilizing structural deviations in the transmembrane. Novel ligand binding residues S/T (6.56) and F (7.35) along with the positional significance of Y (3.28), Y (6.55) in regulating function were identified. Our models have shown that the Phenylalanine at 7.39 in TM7 can favourably interact with positively charged N-methyl group of the catecholamine ligands via hydrophobic contacts rather than 7.38 as reported previously. Furthermore, we are able to correctly show the orientation of Serine at 5.42 and 5.46 and discuss the relevance of residues at position 3.37 and 5.43 in the receptor regulation. We also demonstrate and propose that the orientation of V (2.61)/S should be taken into account in drug/ pharmacophore design specific for α-2 adrenergic receptors. We believe that these findings will open new lead for ligand/ pharmacophore design, in silico leading further to experimental validation using Zebrafish as experimental model.
Cloning and expression of a fish α2-adrenoceptor
British Journal of Pharmacology, 1993
Pigment granule aggregation in specialized cells (melanophores) from the skin of teleost fishes has been shown to be mediated by receptors with an a2-adrenoceptor pharmacology. We now report the cloning of the X2-F, a fish skin a2-receptor from the cuckoo wrasse (Labrus ossifagus). 2 Degenerate oligonucleotides corresponding to conserved regions of the human M2-adrenoceptor subtypes were used in a polymerase chain reaction (PCR) with cDNA prepared from mRNA isolated from the skin of the cuckoo wrasse. An 876 base pair (bp) product was obtained that was homologous with that of the human M2-adrenoceptor and was used to screen a genomic library from the cuckoo wrasse. 3 A clone (pTB17BS) consisting of-5 kb of genomic DNA was obtained which contained the nucleotide sequence of the initial PCR product. In addition, it contained an open reading frame that encoded a protein of 432 amino acids and-2 kb of 5'-untranslated sequence.The deduced amino acid sequence of this protein showed 47-57% identity with the human M2-adrenoceptors and thus appeared to encode a fish aX2-adrenoceptor. 4 In the 5'-untranslated region of the gene, nucleotide sequences were present suggesting that transcription of the 02-F might be regulated by cyclic AMP, calcium and/or steroids. 5 The a2-F was expressed in COS-7 cells and radioligand binding studies were performed with [3H]-rauwolscine. The binding was of high affinity and it was saturable with a KD of 0.8 ± 0.1 nM and a Bmax of 5.7 + 1.0 pmol mg-' of protein. 6 Competition curves for the displacement of specific [3H]-rauwolscine binding showed the following order of potency: for agonists, medetomidine > clonidine >p-aminoclonidine > B-HT 920 > (-)-noradrenaline; for antagonists, rauwolscine > atipamezole > yohimbine > phentolamine > prazosin. 7 These results show that x2-F has characteristics of both the human x2-CIO and a2-C4 and that it might represent an ancestral oc2-adrenoceptor subtype.
In vivo and in vitro assessment of cardiac -adrenergic receptors in larval zebrafish (Danio rerio)
Journal of Experimental Biology, 2011
-Adrenergic receptors (ARs) are crucial for maintaining the rate and force of cardiac muscle contraction in vertebrates. Zebrafish (Danio rerio) have one 1AR gene and two 2AR genes (2aAR and 2bAR). We examined the roles of these receptors in larval zebrafish in vivo by assessing the impact of translational gene knockdown on cardiac function. Zebrafish larvae lacking 1AR expression by morpholino knockdown displayed lower heart rates than control fish, whereas larvae deficient in both 2aAR and 2bAR expression exhibited significantly higher heart rates than controls. These results suggested a potential inhibitory role for one or both 2AR genes. By using cultured HEK293 cells transfected with zebrafish ARs, we demonstrated that stimulation with adrenaline or procaterol (a 2AR agonist) resulted in an increase in intracellular cAMP levels in cells expressing any of the three zebrafish ARs. In comparison with its human AR counterpart, zebrafish 2aAR expressed in HEK293 cells appeared to exhibit a unique binding affinity profile for adrenergic ligands. Specifically, zebrafish 2aAR had a high binding affinity for phenylephrine, a classical -adrenergic receptor agonist. The zebrafish receptors also had distinct ligand binding affinities for adrenergic agonists when compared with human ARs in culture, with zebrafish 2aAR being distinct from human 2AR and zebrafish 2bAR. Overall, this study provides insight into the function and evolution of both fish and mammalian -adrenergic receptors.
A putative β 2 -adrenoceptor from the rainbow trout ( Oncorhynuchus mykiss )
European Journal of Biochemistry, 2001
Extensive molecular characterization of mammalian b-adrenoceptors has revealed complex modes of regulation and interaction. Relatively little attention, however, has focused on adrenoceptors from early branching vertebrates such as fish. Using an RT-PCR approach we have cloned a rainbow trout b 2 -adrenoceptor gene that codes for a 409-amino-acid protein with the same seven transmembrane domain structure as its mammalian counterparts. This rainbow trout b 2 -adrenoceptor shares a high degree of amino-acid sequence conservation with other vertebrate b 2 -adrenoceptors. The conclusion that this sequence is a rainbow trout b 2 -adrenoceptor is further supported by phylogenetic analysis of vertebrate b-adrenoceptor sequences and competitive pharmacological binding data. RNase protection assays demonstrate that the rainbow trout b 2 -adrenoceptor gene is highly expressed in the liver and red and white muscle, with lower levels of expression in the gills, heart, kidney and spleen of the rainbow trout. The lack of regulatory phosphorylation sites within the G-proteinbinding domain of the rainbow trout b 2 -adrenoceptor sequence suggests that the in vivo control of trout b 2 -adrenoceptor signaling differs substantially from that of mammals.