* Department of Pharmacology and Clinical Pharmacology
Turku Graduate School of Biomedical Sciences, University of Turku, Turku, Finland
Department of Biochemistry and Pharmacy, Åbo Akademi University, Turku, Finland
Department of Neuroscience, Unit of Pharmacology, Uppsala University, Uppsala, Sweden
| Institute of Neuroscience, University of Oregon, Eugene, Oregon
Correspondence: E-mail: mschein{at}utu.fi.
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Abstract |
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Key Words: genome duplication synteny fish 2-adrenergic receptor
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Introduction |
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Three different intronless genes encode the three distinct mammalian 2-AR subtypes,
2A,
2B, and
2C. The human
2-AR genes have been designated ADRA2A (
2C10), ADRA2B (
2C2), and ADRA2C (
2C4) and are located on human chromosomes 10, 2, and 4 (Kobilka et al. 1987; Regan et al. 1988; Lomasney et al. 1990; Bylund et al. 1992). Related sets of
2-AR subtype genes or cDNAs have been cloned from some other mammalian species used as experimental animals such as rat, mouse, and guinea pig (for review, see MacDonald, Kobilka, and Scheinin [1997]). The rodent
2A-AR was initially designated
2D-AR on the basis of its ligand-binding profile but was subsequently shown to be orthologous to human
2A-AR (Lanier et al. 1991; Link et al. 1992). Occasionally, it is still misleadingly called
2D. Despite this, we propose that the fourth
2-AR subtype reported in this paper should be called
2D-AR. There are two duplicates of this receptor subtype in zebrafish, encoded by the genes adra2da and adra2db.
Single counterparts of the human 2-AR subtype genes have also been cloned from several mammalian species such as pig (
2A) (Guyer et al. 1990), cow (
2A) (Venkataraman, Duda, and Sharma 1997), tree shrew (
2A and
2C, both are partial clones) (Meyer et al. 2000; Flugge et al. 2003), and opossum (
2C) (Blaxall et al. 1994). Partial
2B-AR genes have also been cloned from a range of mammals for use in molecular phylogeny analysis and to study the molecular evolution of mammalian
2B-ARs (Springer et al. 1997; Stanhope et al. 1998; Madsen et al. 2001; Springer et al. 2001; Teeling et al. 2002; Madsen et al. 2002). A previous study provides evidence for the existence of different classes of adrenergic receptors in nonmammalian vertebrates, too. This cross-hybridization study, using different adrenergic receptor probes, identified sequences homologous to human
2A-AR in goldfish, a frog, a turtle, and chicken (Palacios et al. 1989). A partial cDNA sequence is known for a chicken
2A-AR (Blaxall, Heck, and Bylund 1993). The ligand-binding properties of the
2-AR cloned from the fish cuckoo wrasse (Labrus ossifagus) are intermediate between
2A and
2C, and it was initially thought to represent an "ancestral"
2-AR subtype (Svensson et al. 1993). On the amino acid sequence level, this "
2F-AR" shows the greatest similarity to the
2C-AR subtype. In GenBank, there is a genomic DNA sequence of a putative full-length goldfish
2-AR gene (L09064, unpublished) that does not appear to be orthologous to any of the three mammalian
2-AR subtypes (MacDonald, Kobilka, and Scheinin 1997). In addition, in GenBank, there are fragments of nucleotide sequences of putative
2-AR subtypes from lamprey, several fish species, birds, a reptile, and an amphibian (accession numbers AL606538 to AL606586, unpublished). Eight different
2-AR protein sequences from the pufferfish (Takifugu rubribes), the first fish with a sequenced genome, have been recently listed in the SWISS-PROT protein database (Q8JG00 to Q8JG07, unpublished). Currently no
2-ARs have been definitively identified in invertebrates. Several biogenic amine receptors are predicted in the mosquito and fruit fly genomes (Hill et al. 2002). The fruit fly octopamine/tyramine and snail octopamine receptor are highly similar to the mammalian adrenergic receptors, and they also bind
2-adrenergic drugs (Gerhardt et al. 1997; Chatwin et al. 2003). A tyramine receptor in the nematode C. elegans has also been characterized (Rex and Komuniecki 2002). Several putative "alpha adrenergic" receptors have been annotated in the mosquito, fruit fly, and nematode genomes (genomes browseable via www.ensembl.org), although it is unclear whether any of these species have adrenaline or noradrenaline as natural ligands.
The diversity of GPCR subtypes seems to have arisen in early vertebrate evolution and may thereby be explained by the extensive increase in gene number (Miyata and Suga 2001; Spring 2003) proposed to be the result of two genome duplications before the radiation of jawed vertebrates some 500 MYA (Holland et al. 1994; Vernier et al. 1995; Postlethwait et al. 1998). This evolutionary view is supported by the chromosomal localization of the human AR genes in related chromosomal segments called paralogy groups or paralogons (Pebusque et al. 1998; Wraith et al. 2000). The much-debated HOX-paralogons do not overlap with the ADR-bearing paralogons. It is noteworthy that the human chromosome Hsa2 is a result of a recent fusion of two different chromosomes in the human lineage, the ADRA2B-bearing Hsa2p having a different origin from the HOX-bearing Hsa2q (Larhammar, Lundin, and Hallbook 2002). From this point of view, our starting hypothesis was that the fish genome should have the same three 2-AR subtype genes as mammals (and possibly duplicates of some of these if the teleost fish genome has undergone an additional genome doubling followed by some gene loss [Postlethwait et al. 1998; Taylor et al. 2001]). In addition, a fourth subtype gene might be present, representing the expected fourth product of two sets of genome duplication, currently thought to be missing from mammalian genomes. To study the evolution and structural conservation of
2-ARs and to facilitate further studies on their possible importance in developmental biology, we turned to a genetically well-characterized species of fish, the zebrafish (Danio rerio), which is highly amenable to developmental and genetic studies and useful in studies on brain aminergic systems, too (Guo et al. 1999). Based on the conservation of the HOX-gene clusters across species, it is possible that there has been an extra round of chromosomal duplications in the teleost lineage, leading to two orthologs for a single mammalian gene (Amores et al. 1998; Postlethwait et al. 1998; Naruse et al. 2000). Recently, there has been a debate on the nature of the origin of the duplicated fish genes (i.e., whether they arose from local or large-scale duplications) (Taylor et al. 2001; Taylor, Van de Peer, and Meyer 2001; Robinson-Rechavi et al. 2001a; Robinson-Rechavi 2001b. The
2-ARs belong to another well-conserved gene cluster, and we wished to test the hypothesis that the evolutionary pattern displayed by the HOX-containing chromosome regions can be generalized to other regions of the zebrafish genome.
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Materials and Methods |
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Another zebrafish genomic DNA-library in a Lawrist7 cosmid-vector (from the Resource Center/Primary Database (RZPD), Max-Planck-Institute for Molecular Genetics, Berlin, Germany) was screened with the cDNA clone. Hybridization was carried out as above but at 65°C. The filters were washed twice in 2 x SSC/0.1% SDS for 5 min at 65°C and twice in 0.1 x SSC/0.1% SDS for 30 min at 65°C. A clone containing a 1,299-bp ORF coding for an 2C-like AR was isolated as above, as well as two clones containing identical sequences. The same library was screened for additional subtypes using a probe corresponding to a zebrafish
2B-AR like EST (AI461341, coding for transmembrane [TM] domains 6 and 7 and the carboxyl-terminal tail). This hybridization was carried out in ExpressHyb-buffer (Clontech, Palo Alto, Calif.) for 2 h at 60°C. The filters were washed twice in 2 x SSC/0.1% SDS for 5 min at 60°C, twice in 0.5 x SSC/0.1% SDS for 30 min, and once in 0.5 x SSC/0.1% SDS for 15 min at 60°C. Sixteen positive cosmid clones were isolated and analyzed as above. One clone contained a 1,533-bp ORF coding for an
2B-like AR. Another cosmid clone was directly sequenced further using primer walking. This clone contained a truncated coding sequence for an
2-AR differing from the other zebrafish
2-ARs. The cosmid clone was truncated at its 5' end, lacking the N-terminus and N-terminal half of TM1. A Blast search with this truncated sequence identified a closely related, but not identical, truncated
2-AR sequence (GenBank accession number AL606583). AL606583 spans from the C-terminal end of TM3 to the second half of TM6 (ending with conserved sequence CWF). The other clones contained copies of related genes (see Results) or were false positives.
Inverse PCR
Zebrafish genomic DNA was prepared as described previously (Akimenko 1995). One µg DNA/reaction was digested with eight different restriction enzymes, BamHI, EcoRI, HindIII, KpnI, PstI, SacI, SpeI, and XhoI (New England Biolabs Inc., Beverly, Mass.), overnight at 37°C. The digests were diluted to 350 µl and extracted with an equal volume of phenol:chloroform (1:1). After gentle vortexing (20 s) and centrifugation, the aqueous phase containing the DNA was removed. DNA was precipitated with 0.05 volumes of 3 M sodium acetate and 2 volumes of cold 99% ethanol and centrifugation at 12,000 rpm for 15 min. The precipitate was dissolved in 449 µl nuclease-free water. Ligase buffer, 50 µl, and 1 µl (400 U) T4-ligase (New England Biolabs) were added, and ligation was carried out overnight at 14°C. DNA was precipitated with 150 µl 5 M ammonium acetate and 700 µl 99% ethanol, centrifuged, and washed with 70% ethanol. The pellet was dissolved in 10 µl of nuclease-free water.
Two sets of primers were designed based on the obtained truncated coding sequences from the cosmid clone and the related genomic sequence (AL606583), one inner primer pair and one outer pair for each of these sequences. The primers were synthesized in the reverse complementary direction to amplify the nucleotide sequences upstream and downstream from the known sequences. The primer sequences are provided in the Supplementary Material online.
PCR reactions were run using a DynaZyme EXT kit with proofreading DNA polymerase (FinnZymes, Espoo, Finland) and the following reagents: forward primer 15 pmol, reverse primer 15 pmol, and DMSO 1:20. Primary PCR reactions were run with the inner primer pair and 1 µl of the ligation products as templates, using the following cycling conditions: 94°C for 2 min, 1 cycle; 94°C for 30 s, 53°C for 30 s, and 72°C for 2 min, 40 cycles, and final extension at 72°C for 7 min. One µl of the primary PCR reactions was used as the template in a second PCR using the outer primer pair (conditions as above, except with an annealing temperature of 54°C; for AL606583 the annealing temperature was 59°C for both reactions). The second PCR reactions were analyzed on a 1.5% agarose gel and single bands were identified from the gel, excised, and purified using the High Pure PCR Product Purification Kit (Roche Molecular Biochemicals, Mannheim, Germany). The fragments were either directly sequenced using the outer PCR primers or A/T-ligated to pGEM-T-Easy vector (Promega, Madison, Wis.) according to the manufacturer's instructions, introduced into XL-1 Blue supercompetent cells (Stratagene, La Jolla, Calif.) for plasmid growth, and isolated using the NucleoSpin Plasmid kit (MACHEREY-NAGEL GmbH & Co., Düren, Germany). The plasmid preparations were checked for correct inserts using restriction digestions and subsequently sequenced with the universal primers T7 and SP6.
For the cosmid clone, one of the fragments (EcoRI-digested initial template) contained a 190-bp sequence overlapping with the cosmid clone sequence and 195 bp of good quality upstream sequence, the overlapping parts of the sequences being 100% identical. The nucleotide sequence containing a 1,227-bp ORF for a previously unpublished zebrafish 2-AR (
2Da-AR) was deduced from the sequences of the truncated cosmid clone and the inverse-PCR product.
For AL606583, the full-length coding sequence was constructed in a similar way. Fragments amplified from SacI-digested and EcoRI-digested initial templates contained an upstream sequence with a start codon and 287 bp of 5'-UTR. A fragment from the BamHI-digested initial template contained the downstream sequence, a stop codon, and 38 bp of 3'-UTR. The overlapping parts of the sequences were 100% identical. By combining the sequences of AL606583 and the inverse PCRgenerated fragments, another previously unpublished zebrafish 2-AR gene sequence containing a 1,248-bp ORF was obtained (
2Db-AR). The obtained gene sequences were also used to search the Sanger Institute's zebrafish genome database (www.sanger.co.uk, available via Blast and keywords from www.ensembl.org/Danio_rerio/) to identify possible additional
2-AR sequences and to check for possible sequence variants of the identified genes.
DNA Sequencing and Sequence Analysis
All sequencing was performed using the ABI Prism Automatic sequencer (Applied Biosystems Inc., Foster City, Calif.) and several specific oligonucleotide primers on both DNA strands. Blast searches (from http://www.ncbi.nlm.nih.gov:80/BLAST/ for GenBank entries and from www.ensembl.org for zebrafish genome specific sequences, GENSCAN predictions) using full-length DNA-DNA (BlastN) or protein (BlastP) (sequences spanning TM1 to TM5 and TM6 to TM7) against translated databases; keyword searches for annotated sequences were used to identify related sequences. Raw sequence assembly and more detailed sequence analysis were performed using the Lasergene software package (DNASTAR Inc., Madison, Wis.). Sequences from other species were retrieved from GenBank and nucleotide sequences were translated into putative protein sequences using the Lasergene package. As a result, in addition to the five cloned zebrafish 2-AR receptors, 27 full-length and 46 truncated protein sequences of putative
2-ARs were found. Multiple sequence alignments were constructed using Malign (Johnson and Overington 1993) for (1) the full-length sequences, (2) the regions common to all of the 78 available
2-AR sequences, and (3) separately for sequences from each of the four
2-AR subtypes. These individual alignments were themselves aligned, modified manually over the highly divergent regions, and are presented in the Supplementary Material online. Only unambiguously aligned regions from these alignments were used for molecular phylogeny analysis. Phylogenetic trees were constructed with the Neighbor-Joining and maximum-parsimony methods using the programs SEQBOOT, PROTPARS, PROTDIST, Neighbor, and Consense from the PHYLIP version 3.5c package (Felsenstein 1993) and with the maximum-likelihood method using PROTML form the MOLPHY package. The statistical significance of the presented branching was determined using bootstrap analysis (1,000 data replicates). In contrast to trees based on the ortholog-specific alignments, the tree based on the sequences common to all of the 78 available
2-AR sequences (data not shown) showed low stability, as reflected in the bootstrap analysis, probably because there was an insufficient number of phylogenetically informative sites.
RT-PCR
Total RNA was isolated either from a pool of 30 zebrafish embryos (age 48 to 50 hpf) or an individual whole adult zebrafish using RNAwiz (Ambion, Inc., Austin, Tex.). The RNA samples were DNase treated using DNA-free (Ambion) to remove contaminating genomic DNA. Specific primers for adra2da and adra2db were first tested for performance using genomic DNA as a template. The primers were also checked for specificity using plasmids containing the coding sequence for the other duplicate as a template; no cross-recognition was detected. Primer sequences are provided in the Supplementary Material online. In RT-PCR, 100 ng of total RNA was used as the template for the Access RT-PCR System (Promega) with only slight modifications to the manufacturer's recommendations. The final Mg2+-concentration used was 1.5 mM. First-strand cDNA synthesis was performed at 48°C for 45 min followed by RT-enzyme inactivation at 94°C for 2 min. Second-strand synthesis and PCR amplifications were performed using the following cycling conditions: 94°C for 30 s, 60°C for 1 min, and 68°C for 1 min, 40 cycles, and final extension at 68°C for 7 min. Control reactions were run without the RT-enzyme to detect the possible existence of contaminating genomic DNA.
Southern Hybridizations
Genomic DNA from individual, whole adult zebrafish was prepared as described previously (Akimenko 1995), digested with HindIII restriction enzyme, run on a gel, and blotted on a nylon filter (Amersham Pharmacia Biotech UK). The cloned zebrafish 2-AR genes do not contain HindIII recognition sites within their coding sequences. The same
2B-AR and
2C-AR probes, as well as a zebrafish
2A-AR probe corresponding to TM6, TM7, and surrounding regions, were used as for the initial screenings. The
2Da-AR and
2Db-AR probes correspond to the N-terminal part of the receptor (TM1 to TM4 and surrounding regions). Southern hybridizations of filters were performed while gradually lowering the stringency. All hybridizations were carried out in 25% formamide, 6 x SSC, 10% dextran sulphate, 5 x Denhardt's solution, and 0.1% SDS at 65°C, 55°C, 50°C, or 42°C overnight. The filters were first washed twice in 2 x SSC/0.1% SDS at room temperature for 5 min and, depending on the stringency, twice in 0.2 x SSC/0.1% SDS for 30 min at 65°C (high stringency), twice in 0.5 x SSC/0.1% SDS for 30 min at 55°C (high to intermediate), twice in 0.5 x SSC/0.1% SDS for 30 min at 50°C (intermediate to low) or twice in 2 x SSC/0.1% SDS for 30 min at 42°C (low). After each washing step, the filters were exposed either with films or imaging plates, analyzed with a Fuji PhosphorImager (Fuji Corporation, Tokyo, Japan), and rehybridized.
Chromosomal Mapping of the Cloned Genes and Genome Comparisons
PCR primers were designed to amplify a region near the coding sequences of the cloned zebrafish 2-AR genes (adra2a, adra2b, adra2c, adra2da, and adra2db) containing single-strand conformation polymorphisms. Primer sequences for the different genes are provided in the Supplementary Material online. Primers were first tested for performance with genomic zebrafish DNA as the template. The detected polymorphisms were scored on the HS (heat shock) meiotic mapping panel and linkage analysis was performed to determine their chromosomal location. The method is described in detail elsewhere (Kelly et al. 2000; Woods et al. 2000). Gene locations for chromosomal comparisons between different species were retrieved from NCBI's HomoloGene service (http://www.ncbi.nlm.nih.gov/HomoloGene/) and The Zebrafish Server (http://zfin.org/).
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Results |
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A nucleotide fragment (GenBank accession number AL606583) with high similarity to the 2Da-AR sequence was found using a Blast search. The full-length 1,248-bp sequence for this receptor, the
2Db-AR (gene name adra2db), was subsequently obtained using inverse PCR.
A genomic clone containing a 1,119-bp coding sequence for a zebrafish 2A-AR (gene name adra2a) was isolated from the other genomic DNA library and hybridized with the cuckoo wrasse
2-AR probe under low stringency conditions. The coding sequence was interrupted 9 amino acids after TM7. Another phage clone was sequenced to obtain the complete 1,167-bp ORF of this gene and showed that the carboxyl-terminal tail consists of 24 amino acids, similarly to the mammalian
2A-ARs (all of equal length).
Blast searches with the cloned zebrafish 2-AR sequences resulted in two exact or almost exact matches with ESTs: AW077216 overlaps the N-terminal part of adra2a and has one nucleotide difference leading to one amino acid change in TM1 (I27V, not facing the binding cavity according to the modeled structure). The other EST, AI461341, is 100% identical to the zebrafish
2B-AR over the overlapping sequence at the C-terminus. Other exact or nearly exact matches from GenBank include genomic fragments of
2B-AR (AL606585, 99.1% identical, including one amino acid deletion in the third intracellular loop),
2C-AR (AL606584, 99.2% identical, no amino acid differences),
2Da-AR (AL606586, 98.7% identical, one amino acid difference in the third intracellular loop), and
2Db-AR (AL606583, 99.9% identical, no amino acid differences). The quality of these sequences is not easy to assess, as the method of isolation and sequencing of these entries has not been reported. For this reason, we relied on our own adra2 sequences in the phylogenetic analyses. A 99.5% identical match for the zebrafish adra2a was found in the Sanger Institute's zebrafish genome database (Q90WY4); this gene was annotated on the basis of our GenBank entry for adra2a (AY048971). Differences from our adra2a sequence include the same I27V mutation in TM1 as above and another in the third intracellular (IC3) loop (D240E). The mutation I27V in both AW077216 and Q90WY4 is caused by the same nucleotide difference and is thus very likely to represent a true polymorphism, although the quality and reliability of the EST sequences is difficult to judge. However, no matches for the other genes identified here were found in the zebrafish genome database; searches with the cloned adra2 sequences identified only the adra2a sequence.
RT-PCR
As no information about the expression of the fourth 2-AR subtype genes is present from any species, an RT-PCR screening was performed. It shows that adra2db is expressed both in 48 to 50 hpf embryos and in adult zebrafish (fig. 1). For adra2da, no signal was present in the embryos and only a faint signal was seen in adult zebrafish (not shown), but this will require confirmation by in situ hybridizations.
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The most conserved regions among the 2-AR sequences correspond to the putative TM regions defined by similarity with the TM regions in bovine rhodopsin, whose crystal structure is known (Palczewski et al. 2000). The ligand-binding pocket is largely formed by the TM regions, and key residues implicated in binding interactions with adrenaline and noradrenaline (Nyronen et al. 2001) are all conserved among the
2-ARs. The two cysteine residues forming a disulfide bridge linking TM3 to the second extracellular (EC2) loop in the bovine rhodopsin crystal structure (Palczewski et al. 2000) are present in the
2-ARs. The GPCR consensus sequences DRY (TM3) and EKE (TM6), which are thought to form an ion pair that locks the rhodopsinlike GPCRs into an inactive state (e.g., Ballesteros et al. 2001), are also conserved. In the rhodopsin crystal structure, there are two palmitoylated cysteines located at the C-terminus, following a short cytoplasmic helix. In the human
2A-AR and
2B-AR, a single cysteine is present, but no cysteine is found in this region in the human
2C-AR (e.g., Kennedy and Limbird 1993). All of the zebrafish
2-ARs possess a cysteine at the equivalent position and therefore may be palmitoylated. With the exception of a site within the IC3 loop (E[S,T]4D), identified as a G-protein coupled receptor kinase (GRK) phosphorylation site in human
2A-AR (Eason, Moreira, and Liggett 1995; Jewell-Motz and Liggett 1995) and opossum
2C-AR (Deupree, Borgeson, and Bylund 2002), the regions outside of the TMs are more divergent. The equivalent site in the human
2C-AR (ESSAA) differs, is not phosphorylated, and its equivalent in
2B-ARs from eutherians could not be unambiguously identified, although the human
2B-AR is phosphorylated by GRKs. The zebrafish
2A-AR and
2C-AR possess the complete GRK phosphorylation site, whereas the site is present but incomplete in zebrafish
2B-ARs (ESMSSD),
2Da-AR (ESAASD), and
2Db-AR (ESSVSN). A similar site ((D/E)nxx(S/T)) is phosphorylated by Casein Kinase 1, and this mechanism has been proposed as an alternative pathway for GPCR phosphorylation (Tobin 2002).
In contrast, conserved regions among the sequences of the fish and mammalian orthologs of a given subtype are mainly located within the IC1, IC2, EC1, and EC3 loops, near the C-terminus and within EC2 and IC3 adjacent to the TMs. The intracellular surface of 2-ARs interacts with G-proteins, where IC2 plays a role in selective coupling (Ostrowski et al. 1992), and parts of IC3 are required for Gs-coupling and may be required for Gi-coupling, too (Eason and Liggett 1996). Molecular fingerprints unique to each subtype, located 15 to 25 residues upstream from TM6, can be found within the N-terminal region of IC3 and correspond to the following regions in the zebrafish AR sequences: in
2A, residues 274 to 284 (Kx(K/R)xSQxKPG(D/E)); in
2B, residues 396 to 403 (ATx(K/R)GxxL); in
2C, residues 320 to 335 (RxSx(K/R)Sx(D/E)xFxSRR(K/R)R); in
2D (e.g., in
2Da) residues 280 to 282 (RFS) and residues 299 to 303 (RxSWA) were found common to fish and frog (no mammalian orthologs are known). In addition, a second messenger-regulated phosphorylation site for protein kinase C (PKC) in human
2A-AR has been identified at the C-terminus (Liang et al. 2002) of IC3, and sequences at these sites are conserved in fish
2A-ARs. Mammalian
2B-ARs possess a large stretch of glutamic acid residues within IC3 (discussed extensively in Madsen et al. 2002); a stretch of arginine and lysine residues are present in fish
2B-ARs, whereas a histidine repeat is present in
2C-AR from opossum and in lamprey
2-AR.
The 2-AR sequences are most divergent along TM1 near the extracellular surface, near the N-terminus, within EC2, and within most of the long IC3 loop, showing little conservation even among fish and mammalian orthologs. The N-terminal region is N-glycosylated in the bovine rhodopsin crystal structure; N-glycosylation probably takes place in human
2A-ARs and
2C-ARs but not in
2B-AR, which lacks asparagine in this region (e.g., Keefer, Kennedy and Limbird 1994). All of the cloned zebrafish
2-ARs possess one or more asparagine residues within their N-terminal sequence, but their glycosylation states are unknown.
The phylogenetic tree presented in figure 2 is based on an alignment of the available full-length 2-AR sequences, where only the regions unambiguously aligned among the
2-AR subtypes were considered. Only the tree calculated with the maximum-parsimony method is depicted here, but the Neighbor-Joining and maximum-likelihood methods gave very similar branching orders. The cuckoo wrasse "
2F-AR" is a clear ortholog (high bootstrap support) of the zebrafish and mammalian
2C-AR subtypes. The putative
2-AR gene from goldfish (L09064) together with the zebrafish
2Da-AR and
2Db-AR form a separate, fourth
2-AR branch. The pufferfish
2-ARs also group together with their mammalian and zebrafish orthologs. The phylogenetic trees shown in figure 3 are based on alignments of translated partial nucleotide sequences retrieved from GenBank, whose subtypes were identified on the basis of a single tree constructed over all subtypes (data not shown). The available
2-AR sequences of the cluster comprising zebrafish, herring, pufferfish, toothcarp, and seahorse form two distinct groups within each of the four trees, representing the presence of duplicate subtypes, whereas the eel receptors do not always follow this pattern, although they are duplicated in the
2A-AR,
2C-AR, and
2D-AR subtypes, too. Subtypes A to D are present for frog and chameleon, but no duplicate
2-AR subtypes have been found. A single lamprey
2-AR sequence fragment has been identified (see Supplementary Material online), but this sequence is not clearly orthologous to any one of the four
2-AR subtypes.
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Discussion |
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The lengths of the coding sequences vary considerably between the human and zebrafish orthologs, which can be explained for the most part by differences in the length of the third intracellular loop. For GPCRs in general, IC3 loop varies considerably among species orthologs, for example, between the human and pig Y5 NPY receptors (Wraith et al. 2000), as well as among the 2-AR subtypes. The human
2A and
2B subtypes are phosphorylated by GRKs, whereas the human
2C subtype is not. The phosphorylation site has been identified in human
2A-AR (Eason, Moreira, and Liggett 1995), but the phosphorylated serines/threonines remain to be identified in the human
2B-AR. In contrast to human, the fish
2B-ARs possess sequences compatible with a GRK phosphorylation site. At the equivalent location in the kangaroo and opossum
2B-ARs four serines/threonines of the consensus sequence are present, but the required acidic residue is absent; however, a repeat of acidic residues, equivalent to that found in eutherian mammals, is present about five residues upstream. In a naturally occurring variant of human
2B-AR, a deletion of three glutamate residues from the acidic residue repeat results in reduced phosphorylation and impaired desensitization of the receptor by GRKs (Small et al. 2001); this polymorphism is associated with a lowered basal metabolic rate in obese subjects (Heinonen et al. 1999) and an increased risk of acute coronary events, such as myocardial infarction (Snapir et al. 2001; 2003).
We have identified distinct regions of the IC3-loop (approximately 15 to 25 residues upstream from the N-terminal end of TM6), unique to each subtype, that are conserved among fish and mammals, suggesting that they are functionally conserved and contribute to a distinct structural domain characteristic of each subtype. These sequence patterns may play a subtype-specific role within the 2-ARs related to G-protein specificity and binding or to the binding specificity of other intracellular effectors. In addition, these fingerprints provide strong external support that the classification of the
2-ARs into four subtypes is correct, as these fingerprints are not included in the sequence alignment leading to the phylogenetic tree shown in figure 2.
The branching pattern in the phylogenetic tree (fig. 2) does not show obvious support for the hypothesis of two rounds of genome duplication before the divergence of actinopterygian and sarcopterygian lineages about 420 MYA (Amores et al. 1998), most probably because the first and second rounds of duplication took place very close to each other in time, leading to poor resolution of the root of the tree. It is likely that we do not have a sufficient number of phylogenetically informative sites to resolve this issue, and this is reflected in the relatively low bootstrap values at the root of the tree. The low bootstrap values at the root of the tree suggest that other evidence, for example chromosomal mapping data, is needed to resolve the early events in the phylogenetic history of the 2-ARs. The cuckoo wrasse "
2F-AR" is clearly an ortholog of the zebrafish and mammalian
2C-ARs, and we suggest that it should be renamed as such. Likewise, the goldfish
2-AR should be named as
2D-AR. In naming the fourth subtype as
2D-AR (and the duplicate zebrafish genes as adra2da and adra2db) we have followed the IUPHAR Compendium of Receptor Characterization and Classification 2000 and nomenclature guidelines for naming the zebrafish genes. A potential confusion was initially caused by the inappropriate naming of the mouse/rat
2A-AR, and the misnomer should no longer be used.
The 2D-AR subtype appears to be duplicated in zebrafish as well in pufferfish. The nature and timing of gene/genome duplications resulting in duplicated genes in early teleost evolution has been subject to a recent debate (see below). The fourth
2-AR subtype probably represents a result of the second duplication event, and the ancestral parent of the two zebrafish and pufferfish
2D-AR genes present in the last common ancestor of actinopterygians and sarcopterygians has apparently been lost in the mammalian lineage. There are other examples of this phenomenon, the retention of ancient duplicates in the fish lineage and their loss in the mammalian lineage. For example, there is an EVX gene adjacent to HOXA13 and HOXD13 but not at the corresponding position in the HOXB and HOXC clusters in mammals, suggesting that the pregenome-expansion chromosome had an EVX gene at the 5' end of the HOX cluster, and after duplication events, it has been retained in some but not all of the chromosomes. In zebrafish, there is an additional evx gene adjacent to the hoxb cluster (Postlethwait et al. 1998, 2000). Thus, the last common ancestor of zebrafish and mammals must have had an EVX gene adjacent to the HOXB cluster. This has been retained in the zebrafish lineage but lost in the mammalian lineage.
2D-AR may share the functions of its ancestor (the common ancestor of
2D-AR and another
2-AR subtype) as suggested by the DDC (duplication-degeneration-complementation) hypothesis of the preservation of duplicated genes, according to which degenerative mutations in duplicate genes increase rather than reduce the probability of duplicate gene preservation, with the duplicate genes usually sharing the functions of their common ancestor rather than developing new functions (Force et al. 1999).
As shown in figure 3, orthologs of the zebrafish 2D-ARs are present in higher, tetrapod vertebrates such as frog (Rana esculenta) and chameleon. This is consistent with the presence of this gene in the last common ancestor of actinopterygians and sarcopterygians and the loss of this gene in the mammalian lineage after the divergence of reptiles and mammals. Thus, it appears that the divergence of the actinopterygian and sarcopterygian lineages has occurred shortly after the two duplication events. In figure 3, the different duplicated fish
2-ARs are not always consistently named, and the eel
2-AR duplicates cannot always be classified as orthologs of the other duplicates. The eel
2-ARs display a somewhat conserved branching pattern with the group comprising seahorse, pufferfish, cuckoo wrasse, toothcarp, zebrafish, goldfish, and herring, although some eel
2-AR duplicates show low bootstrap values. The positioning of some
2-ARs is obviously not as expected (see for example the shark and chicken
2A-ARs in figure 3). Some of the duplications in different fish species may have taken place independently, such as with the "
2A1" and "
2A2" sequences in eel, or the number of informative sites in the available sequences may be too few to resolve this issue. Resolution of these questions would require much more sequence data from several different species. Additionally, mapping data and the comparison of the chromosomal arrangements of the genes would obviously be helpful. The identity of the single lamprey
2-AR is also impossible to solve with the currently available sequence data; it can either represent a descendant of a gene ancestral to all or represent just two of the four main
2-AR branches. Taylor et al. (2001) date the proposed third round of genome duplication to between 300 and 450 MYA on the basis of data from zebrafish, frog, chicken, human, and mouse. Critics of the hypothesis of a third round of tetraploidization early in teleost evolution point out the possibility of independent local duplications giving rise the abundance of fish genes (Robinson-Rechavi et al. 2001b) but also accept independent partial chromosomal duplications or whole-genome duplications as another possible explanation. Perhaps most importantly, they suggest that the analysis of several fish species and comparative linkage analysis can help to address this question (Robinson-Rechavi et al. 2001a). Our identification of four
2-AR subtype genes with one duplicated subtype in conserved chromosomal segments (see below) supports block duplications comparable to that of the HOX-clusters (Larhammar, Lundin, and Hallbook 2002). The complete octet of
2-ARs in the pufferfish genome is a particularly strong support for the one-to-four-to-eight duplication scheme for the number of paralogs after three rounds of tetraploidizations (Taylor et al. 2001).
The adra2a gene is in a conserved synteny among zebrafish, human, and mouse. The adra2c gene is in a conserved synteny between human and zebrafish. In the mouse, a considerable amount of reorganization of the mouse orthologs of the syntenic group formed by adra2c and other genes on LG1 and their orthologs on human chromosome 4 seem to have taken place among mouse chromosomes 3, 5, and 8. The adra2b gene is in a conserved synteny between zebrafish and mouse (fig. 5). In mouse and cat, orthologs of human chromosomes 20 and 2p are syntenic, implying that this is the ancestral mammalian condition. In zebrafish, too, orthologs of Hsa20 and Hsa2p are syntenic on several chromosomes, especially the duplicated chromosome pair LG17 and LG20. LG8 has one locus from Hsa20, MMP9, and one from Hsa2p, ADRA2B. The zebrafish configuration with mmp9 and adra2b probably represents the ancestral organization, although there are many loci from several other human chromosomes on LG8. Furthermore, the two additional zebrafish ESTs mapped on LG8 and their possible human and mouse orthologs on Hsa20 and Mmu2 support this scheme (see figure 5). The situation with the EST possibly representing the zebrafish adra1a gene is not clear. Because it is based on a short EST sequence, its classification may not be correct. It is not located very close to adra2b and mmp9, and even if it would really represent adra1d, its association might still be random. The genes adra2da and adra2db are located on LG14 and LG21, respectively. Both LG14 and LG21 show conserved syntenies with Hsa5 and Mmu11 and Mmu18, but also with Hsa4 and Hsa10, so our grouping of portions of LG14, LG21, Hsa5, Mmu11, and Mmu18 into the fourth paralogy group is speculative. However, on the basis of the phylogenetic analyses, we conclude that this arrangement is the most plausible one.
In conclusion, the localization of the cloned zebrafish 2A-AR,
2B-AR,
2C-AR,
2Da-AR, and
2Db-AR genes seems to reflect conserved syntenies between zebrafish and human and/or mouse, which corroborates the sequence-based subtype classification presented in this paper. The molecular phylogenetic analysis and the comparative mapping results are consistent with the hypothesis that the initial events in generating different adrenergic receptor subtypes were local duplications, giving rise to ancestors of the
1-AR,
2-AR, and ß-AR genes. All known
2-AR subtype genes (with the possible exception of the pufferfish "
2D2-AR" gene) and most of the ß-AR subtype genes are intronless, as are most vertebrate GPCRs, which has been suggested to reflect retrotransposition as the mechanism of duplication (Gentles and Karlin 1999). However, as the ancestral
1-AR,
2-AR, and ß-AR genes seem to have been located on the same chromosomal segment, it would seem more plausible that they arose from an ancestral adrenoceptor gene by local tandem duplications. After two rounds of block or large-scale (chromosomal, or even genome) duplication, the subtypes evolved by random genetic drift, accompanied by purifying selection, leading to the divergence of sequences among subtypes or species (fig. 6). If the duplications took place as part of two tetraploidization events, one should expect a fourth
2-AR gene copy, as has been found in zebrafish and pufferfish. Orthologs of
2D-AR also seem to be present in at least some other species of fish and even in some tetrapod vertebrates, but so far they have not been found in mammals. However, it is quite common that duplicate genes are either totally lost or present as nonfunctional pseudogenes, as shown by many other gene families, for instance, the HOX-gene cluster (Sharman and Holland 1998). It is very likely that no such gene will be found in the well-characterized human, mouse, and rat genomes, but the existence of such a gene would require considerable reevaluation of the current concepts concerning the functions and physiological roles of the
2-ARs.
|
It is interesting to note that the duplication scheme for the adrenergic receptors is very similar to that of the neuropeptide Y receptors located on the same chromosomal segments in mammals (Wraith et al. 2000). Although orthologous NPY receptors have not yet been identified in zebrafish (Larhammar et al. 2001), recent observations in a cartilaginous fish, the spiny dogfish (a shark), confirm that the gene duplications took place before the gnasthostome radiation. Furthermore, the shark NPY receptor study showed that the anatomical distribution of receptor mRNA (as detected by RT-PCR) seems to be quite different between sharks and mammals (Salaneck et al. 2003). ESTs representing the zebrafish 2A-AR and
2B-AR, the cDNA representing
2C-AR, and the RT-PCR results for
2Db-AR (and possibly also for
2Da-AR) show only that these receptor genes are transcribed, with no correlation of the expression to anatomical structures or possibility to deduce their functions. Our recent unpublished results from HPLC indicate that the concentration of noradrenaline in the adult zebrafish brain is substantial and comparable to that in rat brain: 4.53 ± 0.97 nmol/mg tissue (mean ± SD, n = 15). Our preliminary binding assays indicate that the
2-AR antagonist radioligand [ethyl-3H]RS-79948-197 displays binding in membranes prepared from zebrafish brain homogenates comparable with that observed for the cloned zebrafish receptors. A receptor density (Bmax) of 475 fmol/mg protein and an affinity constant (Kd) of 0.1 nM was obtained in a single experiment on 21 pooled brains. The Kd values determined for the different zebrafish
2-ARs (subtypes A, B, C, Da, and Db), expressed separately in CHO cells, ranged from 0.1 to 0.7 nM (Ruuskanen and Scheinin, unpublished data). Work is in progress to investigate the tissue distribution patterns of the zebrafish
2-AR subtypes.
In this work, we have shown that the events generating the diverse 2-AR subtypes took place before the divergence of ancestors of teleost fish and tetrapods, and we have identified a fourth, duplicated
2-AR subtype that gives further support for the ancient fish-specific genome duplication hypothesis and opens new insights into the study of the adrenergic receptors. These results indicate evolutionary pressure to maintain a larger set of
2-AR subtypes in the fish genomes than is known for mammals and suggest distinct and important biological functions for each of these receptor subtypes.
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