Evolution of vertebrate opioid receptors Susanne Dreborg, Go¨ rel Sundstro¨ m, Tomas A. Larsson, and Dan Larhammar* Department of Neuroscience, Uppsala University, Box 593, SE-75124 Uppsala, Sweden Edited by Tomas Ho¨kfelt, Karolinska Institutet, Stockholm, Sweden, and approved August 15, 2008 (received for review June 9, 2008) The opioid peptides and receptors have prominent roles in pain Many vertebrate gene families have been found to have transmission and reward mechanisms in mammals. The evolution expanded in the early stages of vertebrate evolution, before the of the opioid receptors has so far been little studied, with only a radiation of jawed vertebrates. However, the high degree of few reports on species other than tetrapods. We have investigated sequence divergence over such large evolutionary distances species representing a broader range of vertebrates and found that often obscures orthology–paralogy relationships. Investigation the four opioid receptor types (delta, kappa, mu, and NOP) are of conserved synteny may facilitate identification of orthologs present in most of the species. The gene relationships were and gives important clues to the mechanisms by which the genes deduced by using both phylogenetic analyses and chromosomal were duplicated. We used this approach to investigate the location relative to 20 neighboring gene families in databases of evolution of a few other gene families, namely the neuropeptide assembled genomes. The combined results show that the verte- Y (NPY) family of peptides (27) and the large family of NPY brate opioid receptor gene family arose by quadruplication of a receptors (28). These families were found to have expanded as large chromosomal block containing at least 14 other gene fami- a result of extensive chromosome duplications, most likely lies. The quadruplication seems to coincide with, and, therefore, resulting from two tetraploidizations, i.e., genome duplications, probably resulted from, the two proposed genome duplications in that occurred early in vertebrate evolution (29). These genome early vertebrate evolution. We conclude that the quartet of opioid duplications, often referred to as 1R and 2R, occurred after the receptors was already present at the origin of jawed vertebrates divergence of tunicates and lancelets (30) from vertebrates but Ϸ450 million years ago. A few additional opioid receptor gene before the divergence of cartilaginous fishes and bony verte- duplications have occurred in bony fishes. Interestingly, the brates (31). For the cyclostomes (lampreys and hagfishes) the ancestral receptor gene duplications coincide with the origin of picture is not completely clear but based on analyses of a limited the four opioid peptide precursor genes. Thus, the complete number of gene families they seem to have undergone the first tetraploidization (1R) but not the second (2R) (32–36). vertebrate opioid system was already established in the first As the opioid receptor genes are located on four different jawed vertebrates. chromosomes in human (1, 6, 8, and 20) we decided to investigate whether they arose by duplication of a single ancestral opioid chromosome ͉ G protein-coupled receptor ͉ gene duplication receptor gene in the two tetraploidizations. Other investigators have also suggested that studies of chromosomal location may everal opioid peptides, including endorphin and enkephalins, shed light on opioid receptor evolution (37). We describe here Sare important regulators of nociceptive neurotransmission an investigation, using a combination of sequence-based phy- and reward mechanisms in mammals. Specific binding sites in the logenies and gene locations for the opioid receptors and their brain for opioid compounds were first reported in 1973 (1–3), neighboring families that shows that they expanded by gene and it was soon evident that more than one type of binding site duplications in conjunction with the proposed tetraploidizations existed (4). Subsequently three distinct opioid receptors were in early vertebrate evolution. identified and designated delta, kappa, and mu. These receptors were cloned and found to be encoded by separate genes belong- Results ing to the superfamily of rhodopsin-like G protein-coupled To investigate whether the opioid receptor genes arose by receptors (GPCRs) (5–8). The genes for the opioid receptors duplications of a single ancestral gene in the two basal vertebrate (OPR) have been named OPRD1 (delta), OPRK1 (kappa), and tetraploidizations we have analyzed the opioid receptor gene OPRM1 (mu) by the HUGO Gene Nomenclature Committee family and 20 of the neighboring gene families phylogenetically. (HGNC). Specifically, we wanted to find out whether these gene families Homology searches resulted in the discovery of a fourth were duplicated in the same time period, i.e., after the diver- receptor in both rodents and humans initially named ORL1 for gence of invertebrate chordates and vertebrates but before the opioid receptor-like (9) or LC132 (10). This receptor shows divergence of bony fishes and tetrapods because this is the time 48–49% identity to the other three human receptors, which span in which the two tetraploidizations took place. The gene display 55–58% identity among one another. The receptor has families were analyzed phylogenetically by making both neigh- been named NOP by the International Union of Basic and bor-joining (NJ) trees and quartet-puzzling maximum likelihood Clinical Pharmacology and its gene has been named OPRL1 by (QP) trees in which species that diverged before 2R were used HGNC. An endogenous peptide ligand with some similarity to as outgroups to provide relative dating of the gene duplications. the other opioid peptides was discovered and named nociceptin The opioid receptors were analyzed in human (Homo sapiens), (11) or orphanin FQ (12). mouse (Mus musculus), dog (Canis familiaris), cow (Bos taurus), The evolution of the endogenous opioid peptide ligands has been studied extensively and the major peptide ligands are Author contributions: S.D., G.S., T.A.L., and D.L. designed research; S.D. and G.S. performed generated from four prepropeptides that are encoded by sepa- research; S.D., G.S., T.A.L., and D.L. analyzed data; and S.D., G.S., T.A.L., and D.L. wrote the rate genes in tetrapods. The genes arose by duplications in the paper. common ancestor of tetrapods and bony fishes (13). Opioid The authors declare no conflict of interest. receptor sequences have been reported for a few nonmammalian This article is a PNAS Direct Submission. tetrapods (14–18) and a few teleost fishes (19–24), and a partial *To whom correspondence should be addressed. E-mail: [email protected]. sequence has been reported for a hagfish (19). Functional studies This article contains supporting information online at www.pnas.org/cgi/content/full/ EVOLUTION in amphibians and bony fishes have shown that the opioid system 0805590105/DCSupplemental. is involved in nociception also in these species (25, 26). © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805590105 PNAS ͉ October 7, 2008 ͉ vol. 105 ͉ no. 40 ͉ 15487–15492 Downloaded by guest on September 25, 2021 Dme.3L Bfl.sc203 Hsa.17.SSTR2 gray short-tailed opossum (Monodelphis domestica), chicken 63 Hsa.22.SSTR3 (Gallus gallus), western clawed frog (Xenopus tropicalis), rough- Hsa.16.SSTR5 Hsa.20.SSTR4 skinned newt (Taricha granulosa), zebrafish (Danio rerio), 95 Hsa.14.SSTR1 71 Hsa.20.NPBWR2 medaka (Oryzias latipes), stickleback (Gasterosteus aculeatus), NPBWR2 71 Bta.Un.b and spotted green pufferfish (Tetraodon nigroviridis). We found Mdo.1.a* at least four opioid receptor genes in the genome databases for Ola.7 Gac.XII.a most of these species. The zebrafish and medaka have duplicates 65 Dre.23..b of the OPRK1 and/or the OPRD1 genes, whereas the OPRL1 52 Xtr.sc1400.a* Hsa.8.NPBWR1 63 gene is missing in medaka and the OPRM1 gene is missing in Bta.14.b NPBWR1 80 Cfa.29.a spotted green pufferfish [Fig. 1 and supporting information (SI) 66 96 Mmu.1.b Fig. S1]. However, this does not necessarily mean that these Mdo.3.b genes have been lost because their absence may simply be due to 57 Cmi.AAVX01065032.1 Xtr.sc83.a* 84 incomplete sequencing of the genomes or poor genome assembly 66 Gga.2.a in the databases. The human opioid receptor sequences were Pma.co2020 97 Pma.co7520 Hsa.20.OPRL1 used for blastp searches of the Florida lancelet (Branchiostoma 90 Bta.13 floridae) database and the elephant shark (Callorhinchus milii) 94 Cfa.24 91 database, but this produced no reasonable hits. Mmu.2 OPRL1 The phylogenetic tree (Fig. 1) shows that the neuropeptide 76 Mdo.1.b 65 Tgr.L B/W (NPBW) receptors are closely related to the opioid recep- 95 Gga.20 57 Xtr.sc1400.b tors, i.e., closer than to any other GPCRs. This is supported by Dre.23.a 94 Tni.Un.b* the chromosomal locations because NPBWR1 is located next to 99 100 Gac.XII.b OPRK1 on human chromosome 8 (287 kb downstream) and Hsa.8.OPRK1 50 Cfa.29.b NPBWR2 is situated on human chromosome 20 next to OPRL1 91 Bta.14.a 78 (only 26 kb downstream) (Fig. 2 and 3). Such close linkage can Mmu.1.a 98 be seen in most of the species that have NPBW receptors. An Mdo.3.a 97 Gga.2.b OPRK1 earlier split of the somatostatin receptors from the opioid/ Tgr.K NPBW receptors is suggested by the fact that the vertebrate 96 Xtr.sc83.b* 85 Ola.20* somatostatin receptors cluster with a Florida lancelet sequence 91 93 Tni.6* and a fruit fly (Drosophila melanogaster) allostatin C receptor Gac.XXI* 52 Ola.17 sequence in the phylogenetic tree (Fig. 1). Both the opioid and 64 92 95 Gac.III the NPBW receptor families seem to have expanded in the time Tni.Un.a* 85 Dre.2 period coinciding with the tetraploidizations in early vertebrate Hsa.6.OPRM1 62 Cfa.1 evolution.