The opioid peptides and receptors have prominent roles in pain transmission and reward mechanisms in mammals. The evolution of the opioid receptors has so far been little studied, with only a few reports on species other than tetrapods. We have investigated species representing a broader range of vertebrates and found that the four opioid receptor types (delta, kappa, mu, and NOP) are present in most of the species. The gene relationships were deduced by using both phylogenetic analyses and chromosomal location relative to 20 neighboring gene families in databases of assembled genomes. The combined results show that the vertebrate opioid receptor gene family arose by quadruplication of a large chromosomal block containing at least 14 other gene families. The quadruplication seems to coincide with, and, therefore, probably resulted from, the two proposed genome duplications in early vertebrate evolution. We conclude that the quartet of opioid receptors was already present at the origin of jawed vertebrates Ϸ450 million years ago. A few additional opioid receptor gene duplications have occurred in bony fishes. Interestingly, the ancestral receptor gene duplications coincide with the origin of the four opioid peptide precursor genes. Thus, the complete vertebrate opioid system was already established in the first jawed vertebrates.chromosome ͉ G protein-coupled receptor ͉ gene duplication S everal opioid peptides, including endorphin and enkephalins, are important regulators of nociceptive neurotransmission and reward mechanisms in mammals. Specific binding sites in the brain for opioid compounds were first reported in 1973 (1-3), and it was soon evident that more than one type of binding site existed (4). Subsequently three distinct opioid receptors were identified and designated delta, kappa, and mu. These receptors were cloned and found to be encoded by separate genes belonging to the superfamily of rhodopsin-like G protein-coupled receptors (GPCRs) (5-8). The genes for the opioid receptors (OPR) have been named OPRD1 (delta), OPRK1 (kappa), and OPRM1 (mu) by the HUGO Gene Nomenclature Committee (HGNC).Homology searches resulted in the discovery of a fourth receptor in both rodents and humans initially named ORL1 for opioid receptor-like (9) or LC132 (10). This receptor shows 48-49% identity to the other three human receptors, which display 55-58% identity among one another. The receptor has been named NOP by the International Union of Basic and Clinical Pharmacology and its gene has been named OPRL1 by HGNC. An endogenous peptide ligand with some similarity to the other opioid peptides was discovered and named nociceptin (11) or orphanin FQ (12).The evolution of the endogenous opioid peptide ligands has been studied extensively and the major peptide ligands are generated from four prepropeptides that are encoded by separate genes in tetrapods. The genes arose by duplications in the common ancestor of tetrapods and bony fishes (13). Opioid receptor sequences have been reported for a few nonmammalian tetrapods (1...

BackgroundThe opioid system is involved in reward and pain mechanisms and consists in mammals of four receptors and several peptides. The peptides are derived from four prepropeptide genes, PENK, PDYN, PNOC and POMC, encoding enkephalins, dynorphins, orphanin/nociceptin and beta-endorphin, respectively. Previously we have described how two rounds of genome doubling (2R) before the origin of jawed vertebrates formed the receptor family.Methodology/Principal FindingsOpioid peptide gene family members were investigated using a combination of sequence-based phylogeny and chromosomal locations of the peptide genes in various vertebrates. Several adjacent gene families were investigated similarly. The results show that the ancestral peptide gene gave rise to two additional copies in the genome doublings. The fourth member was generated by a local gene duplication, as the genes encoding POMC and PNOC are located on the same chromosome in the chicken genome and all three teleost genomes that we have studied. A translocation has disrupted this synteny in mammals. The PDYN gene seems to have been lost in chicken, but not in zebra finch. Duplicates of some peptide genes have arisen in the teleost fishes. Within the prepropeptide precursors, peptides have been lost or gained in different lineages.Conclusions/SignificanceThe ancestral peptide and receptor genes were located on the same chromosome and were thus duplicated concomitantly. However, subsequently genetic linkage has been lost. In conclusion, the system of opioid peptides and receptors was largely formed by the genome doublings that took place early in vertebrate evolution.

Comparative studies of proteins often face the problem of distinguishing a true orthologue (species homologue) from a paralogue (a gene duplicate). This identification task is particularly challenging for endocrine peptides and neuropeptides because they are short and usually have several invariant positions. For some peptide families, this has led to a terminology with peptide names relating to the first species where a specific peptide sequence was determined, such as chicken or salmon gonadotropin-releasing hormone, or names that highlight amino acid differences, e.g., Lys-vasopressin. With accumulating information from multiple species, such a terminology becomes almost impenetrable for nonexperts and difficult even for aficionados. The sequenced genomes offer a new way to distinguish orthologues and paralogues, namely by location of the genes relative to neighboring genes on the chromosomes. In addition, the genome databases can ideally provide a complete listing of the family members in each species. Many vertebrate gene families have expanded in the two basal tetraploidizations (2R) and the teleost fish third tetraploidization (3R), after which some vertebrate lineages have lost some of the duplicates. We review here some peptide families (neuropeptide Y, oxytocin-vasopressin, and somatostatin) where genomic information helps simplify nomenclature. This approach is useful also for other gene families, such as peptide receptors.

The opioid receptor family in mammals has four members called delta, kappa, mu, and NOP (the nociceptin/orphanin receptor). We show here that they arose from a common ancestral gene through quadruplication of a large chromosomal region, presumably in the two basal vertebrate tetraploidizations. The four opioid peptide precursor genes have a more complicated evolutionary history involving chromosomal rearrangements but nevertheless seem to have arisen in the same time period as the receptors. Thus the system of opioid peptides and receptors was already established approximately 450 Ma at the dawn of gnathostome evolution.