Persistence of duplicated PAC1 receptors in the teleost, Sparus auratus

Abstract

Background:Duplicated genes are common in vertebrate genomes. Their persistence is assumed to be either a consequence of gain of novel function (neofunctionalisation) or partitioning of the function of the ancestral molecule (sub-functionalisation). Surprisingly few studies have evaluated the extent of such modifications despite the numerous duplicated receptor and ligand genes identified in vertebrate genomes to date. In order to study the importance of function in the maintenance of duplicated genes, sea bream (Sparus auratus) PAC1 receptors, sequence homologues of the mammalian receptor specific for PACAP (Pituitary Adenylate Cyclase-Activating Polypeptide), were studied. These receptors belong to family 2 GPCRs and most of their members are duplicated in teleosts although the reason why both persist in the genome is unknown.Results:Duplicate sea bream PACAP receptor genes (sbPAC1A and sbPAC1B), members of family 2 GPCRs, were isolated and share 77% amino acid sequence identity. RT-PCR with specific primers for each gene revealed that they have a differential tissue distribution which overlaps with the distribution of the single mammalian receptor. Furthermore, in common with mammals, the teleost genes undergo alternative splicing and a PAC1Ahop1 isoform has been characterised. Duplicated orthologous receptors have also been identified in other teleost genomes and their distribution profile suggests that function may be species specific. Functional analysis of the paralogue sbPAC1s in Cos7 cells revealed that they are strongly stimulated in the presence of mammalian PACAP27 and PACAP38 and far less with VIP (Vasoactive Intestinal Peptide). The sbPAC1 receptors are equally stimulated (LOGEC50 values for maximal cAMP production) in the presence of PACAP27 (-8.74 ± 0.29 M and -9.15 ± 0.21 M, respectively for sbPAC1A and sbPAC1B, P > 0.05) and PACAP38 (-8.54 ± 0.18 M and -8.92 ± 0.24 M, respectively for sbPAC1A and sbPAC1B, P > 0.05). Human VIP was found to stimulate sbPAC1A (-7.23 ± 0.20 M) more strongly than sbPAC1B (-6.57 ± 0.14 M, P < 0.05) and human secretin (SCT), which has not so far been identified in fish genomes, caused negligible stimulation of both receptors.Conclusion:The existence of functionally divergent duplicate sbPAC1 receptors is in line with previously proposed theories about the origin and maintenance of duplicated genes. Sea bream PAC1 duplicate receptors resemble the typical mammalian PAC1, and PACAP peptides were found to be more effective than VIP in stimulating cAMP production, although sbPAC1A was more responsive for VIP than sbPAC1B. These results together with the highly divergent pattern of tissue distribution suggest that a process involving neofunctionalisation occurred after receptor duplication within the fish lineage and probably accounts for their persistence in the genome. The characterisation of further duplicated receptors and their ligands should provide insights into the evolution and function of novel protein-protein interactions associated with the vertebrate radiation.

Differential expression pattern of pituitary adenylate cyclase-activating polypeptide (PACAP) alternative splicing variants and its receptors in the immune system of rainbow trout (Oncorhynchus mykiss)

“…1B). To date, PACAP receptor expression in the brain of vertebrates is well documented (reviewed in [5,40]) and in fish, in concordance with our results, the expression of PAC1 receptor has been detected in the brain of goldfish (Carassius auratus) [41], zebrafish (Danio rerio) [42], sea bream (Sparus auratus) [43] and tilapia (Oreochromisis mossambicus) [44]. In addition, the expression of the VPAC1 and VPAC2-like transcripts in the brain of the rainbow trout (O. mykiss) was recently reported [22].…”

Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: 20 Years after the Discovery

“…9). In addition, a teleost-specific duplication has occurred (Cardoso et al, 2007a) that is in line with the proposed partial or whole genome duplication event in fish (3R).…”

“…6), including sheep (Miyata et al, 1989), rat , human , mouse (Okazaki et al, 1995), pig (Kollers et al, 2006), and cattle (Sayasith et al, 2007). The sequence of PACAP has also been established in representative species of nonmammalian vertebrates, notably the chicken Galus domesticus (McRory and Sherwood, 1997), the lizard Podarcis sicula (Valiante et al, 2007), the frogs R. ridibunda (Chartrel et al, 1991) and Xenopus laevis , the lungfish Protopterus dolloi , the salmon Oncorhynchus nerka (Parker et al, 1993), the catfish Clarias macrocephalus (McRory et al, 1995), the stargazer Uranoscopus japonicus (Matsuda et al, 1997), the channel catfish Ictalurus punctatus (Small and Nonneman, 2001), the Arctic grayling Thymallus arcticus, the yellowtail flounder Pleuronectes ferrugineu, the Atlantic halibut Hippoglossus hippoglossus, the Atlantic cod Gadus morhua (Xu and Volkoff, 2009), the sturgeon Ascipenser transmontanus (Adams et al, 2002), the zebrafish Danio rerio (Krueckl et al, 2003;Wang et al, 2003), the seabream Sparus aurata (Cardoso et al, 2007a), the fugu Takifugu rubripes (Cardoso et al, 2007a), the African cichlid fish Haplochromis burtoni (Grone et al, 2007), and the grass carp Ctenopharyngodon idella (Sze et al, 2007). The N-terminal 1-to-27 region of PACAP, which is responsible for the biological activity of the peptide, has been fully conserved in all vertebrate species, except the chicken, sturgeon, and stargazer/flounder/halibut, with one amino acid substitution at positions 2, 15, and 20, respectively (Fig.…”

Newly-identified receptors for peptide histidine-isoleucine and GHRH-like peptide in zebrafish help to elucidate the mammalian secretin superfamily

“…However, we show here that the PAC1R-short isoform in zebrafish has a significant dose-dependent response to PACAPs and VIP in both cAMP and IP assays. Although VIP is not as potent as the PACAP (EC 50 Z10 K9 M) in activating the PAC1R, the EC 50 value of zebrafish VIP is a physiological K7 M), although less than the PACAPs (EC 50 Z10 K9 M; Cardoso et al 2007b). Relevant also is a report that there are at least 14 variants of the PAC1R in human neuroblastoma cells due to alternative splicing and some of these variants also respond to VIP stimulation in a dose-dependent manner in the cAMP and IP signaling pathways (Lutz et al 2006).…”