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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy General and Comparative Endocrinology 177 (2012) 322–331 Contents lists available at SciVerse ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen Profiles in Comparative Endocrinology Characterization of the neuropeptide Y system in the frog Silurana tropicalis (Pipidae): Three peptides and six receptor subtypes G. Sundström a,1, B. Xu a, T.A. Larsson a,2, J. Heldin a,1, C.A. Bergqvist a, R. Fredriksson a, J.M. Conlon b, a c a, I. Lundell , R.J. Denver , D. Larhammar ⇑ a Department of Neuroscience, Uppsala University, Box 593, SE-75124 Uppsala, Sweden b Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, 17666 Al-Ain, United Arab Emirates c Department of Molecular, Cellular and Developmental Biology, The University of Michigan, 3065C Kraus Building, Ann Arbor, MI 48109-1048, USA article info abstract Article history: Neuropeptide Y and its related peptides PYY and PP (pancreatic polypeptide) are involved in feeding Available online 4 May 2012 behavior, regulation of the pituitary and the gastrointestinal tract, and numerous other functions. The peptides act on a family of G-protein coupled receptors with 4–7 members in jawed vertebrates. We Keywords: describe here the NPY system of the Western clawed frog Silurana (Xenopus) tropicalis. Three peptides, NPY NPY, PYY and PP, were identified together with six receptors, namely subtypes Y1, Y2, Y4, Y5, Y7 and PYY Y8. Thus, this frog has all but one of the ancestral seven gnathostome NPY-family receptors, in contrast Pancreatic polypeptide to mammals which have lost 2–3 of the receptors. Expression levels of mRNA for the peptide and receptor G-protein-coupled receptor Silurana tropicalis genes were analyzed in a panel of 19 frog tissues using reverse transcriptase quantitative PCR. The pep- Xenopus tropicalis tide mRNAs had broad distribution with highest expression in skin, blood and small intestine. NPY mRNA was present in the three brain regions investigated, but PYY and PP mRNAs were not detectable in any of these. All receptor mRNAs had similar expression profiles with high expression in skin, blood, muscle and heart. Three of the receptors, Y5, Y7 and Y8, could be functionally expressed in HEK-293 cells and char- acterized with binding studies using the three frog peptides. PYY had the highest affinity for all three receptors (Ki 0.042–0.34 nM). Also NPY and PP bound to the Y8 receptor with high affinity (0.14 and 0.50 nM). The low affinity of NPY for the Y5 receptor (100-fold lower than PYY) differs from mammals and chicken. This may suggest a less important role of NPY on Y5 in appetite stimulation in the frog com- pared with amniotes. In conclusion, our characterization of the NPY system in S. tropicalis with its six receptors demonstrates not only greater complexity than in mammals but also some interesting differ- ences in ligand–receptor preferences. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction the elephant shark (Callorhincus millii) [27]. In mammals, five of these receptor genes are still present, one of which, Y6, is a pseu- Neuropeptide Y receptors are G-protein coupled receptors in- dogene in several mammals including human. The Y6 gene in volved in numerous physiological processes. In mammals these in- chicken is fully functional [5]. A few NPY receptors have been pre- clude appetite regulation, biological rhythms, anxiety, pain, bone viously reported for frogs after cloning, namely Y1 in the African formation, and release of pituitary hormones [40,50]. We have pre- clawed frog Xenopus laevis [4], and Y7 in the frog Pelophylax escu- viously proposed that the ancestral jawed vertebrate had seven lentus (previously called Rana esculenta) and in the western clawed NPY receptors, Y1, Y2, Y4, Y5, Y6, Y7 and Y8 which can be divided frog Silurana (Xenopus) tropicalis [16]. The decision whether to de- into three distinct subfamilies, the Y1 subfamily (Y1, Y4, Y6 and scribe the western clawed frog as S. tropicalis or Xenopus tropicalis Y8), the Y2 subfamily (Y2 and Y7) and the single gene subfamily remains controversial. Cladistic analysis based upon nucleotide se- Y5 [26,27]. All of these receptors are present in a cartilaginous fish, quences of mitochondrial genes strongly supports the monophyly of Xenopus + Silurana which are united in the Xenopodinae but Corresponding author. the use of two genera ‘‘underscores trenchant biological and his- ⇑ E-mail address: [email protected] (D. Larhammar). torical differences between the two clades’’ [15]. 1 Present address: Department of Medical Biochemistry and Microbiology, Uppsala The ligands are members of the NPY family of peptides, which University, Box 582, SE-75123 Uppsala, Sweden. also includes PYY (peptide YY) and PP (pancreatic polypeptide) in 2 Present address: Developmental Biology Unit/Structural and Computational tetrapods. The peptide genes code for pre-propeptides that are pro- Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 cessed by specific peptidases to generate the active peptides. All of Heidelberg, Germany. 0016-6480/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygcen.2012.04.027 Author's personal copy G. Sundström et al. / General and Comparative Endocrinology 177 (2012) 322–331 323 the peptides examined in the NPY family consist of 36 amino acid algorithm to estimate the random starting tree. The number of residues, except for chicken PYY and Burmese python PP that con- substitution rate categories was set to 8 and amino acid substitu- sist of 37 and 35 amino acids, respectively [9,49]. NPY in mammals tion model parameters were estimated from the dataset. Both the is mainly expressed in the brain and is one of the most orexigenic NNI and SPR tree improvement methods were used together with peptides known. It has been shown to induce feeding in different tree topology and branch length optimization. species including Siberian hamster, guinea pig, the frog X. laevis, and goldfish [11,28,29,38]. PYY and PP are mainly expressed in 2.2. Cloning of partial NPY receptor gene sequences in P. esculentus endocrine cells in the gastrointestinal tract in mammals. PYY in its truncated form PYY3–36 and PP have important roles as inhibi- Genomic DNA for the frog Pelohpylax esculentus was kindly pro- tors of feeding in mammals [2,3,24,39]. All three peptides also have vided by Dr. Isabelle Lihrmann, Universit of Rouen, France. This several other roles in neuronal, cardiovascular or gastrointestinal species was previously called R. esculenta and is considered to be contexts [40,50]. Endogenous truncated PYY is not known outside a hybrid between the pool frog Pelophylax lessonae (synonyms Rana mammals where the peptide is cleaved by dipeptidyl peptidase IV lessonae, Rana esculenta lessonae) and the marsh frog Pelophylax rid- [36]. This peptidase cannot cleave between two proline residues ibundus (Rana ridibunda). Degenerate PCR with primers based on and all known non-mammalian PYY sequences have the amino gene sequences for NPY-family receptors from several mammals acids Tyr-Pro-Pro, suggesting resistance to this type of cleavage. and chicken was run on P. esculentus genomic DNA using the stoffel A PYY peptide was isolated from the frog Phyllomedusa bicolor in Taq polymerase (Applied Biosystems) and the following PCR the 1990s [37]. It was named skin PYY due to its main location, but conditions: 120 s at 95 °C for one cycle, thereafter 30 s at 95 °C, alignment with PYY from other species identified it as a regular touch-down from 55 to 42 °C for 45 s, and 60 s at 72 °C for 20 PYY sequence. Subsequently, all three members of the peptide cycles, followed by 20 cycles with 30 s at 95 °C, 45 s at 42 °C and family have been isolated or cloned from a variety of frog species 60 s at 72 °C finishing with 5 min at 72 °C. The PCR products were [6,7,8,18,35,34,41,46]. cloned using the TOPO-cloning kit (Invitrogen), sequenced using The NPY system has been suggested to be involved in several the BigDye V3 terminator sequencing kit (Applied Biosystems) aspects of frog physiology and behaviors including background and the extension products were analyzed on an ABI 310 automatic adaptation [17], visual neurotransmission [44], antimicrobial sequencer. The sequence was compared to the GenBank database activity [14,48] and feeding [10]. To facilitate more detailed studies using the On-Line BLASTX program. of these and other effects of the NPY-family peptides, we decided to clone the NPY-family receptors in a frog. We initially attempted 2.3. Sequencing and expression constructs for S. tropicalis receptors this in the frog P. esculentus and identified four distinct receptors using degenerate PCR primers. However, these sequences were Sequences corresponding to the entire coding regions of six S. not full-length and therefore did not allow functional expression. tropicalis NPY receptors were amplified from genomic DNA. The We therefore turned to the genome of S. tropicalis [21] and were PCR products were directionally cloned into the pcDNA3 vector able to identify three receptor genes orthologous to those of P.