Cloning and Characterization of a Novel Neuropeptide Y Receptor Subtype in the Zebrafish

Abstract

Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) form a family of structurally related peptides. As we have previously isolated clones for NPY and PYY from the zebrafish (Danio rerio), we wished to clone the receptors for these peptides to allow correlation of ligand and receptor distribution. We describe here the cloning and functional expression of a receptor with equally high identity to the NPY-Y1 receptor as to the recently cloned Y4/PP1 and Y6 receptors with an overall amino acid sequence identity of approximately 50%. Furthermore, the zebrafish receptor gene lacks the intron present in the coding region in vertebrate Y1 genes. These features strongly suggest that the zebrafish receptor represents a separate subtype. Hence, we have named it zYb for zebrafish Y-receptor b. (We have also discovered a unique receptor called zYa.) The zYb receptor has a binding profile that is reminiscent of Y1 with affinities for NPY and PYY in the low picomolar range, whereas affinities for Y2-selective ligands are considerably lower. It couples to adenylyl cyclase by inhibiting cAMP synthesis. Receptor mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR) in brain, eye, and intestine. The binding profile and amino acid identity show that the zebrafish zYb receptor is related to Y1 but represents a distinct subtype that is likely to be present also in mammals.

The Syntenic Relationship of the Zebrafish and Human Genomes

“…For example, our WU-BLAST analysis failed to distinguish between human ACTB (on Hsa1), ACTC (on Hsa15), and ACTG1 (on Hsa17) as the most likely ortholog for zebrafish bact2 (Kelly and Reversade 1997). The map position for bact2 on LG3 (Geisler et al 1999) near Pyy (on Hsa17; Lundell et al 1997) argues that bact2 is the zebrafish ortholog for ACTG1, rather than ACTB or ACTC. Similarly, WU-BLAST analysis fails to unambigu- .…”

Birth and death of neuropeptide Y receptor genes in relation to the teleost fish tetraploidization

“…A preliminary version of a part of this study was previously published (Salaneck et al, 2005) before the zebrafish Y 1 gene was identified. Also, we wanted to better understand the evolutionary history of the two subtypes called Y 8 that have been found in euteleosts, namely Y 8a and Y 8b , initially called Yc and Yb, respectively (Lundell et al, 1997;Ringvall et al, 1997). A single Y 8 gene exists in frogs, mammals and chicken.…”

“…The Acba Y 1 sequence was extended in the 5′ direction using the inverse PCR technique described earlier (Salaneck et al, 2003). BLAST searches using the human NPY receptor sequences and previously identified Danio rerio sequences (Lundell et al, 1997;Ringvall et al, 1997;Starback et al, 1999;Fredriksson et al, 2004;Fredriksson et al, 2006) were used to make a preliminary survey of the NPY receptor repertoire in the fully sequenced genomes of Danio rerio, Oryzias latipes, Tetraodon nigroviridis, Takifugu rubribes and Gasterosteus aculeatus (see Supplementary Table 2). In D. rerio the sequence for Y 1 was obtained by PCR with specific primers based on the predicted coding sequence of the Y 1 sequence found in the zebrafish genome database and cloned into TOPO vector and sequenced as described above.…”

Evolutionary perspective on the NPY-PYY-PP peptides and their receptors

“…Amphibians have all of the mammalian subtypes and in addition Y7, as in chicken, and also the Y1-subfamily receptor previously described in fish, namely Y8, previously called Yb [92]. Only the Y1, Y2 and Y7 sequences have yet been published [93,94] and work is in progress to characterise all receptors.…”

“…Prior to the initiation of these genome sequencing projects three Y1-like receptors had been cloned from zebrafish, but did not seem to represent orthologues to any of the mammalian Y1, Y4 or Y6 genes. These fish sequences were therefore tentatively regarded as novel genes and given distinct gene names, namely Ya, Yb and Yc [92,95,96]. As additional Y receptor sequences were reported, the relationships of these genes have become clearer.…”