Interactions of zebrafish peptide YYb with the neuropeptide Y-family receptors Y4, Y7, Y8a, and Y8b

Abstract

The neuropeptide Y (NPY) system influences numerous physiological functions including feeding behavior, endocrine regulation, and cardiovascular regulation. In jawed vertebrates it consists of 3-4 peptides and 4-7 receptors. Teleost fishes have unique duplicates of NPY and PYY as well as the Y8 receptor. In the zebrafish, the NPY system consists of the peptides NPYa, PYYa, and PYYb (NPYb appears to have been lost) and at least seven NPY receptors: Y1, Y2, Y2-2, Y4, Y7, Y8a, and Y8b. Previously PYYb binding has been reported for Y2 and Y2-2. To search for peptide-receptor preferences, we have investigated PYYb binding to four of the remaining receptors and compared with NPYa and PYYa. Taken together, the most striking observations are that PYYa displays reduced affinity for Y2 (3 nM) compared to the other peptides and receptors and that all three peptides have higher affinity for Y4 (0.028-0.034 nM) than for the other five receptors. The strongest peptide preference by any receptor selectivity is the one previously reported for PYYb by the Y2 receptor, as compared to NPY and PYYa. These affinity differences may be helpful to elucidate specific details of peptide-receptor interactions. Also, we have investigated the level of mRNA expression in different organs using qPCR. All peptides and receptors have higher expression in heart, kidney, and brain. These quantitative aspects on receptor affinities and mRNA distribution help provide a more complete picture of the NPY system.

Keywords: G-protein-coupled receptor; NPY; PYY; evolution; zebrafish.

Figure 1

Alignment of the mature peptides in the NPY family in zebrafish. Amino acid differences are shadowed. Proposed tertiary structures are described at the top.

Figure 2

Binding curves for each receptor. Panels (A–D) are the representative binding curves for Y4, Y7, Y8a and Y8b, respectively. The x-axis is the logarithmic concentration of unlabeled ligand. All experiments were done in duplicate. Note that the curve shown for the binding of zfPYYa to Y8b displays greater difference to the other peptides than if all experiments are considered (see Table 2).

Figure 3

Expression data for neuropeptide Y peptides in a panel of eight zebrafish organs. Error bars display the standard error of the mean. Normalized Ct values were used to calculate the relative expression values. For each transcript the tissue with the lowest expression was used to calculate relative expression. For all genes, the eye had lowest expression. The analysis was performed twice, each time with duplicate samples.

Figure 4

Expression data for neuropeptide Y receptors in a panel of eight zebrafish organs. Error bars display standard error of the mean. Normalized Ct values were used to calculate the relative expression values. For each transcript the tissue with the lowest expression was used to calculate relative expression. For the different genes, the following tissues had lower expression: Y2, eye; Y4, GI; Y7, GI; Y8a, eye; Y8b, muscle. The analysis was performed twice, each time with duplicate samples.