Phylogenetic appearance of neuropeptide S precursor proteins in tetrapods

Abstract

Sleep and emotional behavior are two hallmarks of vertebrate animal behavior, implying that specialized neuronal circuits and dedicated neurochemical messengers may have been developed during evolution to regulate such complex behaviors. Neuropeptide S (NPS) is a newly identified peptide transmitter that activates a typical G protein-coupled receptor. Central administration of NPS produces profound arousal, enhances wakefulness and suppresses all stages of sleep. In addition, NPS can alleviate behavioral responses to stress by producing anxiolytic-like effects. A bioinformatic analysis of current genome databases revealed that the NPS peptide precursor gene is present in all vertebrates with the exception of fish. A high level of sequence conservation, especially of aminoterminal structures was detected, indicating stringent requirements for agonist-induced receptor activation. Duplication of the NPS precursor gene was only found in one out of two marsupial species with sufficient genome coverage (Monodelphis domestica; opossum), indicating that the duplicated opossum NPS sequence might have arisen as an isolated event. Pharmacological analysis of both Monodelphis NPS peptides revealed that only the closely related NPS peptide retained agonistic activity at NPS receptors. The duplicated precursor might be either a pseudogene or could have evolved different receptor selectivity. Together, these data show that NPS is a relatively recent gene in vertebrate evolution whose appearance might coincide with its specialized physiological functions in terrestrial vertebrates.

Figure 1

Alignment of NPS peptide sequences deduced from DNA sequences as described in Table 1. Amino acid residues are given in single letter code and black shaded letters indicate differences from the human NPS sequence.

Figure 2

Phylogenetic tree of NPS precursor protein sequences deduced from GenBank DNA resources as described in Table 1. The length of each pair of branches represents the distance between sequence pairs, while the units at the bottom of the tree indicate the number of nucleotide substitution events. Deviations from common phylogenetic models might be caused by unequal length of the aligned sequences. Calculations were performed using the ClustalW algorithm in MegAline, Lasergene version 7.

Figure 3

Alignment of the two NPS precursor-like proteins deduced from opossum genomic DNA resources. The middle line depicts identical amino acids while “+”-signs indicate conservative substitutions. Dashes (−) were added to optimize the alignment.

Figure 4

Pharmacological activity of opNPS I (■) and opNPS II (▲) at mouse NPSR stably expressed in HEK 293 cells. Incubations were done in triplicate and repeated twice with similar results. Data from a representative experiment are shown as means ± SEM. EC₅₀ values were calculated from fitted curves using nonlinear regression analysis.