The novel neuropeptide spexin (SPX), which is encoded by the C12ORF39 gene, was originally discovered using bioinformatics tools. The predicted mature SPX peptide sequence of 14 amino acids flanked by dibasic cleavage sites is evolutionarily conserved across vertebrate species. SPX expression at the mRNA and/or protein level has been documented in brain regions and peripheral tissues of humans, mice, rats, and goldfish, suggesting multiple physiological functions of SPX. Recently, SPX was implicated in regulation of feeding behaviors and related metabolic processes. SPX mRNA levels are markedly decreased in the fat of obese humans, and administration of SPX leads to weight loss in diet-induced obese rodents. SPX also suppresses appetite in goldfish. In addition, SPX is likely involved in reproduction, cardiovascular/renal function, and nociception. The precise roles of SPX in these processes, however, are not well understood due to a lack of information on the SPX receptor. Recently, we demonstrated that SPX is an endogenous ligand that acts at GALR2 and GALR3 but not at GALR1, while GAL activates all three receptor subtypes with relatively low potency and affinity for GALR3.
The SPX and GAL genes likely emerged through a local duplication from a common ancestor gene, and as a result, their mature peptides share several conserved residues, including Trp2, Thr3, Tyr9, Leu10, and Gly12. Like SPX, GAL is widely expressed in the central nervous system and peripheral tissues. The actions of SPX and GAL in appetite behavior and reproduction, however, appear to oppose each other. For instance, levels of circulating GAL, along with neuropeptide Y and leptin, are significantly higher in obese women, and GAL administration or overexpression in mice results in an increase in food intake. Thus, GAL appears to be orexigenic, while SPX is anorexic. Administration of galanin-like peptide (GALP), a GAL paralog, stimulates luteinizing hormone (LH) secretion in the rat, while SPX administration attenuates LH secretion in the goldfish. These opposing effects are likely due, at least in part, to GAL receptor subtype-specific signaling pathways. Specifically, GALR1 and GALR3 induce inhibitory Gi-coupled signaling, while GALR2 triggers stimulatory Gq-coupled signaling.
Considering the cross-reactivity between SPX and GAL, as well as the GAL receptors and the overall complexity of the GAL receptor-mediated signaling pathway, development of more stable and subtype-selective GAL receptor agonists would lead to marked progress in the elucidation of GAL receptor subtype-specific mediated physiological functions. Thus, we focused our studies on the development of SPX sequence-based GALR2-specific agonists, based on the observations that both SPX and GAL have similarly high affinity for the GALR2 and that GAL exhibited a lower affinity and efficacy for the GALR3 than SPX. These findings led to the hypotheses that SPX-based agonists initially prevent the interaction with GALR1 and that replacement of residues in SPX with those of corresponding GAL will decrease the potency of the ligand for GALR3 without altering the affinity for GALR2. As predicted, the present study showed that replacement of SPX-specific residues Gln5, Met7, Lys11, and Ala13 with those of GAL maintained potency to the GALR2 but significantly decreased the potency toward the GALR3. In addition, replacement and modification of Asn1 greatly increased the stability of the GALR2-specific agonist in 100% fetal bovine serum (FBS). Considering the relevance of GALR2-mediated pathophysiology, such as anxiety-like behavior, and the putative role of SPX in appetite regulation, a GALR2-specific agonist may prove useful for the treatment of obesity and anxiety disorders.
To develop a specific and long-acting GALR2-specific agonist, we compared the amino acid sequences of SPX1, SPX2, GAL, and GALP. These peptides share the residues Trp2, Tyr9, Leu10 and Gly12, while other residues are specific to GAL or SPX or variable across vertebrate species. The SPX-specific residues in SPX1 were replaced with the corresponding GAL-specific residues. D-amino acids were substituted for residues common to SPX and GAL in SPX1.
Conserved residues in the peptides are indicated in yellow. SPX-specific residues are in green, while GAL-specific residues are highlighted in violet. The residues in human SPX1 were changed as indicated.
The generated mutant SPX peptides were incubated with cells expressing GALR2 and GALR3. Because each GAL receptor subtype is coupled to a different G protein (Gi for GALR1 and GALR3; Gq/11 for GALR2), we employed the HEK293-Gqi stable cell line for these experiments. The activities of the receptors were traced using the serum-response element-driven luciferase (SRE-luc) assay system. Of these, the Gly8 substitution resulted in decrease in the potency toward both receptors, while the Gly1, Leu4, and His14 substitutions retained potencies to both receptors that are similar to WT SPX. Interestingly, Asn5-, Ala7-, Leu11-, Phe11-, and Pro13-substituted mutants showed a selective decrease in the potency for the GALR3 without a concomitant effect on the activation profile for the GALR2. This result suggests that SPX residues Gln5, Met7, Lys11, and Ala13 are responsible for the selectivity for the GALR3, while substitutions of these residues are acceptable for activation of the GALR2.
