Human Neuropeptide S Receptor Is Activated via a Gαq Protein-biased Signaling Cascade by a Human Neuropeptide S Analog Lacking the C-terminal 10 Residues

Abstract

Human neuropeptide S (NPS) and its cognate receptor regulate important biological functions in the brain and have emerged as a future therapeutic target for treatment of a variety of neurological and psychiatric diseases. The human NPS (hNPS) receptor has been shown to dually couple to G␣ s -and G␣ q -dependent signaling pathways. The human NPS analog hNPS-(1-10), lacking 10 residues from the C terminus, has been shown to stimulate Ca 2؉ mobilization in a manner comparable with full-length hNPS in vitro but seems to fail to induce biological activity in vivo. Here, results derived from a number of cellbased functional assays, including intracellular cAMP-response element (CRE)-driven luciferase activity, Ca 2؉ mobilization, and ERK1/2 phosphorylation, show that hNPS-(1-10) preferentially activates G␣ q -dependent Ca 2؉ mobilization while exhibiting less activity in triggering G␣ s -dependent CRE-driven luciferase activity. We further demonstrate that both G␣ q -and G␣ s -coupled signaling pathways contribute to full-length hNPS-mediated activation of ERK1/2, whereas hNPS-(1-10)-promoted ERK1/2 activation is completely inhibited by the G␣ q inhibitor UBO-QIC but not by the PKA inhibitor H89. Moreover, the results of Ala-scanning mutagenesis of hNPS-(1-13) indicated that residues Lys 11 and Lys 12 are structurally crucial for the hNPS receptor to couple to G␣ s -dependent signaling. In conclusion, our findings demonstrate that hNPS-(1-10) is a biased agonist favoring G␣ q -dependent signaling. It may represent a valuable chemical probe for further investigation of the therapeutic potential of human NPS receptor-directed signaling in vivo.

Pharmacological profile of the neuropeptide S receptor: Dynamic mass redistribution studies

“…This was largely expected for PTX, since no data in the literature reported NPSR coupling to Gi, but not for rolipram since several papers showed an increase of cAMP levels after the activation of NPSR, thus suggesting that NPSR can couple to Gs proteins. Of note, the majority of these studies have been performed with cells expressing the human isoforms of the receptor . Possible differences in the coupling between the human and the murine NPSR may explain the discrepancy between our data that excludes a Gs dependent component of the NPSergic DMR response and those from literature reporting increase of cAMP levels in response to human NPSR activation.…”

“…The establishment and optimization of reliable in vitro assays to investigate NPSR pharmacology will likely speed up the drug discovery process toward the identification of such molecules. As reported for several GPCRs, the intracellular signaling cascade that follows NPSR activation is complex and involves different parallel pathways such as Gq, Gs, and extracellular signal‐regulated kinases (ERKs)). Additionally, the complexity of NPSR signaling has been recently underlined by the identification of NPSR biased agonists, such as NPS (1‐10) and SFKN‐NH 2, that preferentially signal through IP 3 ‐DAG‐Ca 2+ second messengers.…”

“…As reported for several GPCRs, the intracellular signaling cascade that follows NPSR activation is complex and involves different parallel pathways such as Gq, Gs, and extracellular signal‐regulated kinases (ERKs)). Additionally, the complexity of NPSR signaling has been recently underlined by the identification of NPSR biased agonists, such as NPS (1‐10) and SFKN‐NH 2, that preferentially signal through IP 3 ‐DAG‐Ca 2+ second messengers. This class of NPSR ligands may be useful as innovative anxiolytics .…”

“…Possible differences in the coupling between the human and the murine NPSR may explain the discrepancy between our data that excludes a Gs dependent component of the NPSergic DMR response and those from literature reporting increase of cAMP levels in response to human NPSR activation. It is possible that other signaling pathways activated by NPSR, for example, the ERK pathway, might be responsible for the Gq independent component of the DMR signal elicited by NPS in HEK293 mNPSR cells.…”

Identification of the first biased NPS receptor agonist that retains anxiolytic and memory promoting effects with reduced levels of locomotor stimulation

“…There is some indication that this approach is feasible based on an analog of NPS in which 10 of the C-terminal residues were deleted. (Liao et al, 2016)…”