| Human neuropeptide S (NPS), a novel neuropeptide with a highly conserved serine at the amino-terminal end of the mature 20 amino acid peptide, has been newly identified as an endogenous ligand for the orphan G protein-coupled receptor GPR154, hereafter referred to as the neuropeptide S receptor (NPSR). The NPS and its cognate receptor have been found to regulate important biological functions in the brain and have emerged as a future therapeutic target for treatment of a variety of neurological as well as psychiatric diseases.NPS was dually coupled to Gas- and Gαq-protein to activate the signaling pathway of cAMP/PKA and Ca2+/PKC. However, compared with NPS, we first showed that NPS-(1-10) lacking ten residues from the C-terminal can preferentially stimulate Gaq-dependent Ca2+ mobilization but exhibit less activity in triggering Gas-dependent cAMP production. Our results demonstrated that NPS-(1-10) was a biased ligand which selectively induced stabilization between NPSR and Gα4-protein. Competitive binding analysis showed that NPS and NPS-(1-10) display the similar potency to bind to the receptor and it suggested that deletion of the C-terminal 10 residues has, if any, no significant effect on NPS binding to NPSR. Additionally, NPSR could signal through mitogen-activated protein kinase (MAPK) cascades and we found that activation of ERK1/2 by NPS could be blocked by the PKA inhibitor H89 and the Gαq inhibitor UBO-QIC. It suggested that NPSR activated ERK1/2 involving both Gαs and Gαq-dependent signaling. On the other hand, the activation of ERK1/2 by NPS-(1-10) was absolutely blocked by the Gαq inhibitor UBO-QIC instead of the PKA inhibitor H89. These results indicated that NPS-(1-10)-mediated activation was only related to Gαq-dependent signaling.To investigate the mechanism of biased agonismof NPS-(1-10), alanine-scan mutagenesis were performed to assess roles of residues of NPS in the NPSR activation. Our data showed that two mutants, NPS-K11A and NPS-K12A, preferentially activated Gaq-dependent Ca2+ mobilization like NPS-(1-10), whereas mutant NPS-T13A and NPS-(1-13) exhibited the same ability to induce Gas- and Gaq-coupled signaling pathways. In addition, the activation of ERK1/2 by NPS-K11A and NPS-K12A was inhibited by the Gaq inhibitor UBO-QIC, but not by the PKA inhibitor H89.Taken together, these results suggested that residues Lys11 and Lys12were structurally important for the hNPS receptor to couple to Gas-dependent signaling.To gain insight into structure-function relationships of NPSR, functional assays were performed to evaluate the activities of 9 natural variants of hNPSR. Our results showed that NPSR-N1071 exhibited higher cell surface expression and binding affinity than the wild-type receptor, different from the previous reports, leading to a significant increase in signaling. Mutants NPSR-S241R and NPSR-Q344R showed the similar potential to trigger Gas-and Gaq-coupled signaling cascades comparable to the wild-type, whereas mutants NPSR-D94V and NPSR-C197F were found to be failed to induce intracellular cAMP accumulation and Ca2+ mobilization. Mutants NPSR-R122Q, NPSR-S143G and NPSR-I315T displayed the ability to induce intracellular cAMP accumulation to a certain extent, but no activity in triggering Ca2+ mobilization, on the contrary, mutant NPSR-T212I was found to preferentially activate Gaq-dependent signaling pathway over Gas-coupled cascade. These results provided important clues to understand NPSR structure-function relationships.In conclusion, we have demonstrated that NPS-(1-10) is a biased agonist favoring Gaq-dependent signaling at human NPSR and the residues Lys11 and Lys12 are structurally critical for the hNPS receptor to couple to Gas-dependent signaling. Our findings provide a structural and mechanistic basis for better understanding the NPSR-mediated signaling pathways in both in vitro, and importantly, in vivo systems and will be helpful to facilitate the development of novel drugs with improved efficacy and fewer side effects. |
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