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 Gαs- and Gαq-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 hNPSin vitrobut seems to fail to induce biological activityin vivo Here, results derived from a number of cell-based 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 Gαq-dependent Ca(2+)mobilization while exhibiting less activity in triggering Gαs-dependent CRE-driven luciferase activity. We further demonstrate that both Gαq- and Gαs-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 Gαqinhibitor 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 Gαs-dependent signaling. In conclusion, our findings demonstrate that hNPS-(1-10) is a biased agonist favoring Gαq-dependent signaling. It may represent a valuable chemical probe for further investigation of the therapeutic potential of human NPS receptor-directed signalingin vivo.

Keywords: G protein; G protein-coupled receptor (GPCR); extracellular-signal-regulated kinase (ERK); neuropeptide; signal transduction.

FIGURE 1.

hNPS-mediated CRE-driven luciferase activity and Ca²⁺ mobilization in NPSR-expressing HEK293 cells. A, CRE-driven luciferase activity in HEK293 cells transiently co-transfected with CRE-Luc and NPSR was determined in response to different doses of hNPS. B, effects of different inhibitors on CRE-driven luciferase activity. HEK293 cells were pretreated with a PKA inhibitor (H89, 10 μm) and Gα_q inhibitor (UBO-QIC, 100 nm) for 1 h prior to incubation with hNPS (1 μm) for 4 h. C, HEK293 cells transiently transfected with NPSR were measured in response to different concentrations of hNPS using the fluorescent Ca²⁺ indicator Fura-2. D, effects of pretreatment of the Gα_q inhibitor (UBO-QIC, 100 nm) and the PKA inhibitor (H89, 10 μm) on hNPS-mediated Ca²⁺ influx in HEK293 cells. Data were analyzed by using Student's t test (***, p < 0.001; ns, not significant). All pictures and data shown represent the means ± S.E. (error bars) from at least three independent experiments.

FIGURE 2.

hNPS-(1–10)-mediated CRE-driven luciferase activity and Ca²⁺ mobilization in NPSR-expressing HEK293 cells. A, HEK293 cells transiently transfected with NPSR were measured in response to different concentrations of hNPS-(1–10) using the fluorescent Ca²⁺ indicator Fura-2. B, effects of pretreatment with the Gα_q inhibitor (UBO-QIC, 100 nm) on hNPS-(1–10)-mediated Ca²⁺ influx in HEK293 cells. C, CRE-driven luciferase activity in HEK293 cells transiently co-transfected with CRE-Luc and NPSR was determined in response to different doses of hNPS-(1–10). All pictures and data shown represent the means ± S.E. (error bars) from at least three independent experiments.

FIGURE 3.

Direct interaction of NPSR with hNPS and hNPS-(1–10). A, Cy5-[Lys¹⁹]NPS activity was assayed using the CRE-driven luciferase system. B, binding of 100 nm Cy5-[Lys¹⁹]NPS, in the presence or absence of different concentrations of unlabeled hNPS and hNPS-(1–10), was determined on hNPSR-transfected and untransfected cells. The extent of binding was determined by fluorescence intensity. Nonspecific binding was determined by detecting Cy5-NPS binding in the presence of 10 μm unlabeled NPS, whereas specific binding was calculated by subtracting nonspecific binding from the total. The data shown are presented as a percentage of hNPS binding and are representative of at least three independent experiments.

FIGURE 4.

NPSR activates ERK1/2 mainly via Gα_q-dependent signaling pathway by hNPS and hNPS-(1–10) in HEK293 cells. A and B, time course (A) and concentration dependence (B) of hNPS-stimulated phosphorylation of ERK1/2 in HEK293 cells transiently transfected with NPSR. C and D, time course (C) and concentration dependence (D) of hNPS-(1–10)-stimulated phosphorylation of ERK1/2 in HEK293 cells transiently transfected with NPSR. E and F, time course of PKA inhibitor H89 (10 μm) (E) and Gα_q inhibitor UBO-QIC (100 nm) (F) on hNPS-mediated activation of ERK1/2. G and H, time course of PKA inhibitor H89 (10 μm) (G) and Gα_q inhibitor UBO-QIC (100 nm) (H) on hNPS-(1–10)-mediated activation of ERK1/2. The cells were pretreated with or without (control) inhibitors for 1 h and then stimulated with hNPS or hNPS-(1–10) (1 μm, 5 min). The phospho-ERK (P-ERK) was normalized to a loading control (total ERK (T-ERK)). All pictures and data shown represent the means ± S.E. (error bars) from at least three independent experiments. Statistical analysis was performed by a two-tailed Student's t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001, versus counterpart control).

FIGURE 5.

The hNPS-(1–13) analogs-mediated CRE-driven luciferase activity and Ca²⁺ mobilization in NPSR-expressing HEK293 cells. A, CRE-driven luciferase activity in HEK293 cells transiently co-transfected with CRE-Luc and NPSR was determined in response to different doses of hNPS. B, HEK293 cells transiently transfected with NPSR were measured in response to different concentrations of the hNPS-(1–13) analogs using the fluorescent Ca²⁺ indicator Fura-2. Effects of pretreatment of Gα_q inhibitor (UBO-QIC, 100 nm) on the hNPS-(1–13) analog-mediated Ca²⁺ influx in HEK293 cells (C–F). All pictures and data shown represent the means ± S.E. (error bars) from at least three independent experiments. P-ERK, phospho-ERK; T-ERK, total ERK.

FIGURE 6.

hNPS-(1–13) analogs activate ERK1/2 via different signaling pathways in HEK293 cells. Time course of PKA inhibitor H89 (10 μm) (A, C, E, and F) and Gα_q inhibitor UBO-QIC (100 nm) (B, D, G, and H) on NPS variant-mediated activation of ERK1/2 in the following sequence: hNPS-K11A, hNPS-K12A, hNPS-T13A, hNPS-(1–13). The cells were pretreated with or without (control) inhibitors for 1 h and then stimulated with different hNPS variants (1 μm, 5 min). The phospho-ERK (P-ERK) was normalized to a loading control (total ERK (T-ERK)). The data shown are representative of at least three independent experiments. Statistical analysis was performed by a two-tailed Student's t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001, versus counterpart control).