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.

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.

Bombyx mori prothoracicostatic peptide receptor is allosterically activated via a Gα(i/o)-protein-biased signalling cascade by Drosophila sex peptide.

In insects, molting and metamorphosis are strictly regulated by ecdysteroids. Ecdysteroid synthesis is positively or negatively controlled by several neuropeptides. The prothoracicostatic peptide (PTSP) BmPTSP (Bombyx mori prothoracicostatic peptide), isolated from the larval brain of B. mori, has been demonstrated to inhibit ecdysteroid synthesis in the prothoracic glands (PGs) [Hua et al. (1999) J. Biol. Chem. 274, 31169-31173]. More recently, the newly recognized B. mori receptor for Drosophila melanogaster sex peptide (DmSP) has been identified as a receptor for BmPTSP. However, details on the signalling pathways and physiological functions of this receptor have remained elusive. In the present paper, we report the functional characterization of the BmPTSP receptor (BmPTSPR)/sex peptide (SP) receptor (SPR) using both mammalian and insect cells. Synthetic DmSP shows the potential to inhibit forskolin (FSK) or adipokinetic hormone (AKH)-induced cAMP-response element (CRE)-driven luciferase (Luc) activity in a manner comparable with synthetic BmPTSP1. However, DmSP displayed a much lower activity in triggering Ca²âº mobilization and internalization than did BmPTSP1. Additionally, 6-carboxy-fluorescein fluorophore (FAM)-labelled DmSP and BmPTSP3 were found to bind specifically to BmPTSPR/SPR. The binding of FAM-DmSP was displaced by unlabelled DmSP, but not by unlabelled BmPTSP1 and, vice versa, the binding of FAM-BmPTSP3 was blocked by unlabelled BmPTSP3, but not by unlabelled DmSP. Moreover, internalization assays demonstrated that BmPTSP1, but not DmSP, evoked recruitment of the Bombyx non-visual arrestin, Kurtz, to the activated BmPTSPR/SPR in the plasma membrane. This was followed by induction of internalization. This suggests that BmPTSP1 is probably an endogenous ligand specific for BmPTSPR/SPR. We therefore designate this receptor BmPTSPR. In contrast, DmSP is an allosteric agonist that is biased towards Gα(i/o)-dependent cAMP production and away from Ca²âº mobilization and arrestin recruitment.

Activation of BNGR-A24 by direct interaction with tachykinin-related peptides from the silkworm Bombyx mori leads to the Gq- and Gs-coupled signaling cascades.

Tachykinins constitute one of the largest peptide families in the animal kingdom and exert their diverse actions via G protein-coupled receptors (GPCRs). In this study, the Bombyx tachykinin-related peptides (TKRPs) were identified as specific endogenous ligands for the Bombyx neuropeptide GPCR A24 (BNGR-A24) and thus designated BNGR-A24 as BmTKRPR. Using both mammalian cell line HEK293 and insect cell line Sf21, further characterization demonstrated that BmTKRPR was activated, thus resulting in intracellular accumulation of cAMP, Ca(2+) mobilization, and ERK1/2 phosphorylation in a Gs and Gq inhibitor-sensitive manner. Moreover, quantitative reverse transcriptase polymerase chain reaction analysis and dsRNA-mediated knockdown experiments suggested a possible role for BmTKRPR in the regulation of feeding and growth. Our findings enhance the understanding of the Bombyx TKRP system in the regulation of fundamental physiological processes.