Table 1. List of Drosophila neuropeptide GPCRs. Family Synonyms References NPFR1 FR, FMRFaR AlstR, DAR-1 NepYr ETHR CG16958, CG33639 CcapR, CG14547, CG33344 Takr86C, NKD CCKLR-17D1 CCKLR-17D3, DSK-R1, CG6861, CG6894, CG32540 DmsR-1 AlstR2, DAR-2, AR-2 Lkr, DLKR GRHRII, DCR GRHR, DAKHR CG32547, CG15049, CG15050 star1, Drostar1 NPFR76F, Drm-sNPF-R, short NPF CG18639 Takr99D, DTKR CG No. 1147 2114 2872 4395 5811 5911 5936 6111 6515 6857 6881 6986 7285 7395 7887 8422 8784 8795 8985 9918 10001 10626 10698 10823 11325 12370 12610 13229 13575 13702 13758 13803 13995 14003 14484 14575 14593 16726 17415 30340 A A A B A A A A A A A A A A A B A A A A A A A A A B A A A A B A A A A A A A B A AlCR2, Drostar2 PDFR, groom-of-PDF, Han DmsR-2 allatostatin C PDF [36] [75–77] myosuppressin [23] CG14002, CG31645 CG18192, CG30106 capaR CG14594 CG32047, CG33696 CG17043, CG32843 BACR48G21.1 allatostatin B capa DH 31 AKH, adipokinetic hormone; CCAP, crustacean cardioactive peptide; DH, diuretic hormone; ETH, ecdysis-triggering hormone; NPF, neuropeptide F; PDF, pigment-dispersing factor. doi:10.1371/journal.pone.0003048.t001 Ligands NPF FMRFamide allatostatin A ETH CCAP tachykinin sulfakinin sulfakinin proctolin allatostatin C tachykinin DH 44 pyrokinin-2 pyrokinin-2 myosuppressin pyrokinin-1 allatostatin A leucokinin corazonin SIFamide AKH [55] [56,57] [58,59] [60] [21,61] [24,62] [63,64] [2] [65] [66,67] [36] [68] [64,69] [70] [62,71] [62,71] [23] [72] [59] [73] [62,74] [29] [27,62] [64] [22,62] [78] Bombyx Neuropeptide GPCRs Therefore we purified two sNPF peptides in Bombyx from brain extracts, originally prepared for the purification of BMS and Bommo-FMRFamide (BRFa) [11,12], using an ELISA system. Four HPLC purification steps were required to purify two sNPF peptides into homogeneity, sufficient for MALDI-TOF MS analysis (Figure 5A). Aliquots of the isolated fractions were subjected to MALDI-TOF MS, which yielded the sequences TPVRLRFamide and SMRLRFamide (Figure S1). Based on these amino acid sequences, the cDNA encoding the Bombyx sNPF precursor was obtained and three putative mature peptides were found in the precursor sequence (Figure 5B; the purified SMRLRFamide seems to be the truncated form of sNPF-3). Functional analyses of BNGR-A10 and A11 revealed that these are indeed the sNPF receptors (Figure 4D and Figure 5C). Although these receptors also responded to BMS and BRFa, their sensitivity to sNPFs was more than 1,000 times higher than that to BMS and BRFa (Figure 5C and data not shown). We then investigated the putative regulatory role of sNPFs in JH biosynthesis using in vitro CA culture. Addition of sNPFs to the incubation medium resulted in clear inhibition of JH biosynthesis (Figure 5D), suggesting they likely have allatostatic activity in vivo. The identification of these novel allatostatins again demonstrates the effectiveness of our approach in the investigation of unknown neuropeptide functions. Identification of Unexpected Signaling Network within CC-CA Complex Surprisingly, in the course of detailed expression analysis of the six BNGRs, we found that BNGR-A16 is mainly expressed in the CC, not in the CA (Figure 6A). Considering that many previous studies on AT activity in vitro were conducted using the CC-CA complex, it is of interest to hypothesize that the CC affects JH biosynthesis in the CA, and AT regulates JH production indirectly via the CC. The CC is well known as the source of a peptide hormone, adipokinetic hormone [42], so it is plausible that the CC is producing other peptide ligands for the BNGRs expressed in the CA. Remarkably, RT-PCR and in situ hybridization analyses of sNPF revealed its expression mainly in the CC (Figure 6B). This result was also confirmed by direct MS analysis, which showed the existence of all three mature sNPF peptides in the CC-CA (Figure S2). Finally, double staining using immunohistochemistry and in situ hybridization revealed AT receptor expression in the sNPF- producing cells in the CC (Figure 6C). These unexpected findings suggest that AT regulates the production and/or the release of sNPFs from the CC, and at least some of the AT functions on JH biosynthesis may be mediated by sNPF (Figure 6D). Discussion In this study, we conducted a comprehensive cloning and the systematic expression analysis of neuropeptide GPCRs in the silkworm Bombyx mori. These analyses led to the following major findings: 1) identification and functional characterization of the first AT receptor, 2) purification and identification of novel allatostatic peptides produced in the CC, and 3) elucidation of an unexpected signaling network within CC-CA complex, potentially mediated by the above two factors. Our finding that the AT receptor is predominantly expressed not in the CA but in the sNPF-producing cells of the CC raises an intriguing possibility that the allatotropic activity of AT may be mediated by the CC (Figure 6D). In support of this idea, previous assays for AT activity have been mostly done with CC-CA complexes, rather than the CA alone. If AT indeed has any effect on the production and/or the release of sNPF from the CC, such PLoS ONE | www.plosone.org 4 August 2008 | Volume 3 | Issue 8 | e3048