physiological studies, led to the molecular characterization of the first AT receptor, as well as the identification of novel allatoregulatory peptides. Our results clearly show the effectiveness of the systematic analysis of a receptor transcriptome in neuropeptide research, and have enabled us to formulate a novel hypothesis for how JH biosynthesis is regulated by AT. Results Comprehensive Cloning of Bombyx Neuropeptide GPCR Genes When a newly released genome is screened for a certain family of genes, homology searching using the family members from other well-characterized species is the most powerful and reliable approach [1,2,15–19]. In order to thoroughly identify neuropep- tide GPCR genes encoded in Bombyx genome (Bombyx neuropep- tide GPCR genes or BNGRs), therefore, we sought to identify all the homologs of previously categorized D. melanogaster neuropep- tide GPCRs (see Figure 1 for the whole identification process). Based on earlier reports, 40 Drosophila GPCRs were listed as neuropeptide receptors (Table 1) [1,2,20]. Using the amino acid sequences of these Drosophila neuropeptide GPCRs as queries, Bombyx whole-genome shotgun sequence contigs were screened (TBLASTN analysis) by using KAIKOBLAST (http://kaikoblast. dna.affrc.go.jp/; first screening). This resulted in the identification of 195 contigs, whose E-values against at least one Drosophila neuropeptide GPCR are less than 0.1 (Table S1). Since the gene fragments in these contigs may encode other types of proteins whose sequences are similar to the neuropeptide GPCRs, all the contigs were further screened against Drosophila proteome (BLASTX analysis) using FlyBase BLAST (http://flybase.net/ blast/; second screening). This second screening yielded 139 contigs, whose predicted amino acid sequences showed the highest similarities to one of the 40 Drosophila neuropeptide GPCRs (Table S1). These contigs were termed putative BNGR contigs, and were subjected to further analyses. Considering that the number of BNGRs is likely to be similar to that of Drosophila neuropeptide GPCRs, the large number of putative BNGR contigs clearly suggests that most BNGRs are fragmented into several contigs, making it impossible to predict the whole structure of a BNGR from the published genome sequence. Therefore, the best and perhaps the only way to identify all the BNGRs from this incomplete genome information is to perform rapid amplification of cDNA ends (RACE) from each putative gene fragments. Although this is quite laborious, cloned cDNA sequences are even more reliable than the predicted ones, as evidenced by the repeated correction of Drosophila GPCR gene sequences every when they are actually cloned [21–24]. In Bombyx, 8 genes have been previously reported to encode neuropeptide GPCRs [11,12,25–30]. BLAST searching for the contigs containing the fragments of these genes revealed that fragments of all the 8 genes are included in 23 putative BNGR contigs, yielding an average of 2.9 putative BNGR contigs for each gene (Table S2). This ensures the comprehensiveness of the putative BNGR contig repertoire, because a single fragment of a gene is enough to determine the whole structure of the cDNA by RACE. After removing the contigs attributed to the reported genes, RACE was applied to each of the remaining 116 contigs. Based on the obtained cDNA sequences, the whole coding region of each BNGR was cloned into pME18S, a mammalian expression vector. The cloned BNGR was sequenced, and the predicted amino acid sequence was submitted to TMHMM (http://www.cbs.dtu.dk/ services/TMHMM/) [31] and CD-Search (http://www.ncbi.nlm. nih.gov/Structure/cdd/wrpsb.cgi) [32], respectively, in order to Bombyx Neuropeptide GPCRs check if 7 transmembrane regions and other characteristic features of GPCRs are conserved in each individual BNGR. The certified BNGR coding sequence was then submitted to KAIKOBLAST (BLASTN analysis), to find all the contigs containing the fragments of the cloned BNGR. These identified contigs were removed from the list of putative BNGR contigs, and the next RACE was started from one of the residual contigs. This process was repeated until all the contigs were identified, which resulted in the identification of 39 novel BNGRs, with two splicing variants for BNGR-A6 (Table S3). Among putative BNGR contigs, contig 227253 was shown to contain a fragment of a gene which has high similarity to mammalian melatonin receptors (MTNR). Contig 198123 and 280624 contained fragments of a pseudogene which has multiple stop codons within the conserved domain of GPCRs. Based on the predicted amino acid sequences, phylogenetic analysis was per- formed for Bombyx and Drosophila neuropeptide GPCRs (Figure 2). Quantitative Expression Analysis of BNGRs As a first step toward elucidating neuropeptide signaling pathways, quantitative tissue expression analysis was performed on two larval stages (Figure 3). Among the previously character- ized neuropeptide GPCRs, adipokinetic hormone receptor, diuretic hormone receptor and Bommo-myosuppressin (BMS) receptor were highly expressed in fat body, Malpighian tubules and the prothoracic gland of the last instar larvae, respectively. These expression patterns correspond well to the target tissues of their ligands [11,33,34], suggesting that this type of expression analysis is useful for identifying regulatory circuitry. As for the BMS receptor, significant expression was observed not only in the prothoracic gland, but also in the midgut and Malpighian tubules, as shown in the previous report [11]. Indeed, the tissue expression analysis showed that many receptors are expressed in numerous tissues, suggesting multifunctional features of their ligands. Functional Characterization of CC-CA-Expressed Receptors For detailed studies, we then focused on the six receptors highly expressed in the CC-CA complex: BNGR-A1, A6-A, A10, A11, A16 and B3. There have been two neuropeptides reported to regulate JH production by lepidopteran CA: allatostatin (Manduca-type or C- type; AST-C) and allatotropin (AT) [3,4,35]. In spite of the pivotal role of JH in numerous physiological processes, functions of these regulatory peptides have not been sufficiently analyzed, in part due to the lack of information of their receptors [4]. To begin a characterization of these six BNGRs, each was functionally analyzed using a heterologous expression system. HEK293 cells expressing BNGR-A1 responded to AST-C in a dose-dependent manner (Figure 4A). This result is reasonable, since the Drosophila homologs of BNGR-A1 (CG7285 and CG13702; see Figure 2) have been identified as receptors for AST-C-like peptide [36]. Moreover, BNGR-A16 responded to AT specifically and dose- dependently (Figure 4B), showing that this receptor is the functional AT receptor. No clear homolog of BNGR-A16 was found in the Drosophila genome as expected (Figure 2), since there is no AT neuropeptide gene in the fly [1,37,38]. The identification of the long- sought AT receptor clearly shows the efficiency of our approach in identifying unknown ligands for orphan receptors. Reverse-Physiological Identification of Novel Allatostatic Peptides Another advantage of using Bombyx is that biochemical approaches like peptide purification from organ extracts and physiological techniques like organ culture can be combined with PLoS ONE | www.plosone.org 2 August 2008 | Volume 3 | Issue 8 | e3048