Neuropeptide Receptor Transcriptome Reveals Unidentified Neuroendocrine Pathways

Abstract

Neuropeptides are an important class of molecules involved in diverse aspects of metazoan development and homeostasis. Insects are ideal model systems to investigate neuropeptide functions, and the major focus of insect neuropeptide research in the last decade has been on the identification of their receptors. Despite these vigorous efforts, receptors for some key neuropeptides in insect development such as prothoracicotropic hormone, eclosion hormone and allatotropin (AT), remain undefined. In this paper, we report the comprehensive cloning of neuropeptide G protein-coupled receptors from the silkworm, Bombyx mori, and systematic analyses of their expression. Based on the expression patterns of orphan receptors, we identified the long-sought receptor for AT, which is thought to stimulate juvenile hormone biosynthesis in the corpora allata (CA). Surprisingly, however, the AT receptor was not highly expressed in the CA, but instead was predominantly transcribed in the corpora cardiaca (CC), an organ adjacent to the CA. Indeed, by using a reverse-physiological approach, we purified and characterized novel allatoregulatory peptides produced in AT receptor-expressing CC cells, which may indirectly mediate AT activity on the CA. All of the above findings confirm the effectiveness of a systematic analysis of the receptor transcriptome, not only in characterizing orphan receptors, but also in identifying novel players and hidden mechanisms in important biological processes. This work illustrates how using a combinatorial approach employing bioinformatic, molecular, biochemical and physiological methods can help solve recalcitrant problems in neuropeptide research.

Identification and Functional Characterization of Two Orphan G-protein-coupled Receptors for Adipokinetic Hormones from Silkworm Bombyx mori

“…10, in fifth instar larvae of silkworm, for both BNGR-A28 and -A29 receptors, the highest expression was detected in testes. We could also observe expression at a low level in brain, epidermis, silk gland, ovary, midgut, and fat body for BNGR-A28 but in brain and ovary for BNGR-A29, and this is in agreement with the observations made by Yamanaka et al (20).…”

“…Quantitative Real Time PCR-Quantitative RT-PCR was performed as described previously, with slight modifications (20). Total RNA was extracted from tissue samples as detailed in the manufacturer's protocol (Qiagen).…”

“…Bombyx neuropeptide GPCR A28 and A29 (BNGR-A28 and A29) have been identified as adipokinetic hormone-like receptors in Bombyx by genomic data mining and phylogenetic analysis (20,21). The predicted genes of BNGR-A28 (GenBank TM accession number AB330449) and -A29 (GenBank TM accession number AB330450) were cloned by RT-PCR from brain and testis of silkworm larva, respectively.…”

“…By genomic data mining and phylogenetic analysis, Bombyx neuropeptide GPCR A28 and A29 (BNGR-A28 and -A29) have been identified as adipokinetic hormone-like receptors in Bombyx (20,21). Therefore, in this study, we report on the cloning of cDNAs encoding the putative adipokinetic hormone-like receptors BNGR-A28 and BNGR-A29 from the silkworm B. mori and functional expression of both receptors in HEK293 cells.…”

Genomewide transcriptional signatures of migratory flight activity in a globally invasive insect pest

“…The Bombyx mori homologue of the neuropeptide receptor A16 ( BNGR‐A16 ), downregulated in long‐distance moths from China (FC   =   2.67), is the receptor for the hormone allatotropin (AT) which stimulates juvenile hormone (JH) biosynthesis (Yamanaka et al . 2008). The relationship between AT mRNA expression and JH levels is tightly coupled and injection of AT induces resident behaviour in migrant insects (McNeil et al .…”