Molecular And Functional Characterization Of Corazonin Receptor Of Silkworm, Bombyx Mori

Neuropeptides are neuronal signaling molecules consisting of short chains of amino acids used for cell-to-cell communication. Neuropeptides have been identified from all levels of organism ranging from hydrozoans to Drosophila to humans, and play essential roles in the regulation of a wide range of physiological functions including growth, development, food intake, metabolism, reproduction, social behaviors, learning and memory. The silkworm, Bombyx mori, in addition to its utility as an economically important insect for silk production, has been served as an important model for biochemical, genetic and genomic studies. The availability of complete genome sequences, a relatively large body size, and obvious developmental markers make the silkworm well-suited for elucidation of novel functions and mechanisms of neuropeptides in the regulation of growth and development. Corazonin (Crz) is one of the important neuropeptides in insects, which is playing different physiological roles in the regulation of heart contraction rates, silk spinning rates, the induction of dark color and morphometric phase changes, and ecdysis. It has been identified in most arthropods examined with the notable exception of beetles and an aphid. As a conserved oligopeptide comprised by11amino acids, Crz mostly distributes in neurosecretory neurons of the protocerebrum with some in abdominal and suboesophageal ganglia. However, its signaling pathways and physiological function in Bombyx mori remian obscure so far.Based on the database of Bombyx mori genome, we made sequence alignment with pubulished genome of different insects. Results showed that insects corazonin receptor (including Manduca sexta and Drosophila melanogaster) had the high homologies with Bombyx mori neuropeptide receptor A21(BNGR-A21). And they all have the typical seven-transmembrane domain as the G-protein-coupled receptor (GPCR). Thus we proposed that BNGR-A21might be the corazonin receptor in silkworm. Based on the assumption, a systematic and intensive research was made on BNGR-A21mediated signaling pathway and its probable physiological roles by performing a series of experiments (including cAMP measurement, internalization, ERK phosphorylation). In the current study, using both the mammalian cell line HEK293and insect cell lines BmN and Sf21, we paired the Bombyx corazonin neuropeptide as a specific endogenous ligand for BNGR-A21, and we therefore designated this receptor as BmCrzR. The direct interaction of BmCrzR with BmCrz was confirmed by a Rhodamine-labeled BmCrz peptide, indicating that synthetic BmCrz demonstrated a high affinity for and activated BmCrzR. The activated receptor mediated the intracellular cAMP accumulation by activation of adenylate cyclase. Besides, the induced cAMP could not be inhibited by PTX (Pertussis toxin), nor saturated by CTX (Choleratoxin). When stimulated by Crz, BmCrzR could also mobilized Ca2+, which could be blocked by Gaq inhibitor YM-254890. Above results showed that cAMP and Ca2+participated in the process of downstream cascades, indicating that both Gaq and Gas involved in the BmCrzR mediated signal transduction.Mitogen-activated protein kinase pathways (MAPK) play an important role in regulation of physiological process. Our research demonstrated that activated BmCrzR can evoke ERK1/2phosphorylation in time-and Crz dose-dependent ways, and the phosphorylation was regulated by cAMP/PKA and Ca2+/PKC signaling pathway.Internalization is an essential mechanism for GPCR desensitization. Our results revealed that the internalization of BmCrzR stimulated by BmCrz was in the time-and dose-dependent manner and could be blocked by inhibitors of PKA、PKC and chelator of extracecullar Ca2+. As β-arrestins involved in the internalization of many GPCR, we cloned kurtz gene, an non-visual arrestin in Drosophila melanogaster from Bombyx mori, Bm-kurtz.When BmCrzR internalized in cytoplasm, β-arrestins and kurtz were recruited on the cell membrane. Compared with β-arrestin1, BmCrzR had a higher affinity with β-arrestin2and kurtz. The internalization of BmCrzR was not influenced when only β-arrestinl or β-arrestin2was silenced by siRNA. However, simultaneous knockdown of β-arrestins1and β-arrestins2notably inhibited the internalizaiton of BmCrzR, suggesting that both arrestins participating in the process of BmCrzR internalization. Besides, knockdown of clathrin indicated that it is not essential for BmCrzR internalization. Further investigation demonstrated that internalized BmCrzR was sorted in endosomes and recycled to the cell surface after the removal of agonist. In general, C-terminal is very important in GPCR signaling. In order to clarifying the relationship of structure and function, we constructed four mutants through deleting some sequences in C-terminal of BmCrzR. Results indicated that the internalization of BmCrzR (Δ408-422) was significantly blocked, suggesting the sequence (408-422) contains some sites or domains responsible for internalization. Further exploration proposed that it was the411-413Ser/Thr cluster that is responsable for the inhibited internalization of BmCrzR.Results from quantitative RT-PCR showed that BmCrzR expression was detectable in most of the tissues, of which the silk gland was found as a major expression site, whereas other tissues, including brain, fat body, epidermis, midgut, Malpighian tubule, testis and ovary, expressing BmCrzR at a lower level.siRNA has been used as one of effective methods to knockdown of the target gene expression in recent years. In order to access the physiological processes of BmCrzR, we used in vitro siRNA injection to inhibit the BmCrzR expression in silkworm. Experiments with dsRNA and synthetic peptide injection suggested a possible role of BmCrz/BmCrzR in the regulation of larval growth and spinning rate.Our present results provide the first in-depth information on BmCrzR-mediated signaling, inculding intracellcular accumulation of cAMP, Ca2+, ERK1/2phosphoylation and receptor internalization. Combined with the study of physiology, these results will lead to a better understanding of the BmCrz/BmCrzR system in the regulation of fundamental physiological processes.