The Mechanism Of Signal Transduction Of Adipokinetic Hormone Receptor In Bombyx Mori

Insects, the largest group of animals on earth, play very important roles in their ecosystems through plant pollination, nutrient recycling, and maintenance of plant community composition and structure. In addition, they provide us with many useful materials, such as honey, silk, and varnish. The importance of insects as biomedical models is evident by the fact that many discoveries in digestion, muscle contraction, and important metabolic and developmental pathways in insects are applicable to vertebrate systems.Adipokinetic hormones (AKHs) produced by the insect corpora cardiaca are among the most extensively characterized peptide hormones with almost 40 family members from most of the major insect orders. AKH is normally 8-10 amino acids long with a pyroglutamate at the N-terminus and an amidated C-terminus. In addition to the essential role of mobilization of metabolites during energy-expensive activities such as flight and locomotion, AKH is involved in the control of carbohydrate homeostasis in the haemolymph of Drosophila and Bombyx larvae. As shown in Table1, in Bombyx, a nonapeptide identical with Manduca AKH (AKH1) has been chemically identified, and recently another two cDNAs encoding the prepro-Bombyx AKH2 and AKH3 have been annotated and identified by combining homology search with cDNA cloning.The receptor of AKH was first identified as a typical G protein-coupled receptor from the fruitfly Drosophila melanogaster and the silkworm Bombyx mori in 2002, and then from the cockroach Periplaneta americana and African malaria mosquito Anopheles gambiae. Previous biochemical characterization with isolated fat body suggested that AKH binds to its receptor and activates adenylyl cyclase via the Gs protein, which results in an increase of intracellular cAMP levels. In addition, AKH activates phospholipase C (PLC) to induce the release of Ca2+ from intracellular Ca2+ stores. However, the mechanistic details of AKHR signaling remain to be further elucidated.In this present study, we cloned the AKHR from the fat body of the silkworm Bombyx mori and further functionally characterized it and its peptide ligands in HEK293 cells. We conclude that after activation of AKHR, in addition to cAMP accumulation and Ca2+ release from Ca2+ stores, the mitogen-activated protein kinase (MAPK) pathway is subsequently activated and AKHRs are rapidly internalized from the plasma membrane upon agonist stimulation. AKH1 and AKH2 activated AKHR with similar affinity, but AKH3 exhibits almost much less activity on AKHR. These findings strongly implying that it is more likely that a second intrinsic AKHR exists as a high affinity receptor for AKH3 in Bombyx.Mitogen-activated protein kinase (MAPK) pathways regulate diverse processes ranging from proliferation and differentiation to apoptosis. Although it is well established that GPCRs play important roles in the regulation of intermediary metabolism, they have only recently been recognized as important mediators of cellular growth and differentiation via the MAPK pathway. To know the mechanistic details of AKH-mediated ERK1/2 activation, Using HEK293 cells stably or transiently expressing AKHR, we demonstrated that activation of AKHR elicited transient phosphorylation of ERK 1/2. Our investigation indicated that AKHR-mediated activation of ERK1/2 was significantly inhibited by H-89 (Protein kinase A inhibitor), Go6983, and GF109203X (Protein kinase C inhibitors), but not by U73122 (PLC inhibitor) or FIPI (PLD inhibitor). Moreover, AKHR-induced ERK1/2 phosphorylation was blocked by the calcium chelators EGTA and BAPTA-AM. Furthermore, ERK1/2 activation in transiently AKHR expressing HEK293 cells was found to be sensitive to pretreatment of pertussis toxin, whereas AKHR-mediated ERK1/2 activation was insensitive to siRNA-induced knockdown of p-arrestin and to pretreatment of inhibitors of EGFR, Src and PI3K. Based on our data, we propose that activated AKHR signals to ERK 1/2 primarily via PKA-and extracellular calcium-involved PKC- dependent pathways. Our current study provides the first in-depth study defining the mechanisms of AKH-mediated ERK activation through the Bombyx AKHR. Internalization is an important pattern to regulate the activity of GPCR, which are against-activated receptors from the surface into the intracellular membrane compartments of the cell. A large volume of data has accumulated regarding the mechanisms regulating the endocytosis of a wide variety of different GPCRs. Therefore, to identify the internalization details of GPCR is important to reveal its mechanism of signaling. We established a stable HEK293 cell line expressing AKHR-EGFP. Upon activation of AKHR-EGFP with ligand, the receptor was rapidly and dramatically redistributed in the cytoplasm with distinct perinuclear accumulation. From the results of siRNA, we can deduce GRK2, GRK5, and arrestin3 are involved in the internalization process of AKHR. Sucrose experiment suggests AKH-induced internalization via clathrin coated pits. Receptor recycling assay, we found that after internalization of AKHR, only partial receptors can recycle to membrane, and other receptors maybe degraded.In general, C-terminal is very important in GPCR signaling. To make clear the relationship of structure and function, we have constructed several mutants through delete some sequences in C-terminal of AKHR. We found AKHR (â–³343-363) and AKHR (â–³322-342) have affected on the internalization and location, respectively. The internalization of AKHR (â–³343-363) was significantly blocked, suggest in the sequence (343-363) contains some sites or domains responsible for internalization. Compared with wild type and AKHR (â–³322-342), we found the AKH-induced cAMP accumulation was mostly inhibited, because most AKHR mutant AKHR (â–³322-342) mainly located in endoplasmic reticulum (ER).