The insect adipokinetic hormones (AKHs) are a large family of peptide hormones that are involved in the mobilization of sugar and lipids from the insect fat body during energy-requiring activities such as flight and locomotion, but that also contribute to hemolymph sugar homeostasis. Here, we have identified the first insect AKH receptors, namely those from the fruitfly Drosophila melanogaster and the silkworm Bombyx mori. These results represent a breakthrough for insect molecular endocrinology, because it will lead to the cloning of all AKH receptors from all model insects used in AKH research, and, therefore, to a better understanding of AKH heterogeneity and actions. Interestingly, the insect AKH receptors are structurally and evolutionarily related to the gonadotropin-releasing hormone receptors from vertebrates. Insects constitute the largest animal group on earth and are economically and ecologically extremely important, because most flowering plants depend on insects for their pollination, and insects can be serious pests. Despite the importance of insects, however, our knowledge of their endocrinology is still incomplete. Although in the last 20 years considerable progress has been made with the isolation and identification of peptide hormones from insects (1, 2), the identification of their receptors has remarkably lagged behind (1-4). The adipokinetic hormones (AKHs) are one of the best studied insect neurohormones with more than 30 different family members isolated from over 70 species (1, 2, 4-11). The action of AKH is comparable to that of glucagon from mammals. It contributes to hemolymph sugar homeostasis, but it is also involved in the mobilization of sugar and lipids from the fat body during energy-requiring activities, such as flight or locomotion (1, 2, 4-11). Here we describe the identification of an AKH receptor from the fruitfly Drosophila melanogaster and that from another model insect, the silkworm Bombyx mori. These findings will provide an important lead to find additional insect AKH receptors, which will help us to understand AKH heterogeneity and actions. Materials and MethodsExtraction of the Receptor Ligand. A total of 400 g of third-instar larvae from D. melanogaster (Canton S.) were ground to powder under liquid nitrogen, boiled in 3 vol of deionized water for 20 min, and cooled to 0°C. After acetic acid addition (final pH, 3.0) and homogenization with a Braun food processor, the mixture was centrifuged, and the supernatant was brought to pH 7.0 with a diluted NaOH solution. The extract was then desalted by using several SepPak C18 cartridges (Waters). After being rinsed with 5 ml of H 2 O, each cartridge was eluted with 4 ml of 50% acetonitril in 0.1% trifluoroacetic acid. All eluates were lyophilized and used as a starting material for HPLC (Table 1).HPLC of the Extracts. The HPLC system used was from Shimazu (LC-6A; SPD-6AV; SCL-6B; C-R6A). Columns 1, 2, 5, and 7 (see Table 1) were purchased from Latek (Heidelberg), columns 4 and 6 were purchased from Macherey-Nagel (Düren, Ge...

The occurrence of a deoxyribonucleoside kinase in Drosophila melanogaster (Dm-dNK) with remarkably broad substrate specificity has recently been indicated (Munch-Petersen, B., Piskur, J., and Søndergaard, L. (1998) J. Biol. Chem. 273, 3926 -3931). To prove that the capacity to phosphorylate all four deoxyribonucleosides is in fact associated to one polypeptide chain, partially sequenced cDNA clones, originating from the Berkeley Drosophila genome sequencing project, were searched for homology with human deoxyribonucleoside kinases. The total sequence of one cDNA clone and the corresponding genomic DNA was determined and expressed in Escherichia coli as a glutathione S-transferase fusion protein. The purified and thrombin cleaved recombinant protein phosphorylated the four deoxyribonucleosides with high turnover and K m values similar to those of the native Dm-dNK, as well as the four ribonucleosides and many therapeutical nucleoside analogs. DmdNK has apparently the same origin as the mammalian kinases, thymidine kinase 2, deoxycytidine kinase, deoxyguanosine kinase, and the herpesviral thymidine kinases, but it has a unique C terminus that seems to be important for catalytic activity and specificity. The Cterminal 20 amino acids were dispensable for phosphorylation of deoxyribonucleosides but necessary for full activity with purine ribonucleosides. Removal of the C-terminal 20 amino acids increased the specific activity 2-fold, but 99% of the activity was lost after removal of the C-terminal 30 amino acids.

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The cockroach-type or A-type allatostatins are inhibitory insect neuropeptides with the C-terminal sequence Tyr/Phe-X-Phe-Gly-Leu-NH 2 . Here, we have cloned an A-type allatostatin receptor from the silkworm Bombyx mori (BAR). BAR is 361 amino acid residues long, has seven transmembrane domains, shows 60% amino acid residue identity with the first Drosophila allatostatin receptor (DAR-1), and 48% identity with the second Drosophila allatostatin receptor (DAR-2). The BAR gene has two introns and three exons. These two introns coincide with and have the same intron phasing as two introns in the DAR-1 and DAR-2 genes, showing that the three receptors are not only structurally but also evolutionarily related. Furthermore, we have cloned a Bombyx allatostatin preprohormone that contains eight different Atype allatostatins. Chinese hamster ovary cells permanently transfected with BAR DNA react on the addition of 4 ؋ 10 ؊9 M Bombyx A-type allatostatins with a second messenger cascade (measured as bioluminescence), showing that BAR is a functional A-type allatostatin receptor. Southern blots suggest that Bombyx has at least one other BAR-related gene in addition to the BAR gene described in this paper. Northern blots and quantitative reverse transcriptase-polymerase chain reaction of different larval tissues show that BAR mRNA is mainly expressed in the gut and to a much lesser extent in the brain. To our knowledge, this is the first report on the molecular cloning and functional expression of an insect gut/brain peptide hormone receptor.Insects constitute a large group of animals (more than 70% of all animal species are insects) and are ecologically and economically extremely important, because most flowering plants depend on insects for their pollination (honeybees alone pollinate 20 billion dollars worth of crop yearly in the United States). But insects can also be severe pests in agriculture, destroying 30% of our potential annual harvest, and can be vectors for major diseases such as malaria, sleeping disease, and yellow fever. Despite the importance of insects, however, their molecular endocrinology is not well understood. This will certainly change after the recent publication of the Drosophila genome (1, 2), which represents a breakthrough for our understanding of virtually all aspects of insects. For the insect molecular endocrinology field, e.g. one can anticipate that the major future advances will occur by "mining" of the "Drosophila Genome Project" data base and cloning of important key proteins involved in insect endocrinology (3-9).Although most initial findings will be made in Drosophila, one can expect that these discoveries also will have their impacts on our understanding of the molecular endocrinology of other insects. Although Drosophila offers many important advantages for studying the molecular biology and genetics of an insect (a sequenced genome; the presence of numerous mutants, including knock-out mutants (7); and the possibility of creating transgenic animals), it is relatively small, which m...

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The insect allatostatins obtained their names because they block the biosynthesis of juvenile hormone (a terpenoid) in the corpora allata (two endocrine organs near the insect brain). Chemically, the allatostatins can be subdivided into three different peptide groups: the A-type allatostatins, first discovered in cockroaches, which have the C-terminal sequence Y/FXFGLamide in common; the B-type allatostatins, first discovered in crickets, which all have the C-terminal sequence W(X)(6)Wamide; and the C-type allatostatins, first discovered in the moth Manduca sexta, which have an unrelated and nonamidated C terminus. We have previously reported the structure of an A-type allatostatin preprohormone from the fruitfly Drosophila melanogaster. Here we describe the molecular cloning of a B-type prepro-allatostatin from Drosophila (DAP-B). DAP-B is 211 amino acid residues long and contains one copy each of the following putative allatostatins: AWQSLQSSWamide (drostatin-B1), AWKSMNVAWamide (drostatin-B2),

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