ABSTRACT
In order to better understand the relationship between dopamine and the release of diapause hormone into the blood, we cloned and characterized cDNAs encoding Bombyx mori dopamine receptor-1 and -2 (BmDopR1 and 2) from the pupal brain-suboesophageal ganglion. BmDopR1 and 2 had high similarities to group 1 (Drosophila melanogaster DOP1 and Apis mellifera DOP1) and group 2 (D. melanogaster DopR99B, A. mellifera DOP2 and Papilio xuthus DOP1), respectively. When BmDopR1 and 2 were expressed in human embryonic kidney (HEK) cells, they responded to dopamine by increasing intracellular cAMP levels, thus indicating the presence of D1-like receptors. There were no clear differences in BmDopR1 and 2 mRNA levels between brain-suboesophageal ganglion complexes of diapause and nondiapause egg producers during pupal-adult development. BmDopR1 and 2 mRNAs were concentrated in the mushroom body calyx rather than in the suboesophageal ganglion. Taking into account the results of earlier experiments on excised regions corresponding to mushroom bodies, BmDopR1 and 2 in the mushroom body apparently play a role in the release of diapause hormone.
Subject(s)
Bombyx/genetics , Receptors, Dopamine D1/genetics , Receptors, Dopamine D2/genetics , Amino Acid Sequence , Animals , Base Sequence , Brain/metabolism , Cell Line , Cloning, Molecular , DNA, Complementary/genetics , Female , Ganglia, Invertebrate/chemistry , Ganglia, Invertebrate/metabolism , Humans , In Situ Hybridization , Molecular Sequence Data , Neuropeptides/physiology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , TransfectionABSTRACT
The main blood sugar in insects, trehalose, differs from glucose in mammals. To incorporate trehalose into cells and utilize it, tissue cells possess the enzyme trehalase (EC3.2.1.28), which catalyses trehalose into glucose, in the organellar membrane or in the cytoplasm. Soluble and membrane-bound trehalase proteins have been isolated from insects. To date, however, only genes encoding the soluble trehalase have been reported in insects. Soluble trehalase is therefore believed to become localized on the cell surface via modification. In contrast, cDNAs encoding trehalase localized on the apical cell surface via the glycosylphosphatidylinositol-anchor have been isolated from mammalian small intestines. The amino acid sequence contains a specific hydrophobic region and an upstream omega site, which is cleaved for glycosylphosphatidylinositol-attachment, at the C-terminus. Here, we describe a cDNA from the silkworm Bombyx mori that encodes a novel trehalase (type-2) with one transmembrane domain and lacking the omega site. Immunoblotting and immunohistochemical analyses demonstrated that in the midgut tissue of Bombyx larvae, soluble trehalase-1 is present mainly in goblet cell cavities, but membrane-bound trehalase-2 is predominantly seen on the visceral muscle surrounding the midgut. To our knowledge, this is the first report of a cDNA encoding trehalase that penetrates the cell membrane in insects and its cellular localization.
Subject(s)
Bombyx/enzymology , Insect Proteins/biosynthesis , Trehalase/chemistry , Trehalase/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Gastrointestinal Tract/metabolism , Larva/metabolism , Molecular Sequence Data , Pupa/metabolism , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
A neuropeptide from brain-suboesophageal ganglion (Br-SG) complexes of the silkmoth, Bombyx mori, shows summer-morph-producing hormone (SMPH) activity in the Asian comma butterfly, P. c-aureum. The SMPH-active peptide was extracted and demonstrated to be almost the same molecular size as bombyxin (4-5kD), a nueropeptide which shows prothoracicotropic hormone (PTTH) activity when assayed in vitro with prothoracic glands (PGs) of 4th-instar B. mori larvae in vitro. A Sephadex G-50 fraction of 3-8kD molecules prepared from Br-SG complexes of B. mori adults was applied to CM-, SP-, DEAE- or QAE- Toyoperal columns at pH 5.6 (or pH 6.9). The SMPH-activity could be separated from the PTTH-activity (or bombyxin) by subjecting a SMPH- and PTTH-active preparation of B. mori to anion-exchange chromatography at pH 6.9. By reversed-phase HPLC following an anion-exchange chromatography, SMPH-activity was recovered in two fractions of 40-45% acetonitril. Results demonstrate that the B. mori peptide showing the SMPH-activity in P. c-aureum is a different molecule than bombyxin.