Expression and functional characterization of a Drosophila neuropeptide precursor with homology to mammalian preprotachykinin A

Peptides structurally related to mammalian tachykinins have recently been isolated from the brain and intestine of several insect species, where they are believed to function as both neuromodulators and hormones. Further evidence for the signaling role of insect tachykinin-related peptides was provided by the cloning and characterization of cDNAs for two tachykinin receptors from Drosophila melanogaster. However, no endogenous ligand has been isolated for the Drosophila tachykinin receptors to date. Analysis of the Drosophila genome allowed us to identify a putative tachykinin-related peptide prohormone (prepro-DTK) gene. A 1.5-kilobase pair cDNA amplified from a Drosophila head cDNA library contained an 870-base pair open reading frame, which encodes five novel Drosophila tachykinin-related peptides (called DTK peptides) with conserved C-terminal FXGXR-amide motifs common to other insect tachykinin-related peptides. The tachykinin-related peptide prohormone gene (Dtk) is both expressed and post-translationally processed in larval and adult midgut endocrine cells and in the central nervous system, with midgut expression starting at stage 17 of embryogenesis. The predicted Drosophila tachykinin peptides have potent stimulatory effects on the contractions of insect gut. These data provide additional evidence for the conservation of both the structure and function of the tachykinin peptides in the brain and gut during the course of evolution.     

Functional comparison of two evolutionary conserved insect neurokinin-like receptors

Tachykinins are multifunctional neuropeptides that have been identified in vertebrates as well as invertebrates. The C-terminal FXGXRa-motif constitutes the consensus active core region of invertebrate tachykinins. In Drosophila, two putative G protein-coupled tachykinin receptors have been cloned: DTKR and NKD. This study focuses on the functional characterization of DTKR, the Drosophila ortholog of the stable fly's tachykinin receptor (STKR). Tachykinins containing an alanine residue instead of the highly conserved glycine (FXAXRa) display partial agonism on STKR-mediated Ca(2+)-responses, but not on cAMP-responses. STKR therefore seems to differentiate between a number of tachykinins. Gly- and Ala-containing tachykinins are both encoded in the Drosophila tachykinin precursor, thus raising the question of whether DTKR can also distinguish between these two tachykinin types. DTKR was activated by all Drosophila tachykinins and inhibited by tachykinin antagonists. Ala-containing analogs did not produce the remarkable activation behavior previously observed with STKR, suggesting different mechanisms of discerning ligands and/or activating effector pathways for STKR and DTKR.     

Widely distributed Drosophila G-protein-coupled receptor (CG7887) is activated by endogenous tachykinin-related peptides

Neuropeptides related to vertebrate tachykinins have been identified in Drosophila. Two Drosophila G-protein-coupled receptors (GPCRs), designated NKD (CG6515) and DTKR (CG7887), cloned earlier, display sequence similarities to mammalian tachykinin receptors. However, they were not characterized with the endogenous Drosophila tachykinins (DTKs). The present study characterizes one of these receptors, DTKR. We determined that HEK-293 cells transfected with DTKR displayed dose-dependent increases in both intracellular calcium and cyclic AMP levels in response to the different DTK peptides. DTK peptides also induced internalization of DTKR-green fluorescent protein (GFP) fusion constructs in HEK-293 cells. We generated specific antireceptor antisera and showed that DTKR is widely distributed in the adult brain and more scarcely in the larval CNS. The distribution of the receptor in brain neuropils corresponds well with the distribution of its ligands, the DTKs. Our findings suggest that DTKR is a DTK receptor in Drosophila and that this ligand-receptor system plays multiple functional roles.     

Neuronal expression of a newly identified Drosophila melanogaster G protein alpha 0 subunit.

Guanine nucleotide-binding proteins (G proteins) mediate signals between activated cell-surface receptors and cellular effectors. A bovine G-protein alpha-subunit cDNA has been used to isolate similar sequences from Drosophila genomic and cDNA libraries. One class, which we call DG alpha 0, hybridized to position 47A on the second chromosome of Drosophila. The nucleotide sequence of the protein coding region of one cDNA has been determined, revealing an alpha subunit that is 81% identical with rat alpha 0. The cDNA hybridizes strongly to a 3.8 kb mRNA and weakly with a 5.3 kb message. Antibodies raised against a trp-E-DG alpha 0 fusion protein recognized a 39,000 Da protein in Drosophila extracts. In situ hybridization to adult Drosophila sections combined with immunohistochemical studies revealed expression throughout the optic lobes and central brain and in the thoracic and abdominal ganglia. DG alpha 0 message and protein were also detected in the antennae, oocytes, and ovarian nurse cells. The neuronal expression of this gene is similar to mammalian alpha 0, which is most abundantly expressed in the brain.     

Molecular cloning and embryonic expression of dFKBP59, a novel Drosophila FK506-binding protein

A Drosophila cDNA encoding a structural homolog of mammalian FKBP59 (also identified as FKBP52), a member of the FK506-binding protein (FKBP) class of immunophilins, was isolated. The gene dFKBP59 corresponding to this cDNA has been characterized and mapped to the 30D3-4 region. The predicted amino acid sequence of this cDNA shows that the dFKBP59 protein contains one highly conserved FKBP12-like domain followed by two others with less conservation. Northern hybridization reveals that the dFKBP59 mRNA is expressed throughout the Drosophila life-cycle. In contrast to its mammalian homologs, in situ hybridization detected dFKBP59 expression in specific tissues: the lymph glands, Garland cells and oenocyte cells, which are all specialized tissues in which intensive exocytic/endocytic cycling takes place. Garland cells and oenocytes (also called Drosophila nephrocytes) function in taking up waste material from the hemolymph. Finally, I have mapped an enhancer trap element within the 5' region of dFKBP59 which may help in future studies to address the question of its function during Drosophila development.     

A novel member of murine Polycomb-group proteins, Sex comb on midleg homolog protein, is highly conserved, and interacts with RAE28/mph1 in vitro

The Polycomb group of (PcG) genes were originally described in Drosophila, but many PcG genes have mammalian homologs. Genetic studies in flies and mice show that mutations in PcG genes cause posterior transformations caused by failure to maintain repression of homeotic loci, suggesting that PcG proteins have conserved functions. The Drosophila gene Sex comb on midleg (Scm) encodes an unusual PcG protein that shares motifs with the PcG protein polyhomeotic, and with a Drosophila tumor suppressor, lethal(3)malignant brain tumor (l(3)mbt). Expressed sequence tag (EST) databases were searched to recover putative mammalian Scm homologs, which were used to screen murine cDNA libraries. The recovered cDNA encodes two mbt repeats and the SPM domain that characterize Scm, but lacks the cysteine clusters and the serine/threonine-rich region found at the amino terminus of Scm. Accordingly, we have named the gene Sex comb on midleg homolog 1 (Scmh1). Like their Drosophila counterparts, Scmh1 and the mammalian polyhomeotic homolog RAE28/mph1 interact in vitro via their SPM domains. We analyzed the expression of Scmh1 and rae28/mph1 using northern analysis of embryos and adult tissues, and in situ hybridization to embryos. The expression of Scmh1 and rae28/mph1 is well correlated in most tissues of embryos. However, in adults, Scmh1 expression was detected in most tissues, whereas mph1/rae28 expression was restricted to the gonads. Scmh1 is strongly induced by retinoic acid in F9 and P19 embryonal carcinoma cells. Scmh1 maps to 4D1-D2.1 in mice. These data suggest that Scmh1 will have an important role in regulation of homeotic genes in embryogenesis and that the interaction with RAE28/mph1 is important in vivo.     

RTVP-1, a novel human gene with sequence similarity to genes of diverse species, is expressed in tumor cell lines of glial but not neuronal origin

A novel gene, RTVP-1, which shows significant sequence identity to the mammalian testis-specific proteins, a family of plant pathogenesis-related proteins and the vespid venom allergen, antigen-5, has been isolated from a cDNA library of the human glioblastoma brain tumor cell line, U-251 MG. The highest degree of sequence identity was with the human testis-specific protein, TPX1 (38.7% over 119 amino acids). Northern hybridization analysis revealed that in fetal tissue RTVP-1 RNA was detected only in the kidney, but its expression was ubiquitous in adult tissues including brain. Multiple mRNAs encoded by RTVP-1 were highly expressed in a panel of cell lines from nervous system tumors arising from glia, although expression was low or absent in non-glial-derived nervous system tumour cell lines. The GenBank DNA database accession number for this sequence is X91911.     

On a potential global role for vitamin K-dependent gamma-carboxylation in animal systems. Evidence for a gamma-glutamyl carboxylase in Drosophila

The vitamin K-dependent gamma-carboxylation of glutamate to gamma-carboxyglutamate was originally well characterized in the mammalian blood clotting cascade. gamma-Carboxyglutamate has also been found in a number of other mammalian proteins and in neuropeptides from the venoms of marine snails belonging to the genus Conus, suggesting wider prevalence of gamma-carboxylation. We demonstrate that an open reading frame from a Drosophila melanogaster cDNA clone encodes a protein with vitamin K-dependent gamma-carboxylase activity. The open reading frame, 670 amino acids in length, is truncated at the C-terminal end compared with mammalian gamma-carboxylase, which is 758 amino acids. The mammalian gene has 14 introns; in Drosophila there are two much shorter introns but in positions precisely homologous to two of the mammalian introns. In addition, a deletion of 6 nucleotides is observed when cDNA and genomic sequences are compared. In situ hybridization to fixed embryos indicated ubiquitous presence of carboxylase mRNA throughout embryogenesis. Northern blot analysis revealed increased mRNA levels in 12-24-h embryos. The continued presence of carboxylase mRNA suggests that it plays an important role during embryogenesis. Although the model substrate FLEEL is carboxylated by the enzyme, a substrate containing the propeptide of a Conus carboxylase substrate, conantokin G, is poorly carboxylated. Its occurrence in vertebrates, molluscan systems (i.e. Conus), and Drosophila and the apparently strong homology between the three systems suggest that this is a highly conserved and widely distributed post-translational modification in biological systems.