Molecular identification of the first insect ecdysis triggering hormone receptors

The Drosophila Genome Project website (www.flybase.org) contains an annotated gene sequence (CG5911), coding for a G protein-coupled receptor. We cloned the cDNA corresponding to this sequence and found that the gene has not been correctly predicted. The corrected gene CG5911 has five introns and six exons (1-6). Alternative splicing yields two cDNAs called A (containing exons 1-5) and B (containing exons 1-4, 6). We expressed these splicing variants in Chinese hamster ovary cells and found that the corrected CG5911-A and -B cDNAs coded for two different G protein-coupled receptors that could be activated by low concentrations of Drosophila ecdysis triggering hormones-1 and -2. Ecdysis (cuticle shedding) is an important behaviour, allowing growth and metamorphosis in insects and other arthropods. Our paper is the first report on the molecular identification of ecdysis triggering hormone receptors from insects.     

A new family of insect tyramine receptors

The Drosophila Genome Project database contains a gene, CG7431, annotated to be an "unclassifiable biogenic amine receptor." We have cloned this gene and expressed it in Chinese hamster ovary cells. After testing various ligands for G protein-coupled receptors, we found that the receptor was specifically activated by tyramine (EC(50), 5x10(-7)M) and that it showed no cross-reactivity with beta-phenylethylamine, octopamine, dopa, dopamine, adrenaline, noradrenaline, tryptamine, serotonin, histamine, and a library of 20 Drosophila neuropeptides (all tested in concentrations up to 10(-5) or 10(-4)M). The receptor was also expressed in Xenopus oocytes, where it was, again, specifically activated by tyramine with an EC(50) of 3x10(-7)M. Northern blots showed that the receptor is already expressed in 8-hour-old embryos and that it continues to be expressed in all subsequent developmental stages. Adult flies express the receptor both in the head and body (thorax/abdomen) parts. In addition to the Drosophila tyramine receptor gene, CG7431, we found another closely related Drosophila gene, CG16766, that probably also codes for a tyramine receptor. Furthermore, we annotated similar tyramine-like receptor genes in the genomic databases from the malaria mosquito Anopheles gambiae and the honeybee Apis mellifera. These four tyramine or tyramine-like receptors constitute a new receptor family that is phylogenetically distinct from the previously identified insect octopamine/tyramine receptors. The Drosophila tyramine receptor is, to our knowledge, the first cloned insect G protein-coupled receptor that appears to be fully specific for tyramine.     

Molecular cloning and genomic organization of a second probable allatostatin receptor from Drosophila melanogaster

We (C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 269, 91-96) and others (N. Birgül et al. (1999) EMBO J. 18, 5892-5900) have recently cloned a Drosophila receptor that was structurally related to the mammalian galanin receptors, but turned out to be a receptor for a Drosophila peptide belonging to the insect allatostatin neuropeptide family. In the present paper, we screened the Berkeley "Drosophila Genome Project" database with "electronic probes" corresponding to the conserved regions of the four rat (delta, kappa, mu, nociceptin/orphanin FQ) opioid receptors. This yielded alignment with a Drosophila genomic database clone that contained a DNA sequence coding for a protein having, again, structural similarities with the rat galanin receptors. Using PCR with primers coding for the presumed exons of this second Drosophila receptor gene, 5'- and 3'-RACE, and Drosophila cDNA as template, we subsequently cloned the cDNA of this receptor. The receptor cDNA codes for a protein that is strongly related to the first Drosophila receptor (60% amino acid sequence identity in the transmembrane region; 47% identity in the overall sequence) and that is, therefore, most likely to be a second Drosophila allatostatin receptor (named DAR-2). The DAR-2 gene has three introns and four exons. Two of these introns coincide with two introns in the first Drosophila receptor (DAR-1) gene, and have the same intron phasing, showing that the two receptor genes are clearly evolutionarily related. The DAR-2 gene is located at the right arm of the third chromosome, position 98 D-E. This is the first report on the existence of two different allatostatin receptors in an animal.     

Molecular identification of a myosuppressin receptor from the malaria mosquito Anopheles gambiae

The insect myosuppressins (X1DVX2HX3FLRFamide) are neuropeptides that generally block insect muscle activities. We have used the genomic sequence information from the malaria mosquito Anopheles gambiae Genome Project to clone a G protein-coupled receptor that was closely related to the two previously cloned and characterized myosuppressin receptors from Drosophila [Proc. Natl. Acad. Sci. USA 100 (2003) 9808]. The mosquito receptor cDNA was expressed in Chinese hamster ovary cells and was found to be activated by low concentrations of Anopheles myosuppressin (TDVDHVFLRFamide; EC50, 1.6 x 10(-8)M). The receptor was not activated by a library of 35 other insect neuropeptides and monoamines, including neuropeptides that resembled myosuppressin in their C-terminal moiety, such as PDRNFLRFamide (Anopheles FMRFamide-3), other Anopheles FMRFamide peptides, or neuropeptide F-like peptides, showing that the receptor was quite selective for myosuppressin. These results also showed that the myosuppressin receptor needs a much larger portion than the C-terminal FLRFamide sequence for its activation. The insect myosuppressins are often grouped together with the insect FMRFamides under the name FaRPs (FMRFamide-related peptides). However, this is not justified anymore, because the insect myosuppressin receptor/ligand couple is both functionally and evolutionarily fully unrelated to the insect FMRFamide receptor/ligand couple. To our knowledge, this is the first report on the molecular identification of a mosquito neuropeptide receptor.     

Molecular identification of a Drosophila G protein-coupled receptor specific for crustacean cardioactive peptide

The Drosophila Genome Project website (www.flybase.org) contains the sequence of an annotated gene (CG6111) expected to code for a G protein-coupled receptor. We have cloned this receptor and found that its gene was not correctly predicted, because an annotated neighbouring gene (CG14547) was also part of the receptor gene. DNA corresponding to the corrected gene CG6111 was expressed in Chinese hamster ovary cells, where it was found to code for a receptor that could be activated by low concentrations of crustacean cardioactive peptide, which is a neuropeptide also known to occur in Drosophila and other insects (EC(50), 5.4 x 10(-10)M). Other known Drosophila neuropeptides, such as adipokinetic hormone, did not activate the receptor. The receptor is expressed in all developmental stages from Drosophila, but only very weakly in larvae. In adult flies, the receptor is mainly expressed in the head. Furthermore, we identified a gene sequence in the genomic database from the malaria mosquito Anopheles gambiae that very likely codes for a crustacean cardioactive peptide receptor.     

Molecular cloning and genomic organization of an allatostatin preprohormone from Drosophila melanogaster

The insect allatostatins are neurohormones, acting on the corpora allata (where they block the release of juvenile hormone) and on the insect gut (where they block smooth muscle contraction). We screened the "Drosophila Genome Project" database with electronic sequences corresponding to various insect allatostatins. This resulted in alignment with a DNA sequence coding for some Drosophila allatostatins (drostatins). Using PCR with oligonucleotide primers directed against the presumed exons of this Drosophila allatostatin gene and subsequent 3'- and 5'-RACE, we were able to clone its cDNA. The Drosophila allatostatin preprohormone contains four amino acid sequences that after processing would give rise to four Drosophila allatostatins: Val-Glu-Arg-Tyr-Ala-Phe-Gly-Leu-NH(2) (drostatin-1), Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu-NH(2) (drostatin-2), Ser-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH(2) (drostatin-3), and Thr-Thr-Arg-Pro-Gln-Pro-Phe-Asn-Phe-Gly-Leu-NH(2) (drostatin-4). Drostatin-2 is identical to helicostatin-2 (11-18) and drostatin-3 to helicostatin-3, two neurohormones previously isolated from the moth Helicoverpa armigera. Furthermore, drostatin-3 has previously been isolated from Drosophila itself. Drostatins-1 and -4 are novel members of the insect allatostatin neuropeptide family. The Drosophila allatostatin preprohormone gene contains two introns and three exons. The gene is located on the right arm of the third chromosome, position 96A-B. The existence of at least four different Drosophila allatostatins opens the possibility of a differential action of some of these hormones on the two recently cloned Drosophila allatostatin receptors, DAR-1 and -2. This is the first report on an allatostatin preprohormone from Drosophila.     

Two nitridergic peptides are encoded by the gene capability in Drosophila melanogaster

A Drosophila gene (capability, capa) at 99D on chromosome 3R potentially encodes three neuropeptides: GANMGLYAFPRV-amide (capa-1), ASGLVAFPRV-amide (capa-2), and TGPSASSGLWGPRL-amide (capa-3). Capa-1 and capa-2 are related to the lepidopteran hormone cardioacceleratory peptide 2b, while capa-3 is a novel member of the pheromone biosynthesis-activating neuropeptide/diapause hormone/pyrokinin family. By immunocytochemistry, we identified four pairs of neuroendocrine cells likely to release the capa peptides into the hemolymph: one pair in the subesophageal ganglion and the other three in the abdominal neuromeres. In the Malpighian (renal) tubule, capa-1 and capa-2 increase fluid secretion rates, stimulate nitric oxide production, and elevate intracellular Ca(2+) and cGMP in principal cells. Capa-stimulated fluid secretion, but not intracellular Ca(2+) concentration rise, is inhibited by the guanylate cyclase inhibitor methylene blue. The actions of capa-1 and capa-2 are not synergistic, implying that both act on the same pathways in tubules. The capa gene is thus the first to be shown to encode neuropeptides that act on renal fluid production through nitric oxide.     

Cloning and expression of Drosophila melanogaster UDP-GlcNAc:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I

A TBLASTN search of the Drosophila melanogaster expressed sequence tag (EST) database with the amino acid sequence of human UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I, EC 2.4.1.101) as probe yielded a clone (GM01211) with 56% identity over 36 carboxy-terminal amino acids. A 550 base pair (bp) probe derived from the EST clone was used to screen a Drosophila cDNA library in lambda-ZAP II and two cDNAs lacking a start ATG codon were obtained. 5'-Rapid amplification of cDNA ends (5'-RACE) yielded a 2828 bp cDNA containing a full-length 1368 bp open reading frame encoding a 456 amino acid protein with putative N-terminal cytoplasmic (5 residues) and hydrophobic transmembrane (20 residues) domains. The protein showed 52% amino acid sequence identity to human GnT I. This cDNA, truncated to remove the N-terminal hydrophobic domain, was expressed in the baculovirus/Sf9 system as a secreted protein containing an N-terminal (His)6 tag. Protein purified by adsorption to and elution from nickel beads converted Man alpha1-6(Man alpha1-3)Man beta-octyl (M3-octyl) to Man alpha1-6(GlcNAc beta1-2Man alpha1-3)Man beta-octyl. The Km values (0.7 and 0.03 mM for M3-octyl and UDP-GlcNAc respectively), temperature optimum (37 degrees C), pH optimum (pH 5 to 6) and divalent cation requirements (Mn > Fe, Mg, Ni > Ba, Ca, Cd, Cu) were similar to mammalian GnT I. TBLASTN searches of the Berkeley Drosophila Genome Project database with the Drosophila GnT I cDNA sequence as probe allowed localization of the gene to chromosomal region 2R; 57A9. Comparison of the cDNA and genomic DNA sequences allowed the assignment of seven exons and six introns; all introns showed GT-AG splice site consensus sequences. This is the first insect GnT I gene to be cloned and expressed.     

NKD, a developmentally regulated tachykinin receptor in Drosophila.

A number of neuropeptides have been described which are present in the insect nervous system. The physiological role of these neuropeptides has not yet been clarified. We have characterized a Drosophila melanogaster cDNA coding for a protein, NKD, whose sequence resembles that of mammalian G protein-coupled neuropeptide receptors. This protein shows 38% homology with the mammalian tachykinin NK3 receptor within the transmembrane domain region. Stable cell lines expressing this cDNA are responsive to Locusta migratoria tachykinin but not to other peptides of the tachykinin family. The expression of this gene is detected principally in adult fly heads, but also in the adult body and in embryos. Interestingly, NKD mRNA is detected at very early stages of Drosophila embryonic development (3 h) and reaches the highest level of expression at 12-16 h, a time which correlates with the period of major neuronal development. In situ hybridization experiments demonstrate that NKD is expressed in the central nervous system, as well as in subsets of neurons in each segment of the developing ventral ganglia. The cytological localization of this gene is at position 86C on the Drosophila third chromosome.     

Molecular identification of the first SIFamide receptor

SIFamide is the short name and also the C terminus of the Drosophila neuropeptide AYRKPPFNGSIFamide. SIFamide has been isolated or predicted from various insects and crustaceans, and appears to be extremely well conserved among these arthropods. However, the function of this neuropeptide is still enigmatic. Here, we have identified the Drosophila gene (CG10823) coding for the SIFamide receptor. When expressed in Chinese hamster ovary cells, the receptor is only activated by Drosophila SIFamide (EC(50), 2x10(-8)M) and not by a library of 32 other insect neuropeptides and eight biogenic amines. Database searches revealed SIFamide receptor orthologues in the genomes from the malaria mosquito Anopheles gambiae, the silkworm Bombyx mori, the red flour beetle Tribolium castaneum, and the honey bee Apis mellifera. An alignment of the five insect SIFamide or SIFamide-like receptors showed, again, an impressive sequence conservation (67-77% amino acid sequence identities between the seven-transmembrane areas; 82-87% sequence similarities). The identification of well-conserved SIFamide receptor orthologues in all other insects with a sequenced genome, suggests that the SIFamide/receptor couple must have an essential function in arthropods. This paper is the first report on the identification of a SIFamide receptor.     

Isolation and characterization of the Drosophila translational elongation factor 2 gene.

We have isolated a cDNA clone that encodes the Drosophila melanogaster elongation factor 2 (EF2), a protein involved in the elongation step of protein synthesis. This identification was based on the high degree of its amino acid sequence identity (greater than 80%) to that of hamster EF2. The gene encoding Drosophila EF2 is found at position 39E-F of the 2L chromosomal arm and maybe identical to the M(2)H locus, which produces a Minute phenotype when mutated. The genomic organization of the locus includes four exons. Conserved sequence segments shared with a variety of GTP binding proteins are found in the amino terminal third of the protein, and segments unique to EF2 and its prokaryotic functional homolog, EF-G, are in the carboxy terminal half; these two regions are segregated in two respective exons.     

Molecular cloning and genomic organization of a novel receptor from Drosophila melanogaster structurally related to mammalian galanin receptors

We screened the Berkeley "Drosophila Genome Project" database with "electronic probes" corresponding to conserved amino acid sequences from the five known rat somatostatin receptors. This yielded alignment with a Drosophila genomic clone that contained a DNA sequence coding for a protein, having amino acid sequence identities with the rat galanin receptors. Using PCR with Drosophila cDNA as a template, and oligonucleotide probes coding for the exons of the presumed Drosophila gene, we were able to clone the cDNA for this receptor. The Drosophila receptor has most amino acid sequence identity with the three mammalian galanin receptors (37% identity with the rat galanin receptor type-1, 32% identity with type-2, and 29% identity with type-3). Less sequence identity exists with the mammalian opioid/nociceptin-orphanin FQ receptors (26% identity with the rat micro opioid receptor), and mammalian somatostatin receptors (25% identity with the rat somatostatin receptor type-2). The novel Drosophila receptor gene contains ten introns and eleven exons and is located at the distal end of the X chromosome.     

The novel exon, exon T, of the human progesterone receptor gene and the genomic organization of the gene

Recently, we have cloned the novel isoform of the progesterone receptor (PR) cDNA (PR isoform S cDNA) from the human testicular cDNA library. The isoform S cDNA consists of the novel exon (termed the exon S of the PR gene) and the exons 4-8 of the PR gene. In order to investigate the existence of the other isoform of the human PR cDNA, the human testicular cDNA library was screened by the exons 4-8 corresponding sequence of the human PR cDNA in the present study. As a result, we have identified a novel isoform of the PR cDNA (termed the PR isoform T cDNA (PR-T cDNA)), which consisted of a previously unidentified 5'-sequence and the exons 4-8 of the PR gene. The structure of this isoform T cDNA is essentially similar to that of the isoform S cDNA. By the genomic cloning, the 5'-sequence of the PR isoform T mRNA was demonstrated to originate from a novel independent exon, exon T, which was located in the 5'-upstream region of the exon S.     

Cloning of the novel isoform of the estrogen receptor beta cDNA (ERbeta isoform M cDNA) from the human testicular cDNA library

Our recent report has revealed the existence of the progesterone receptor (PR) isoform S, which consists of the novel PR exon S and exons 4-8 of the PR gene in the human testicular cDNA library. More recently, we have cloned the human estrogen receptor alpha (ERalpha) isoform S cDNA from the library. The ERalpha isoform S cDNA also contains the novel ERalpha exon S and exons 4-8 of the ERalpha cDNA. Based on these findings, we assumed that the novel isoform of cDNA like the PR- and ERalpha isoforms might exist in the human ER beta (ERbeta). In order to investigate this possibility, we have screened the human testicular cDNA library using the exons 4-8 corresponding sequence of the human ERbeta cDNA. Consequently, we have cloned a novel isoform of the ERbeta cDNA that consists of a previously unidentified 5'-sequence and the exons 5-8 of the ERbeta gene. We termed this isoform cDNA the "ERbeta isoform M cDNA". The 5'-sequence of the ERbeta isoform M cDNA was confirmed to be derived from a novel exon (termed the "exon M") by analysis of the genomic DNA. Moreover, we have analyzed the molecular size of the ERbeta isoform M encoded by the ERbeta isoform M mRNA by transient expression of the ERbeta isoform M cDNA in the 293T cell. The approximately 28 kDa protein, which was recognized by the anti-rat ERbeta antibody against the carboxyl-terminal region, was synthesized in the cells. Thus, we concluded that the ATG in the exon M could be used as the translation initiation codon. This report revealed for the first time the existence of the ERbeta mRNA isoform that is not caused by the skipping of one or more exons, by the alternative usage of the multiple exon 8s, nor by the alternative utilization of the untranslated 5'-exons located on the upstream region of the exon 1.     

Identification of four Drosophila allatostatins as the cognate ligands for the Drosophila orphan receptor DAR-2

The allatostatins are generally inhibitory insect neuropeptides. The Drosophila orphan receptor DAR-2 is a G-protein-coupled receptor, having 47% amino acid residue identity with another Drosophila receptor, DAR-1 (which is also called dros. GPCR, or DGR) that was previously shown to be the receptor for an intrinsic Drosophila A-type (cockroach-type) allatostatin. Here, we have permanently expressed DAR-2 in CHO cells and found that it is the cognate receptor for four Drosophila A-type allatostatins, the drostatins-A1 to -A4. Of all the drostatins, drostatin-A4 (Thr-Thr-Arg-Pro-Gln-Pro-Phe-Asn-Phe-Gly-Leu-NH(2)) is the most effective in causing a second messenger cascade (measured as bioluminescence; threshold, 10(-9) M; EC(50), 10(-8) M), whereas the others are less effective and about equally potent (EC(50), 8 x 10(-8) M). Northern blots showed that the DAR-2 gene is expressed in embryos, larvae, pupae, and adult flies. In adult flies, the receptor is more strongly expressed in the thorax/abdomen than in the head parts, suggesting that DAR-2 is a gut receptor. This is confirmed by Northern blots from 3rd instar larvae, showing that the DAR-2 gene is mainly expressed in the gut and only very weakly in the brain. The Drosophila larval gut also contains about 20-30 endocrine cells, expressing the gene for the drostatins-A1 to -A4. We suggest, therefore, that DAR-2 mediates an allatostatin (drostatin)-induced inhibition of gut motility. This is the first report on the permanent and functional expression of a Drosophila gut neurohormone receptor.     

果蝇程序化死亡基因5(PDCD5)同源cDNA的克隆和序列分析

 为了解人类白血病细胞凋亡相关新基因 TFAR1 9(PDCD5,programmed cell death5)在不同种属间的序列同源性 ,利用 EST(expression sequence tag)拼接、RT- PCR、DNA序列测定技术及计算机分析技术 ,首次成功地进行了果蝇 PDCD5同源 c DNA编码区基因克隆和序列分析 .发现果蝇与小鼠及果蝇与人 PDCD5在核苷酸水平上分别有 57.5%和 57.1 %的同源性 ,在氨基酸水平上分别有 46.8%和 46.4%的同源性 .功能区分析发现 ,果蝇 PDCD5c DNA编码 1 33个氨基酸 ,计算机预测可能是一种核蛋白 ,含 5个可能的酪蛋白激酶 (casein kinase )磷酸化位点 ,2个可能的 PKC磷酸化位点 ,与人 PDCD5的功能区类似 .因而果蝇 PDCD5是与人 PDCD5同源的新基因 ,可能都与细胞程序化死亡相关 .