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.
Lenz
C
C
Department of Cell Biology, Zoological Institute, University of Copenhagen, Universitetsparken 15, Copenhagen, DK-2100, Denmark.
Williamson
M
M
Hansen
G N
GN
Grimmelikhuijzen
C J
CJ
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004330:Drosophila / Q000737:chemistry*; D029721:Drosophila Proteins; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D017403:In Situ Hybridization; D019476:Insect Proteins; D007413:Intestinal Mucosa / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000737:chemistry*; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000737:chemistry*; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D013997:Time Factors
C096426:AstA-R1 protein, Drosophila; C543272:AstA-R2 protein, Drosophila; D029721:Drosophila Proteins; D019476:Insect Proteins; D008024:Ligands; D009479:Neuropeptides; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; C076523:allatostatin