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

The allatostatins are generally inhibitory insect neuropeptides. The Drosop hila orphan receptor DAR-2 is a G-protein-coupled receptor, having 47% amin o acid residue identity with another Drosophila receptor, DAR-1 (which is a lso 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 cog nate receptor for four Drosophila A-type allatostatins, the drostatins-A1 t o -A4. Of all the drostatins, drostatin-A4 (Thr-Thr-Arg-Pro-Gln-Pro-Phe-Asn -Phe-Gly-Leu-NH2) is the most effective in causing a second messenger casca de (measured as bioluminescence; threshold, 10(-9) M; EC50, 10(-8) M), wher eas the others are less effective and about equally potent (EC50, 8 x 10(-8 ) M). Northern blots showed that the DAR-2 gene is expressed in embryos, la rvae, pupae, and adult flies. In adult flies, the receptor is more strongly expressed in the thorax/abdomen than in the head parts, suggesting that DA R-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 s uggest, therefore, that DAR-2 mediates an allatostatin (drostatin)induced i nhibition of gut motility. This is the first report on the permanent and fu nctional expression of a Drosophila gut neurohormone receptor. (C) 2001 Aca demic Press.