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

We (C. Lent ct al. (2000) Biochem. Biophys. Res. Commun. 269, 91-96) and ot hers (N. Birgul ct 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 belong ing to the insect allatostatin neuropeptide family. In the present paper, w e screened the Berkeley "Drosophila Genome Project" database with "electron ic probes" corresponding to the conserved regions of the four rat (delta, k appa, mu, nociceptin/orphanin FQ) opioid receptors. This yielded alignment with a Drosophila genomic database clone that contained a DNA sequence codi ng for a protein having, again, structural similarities with the rat galani n receptors. Using PCR with primers coding for the presumed exons of this s econd Drosophila receptor gene, 5'- and 3'-RACE, and Drosophila cDNA as tem plate, we subsequently cloned the cDNA of this receptor. The receptor cDNA codes for a protein that is strongly related to the first Drosophila recept or (60% amino acid sequence identity in the transmembrane region; 47% ident ity in the overall sequence) and that is, therefore, most likely to be a se cond Drosophila allatostatin receptor (named DAR-2). The DAR-2 gene has thr ee introns and four exons. Two of these introns coincide with two introns i n the first Drosophila receptor (DAR-1) gene, and have the same intron phas ing, showing that the two receptor genes are clearly evolutionarily related . The DAR-2 gene is located at the right arm of the third chromosome, posit ion 98 D-E. This is the first report on the existence of two different alla tostatin receptors in an animal. (C) 2000 Academic Press.