title: Functional annotation of two orphan G-protein-coupled receptors, Drostar1 and -2, from Drosophila melanogaster and their ligands by reverse pharmacology.
abstract: By combining a Drosophila genome data base search and reverse transcriptase-PCR-based cDNA isolation, two G-protein-coupled receptors were cloned, which are the closest known invertebrate homologs of the mammalian opioid/somatostatin receptors. However, when functionally expressed in Xenopus oocytes by injection of Drosophila orphan receptor RNAs together with a coexpressed potassium channel, neither receptor was activated by known mammalian agonists. By applying a reverse pharmacological approach, the physiological ligands were isolated from peptide extracts from adult flies and larvae. Edman sequencing and mass spectrometry of the purified ligands revealed two decapentapeptides, which differ only by an N-terminal pyroglutamate/glutamine. The peptides align to a hormone precursor sequence of the Drosophila genome data base and are almost identical to allatostatin C from Manduca sexta. Both receptors were activated by the synthetic peptides irrespective of the N-terminal modification. Site-directed mutagenesis of a residue in transmembrane region 3 and the loop between transmembrane regions 6 and 7 affect ligand binding, as previously described for somatostatin receptors. The two receptor genes each containing three exons and transcribed in opposite directions are separated by 80 kb with no other genes predicted between. Localization of receptor transcripts identifies a role of the new transmitter system in visual information processing as well as endocrine regulation.
authors.affiliation: Institut für Zellbiochemie und Klinische Neurobiologie, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
authors.forename: Hans-Jurgen
authors.initials: HJ
authors.lastname: Kreienkamp
authors.forename: Hannes Jon
authors.initials: HJ
authors.lastname: Larusson
authors.forename: Ines
authors.initials: I
authors.lastname: Witte
authors.forename: Thomas
authors.initials: T
authors.lastname: Roeder
authors.forename: Necla
authors.initials: N
authors.lastname: Birgul
authors.forename: Hans-Hinrich
authors.initials: HH
authors.lastname: Honck
authors.forename: Sonke
authors.initials: S
authors.lastname: Harder
authors.forename: Gunter
authors.initials: G
authors.lastname: Ellinghausen
authors.forename: Friedrich
authors.initials: F
authors.lastname: Buck
authors.forename: Dietmar
authors.initials: D
authors.lastname: Richter
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics; D004331:Drosophila melanogaster / Q000737:chemistry* / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008024:Ligands; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010802:Phylogeny; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014981:Xenopus
chemical_list: C469361:AstC-R1 protein, Drosophila; C469362:AstC-R2 protein, Drosophila; D018076:DNA, Complementary; D029721:Drosophila Proteins; D008024:Ligands; D010455:Peptides; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D019204:GTP-Binding Proteins