Functional annotation of two orphan G-protein-coupled receptors, Drostar1 and -2, from Drosophila melanogaster and their ligands by reverse pharmacology.
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.
Kreienkamp
Hans-Jurgen
HJ
Institut für Zellbiochemie und Klinische Neurobiologie, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
Larusson
Hannes Jon
HJ
Witte
Ines
I
Roeder
Thomas
T
Birgul
Necla
N
Honck
Hans-Hinrich
HH
Harder
Sonke
S
Ellinghausen
Gunter
G
Buck
Friedrich
F
Richter
Dietmar
D
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
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