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Functional Annotation of Two Orphan G-protein-coupled Receptors, Drostar1 and -2, from Drosophila melanogaster and Their Ligands by Reverse Pharmacology
- Source :
- Journal of Biological Chemistry. 277:39937-39943
- Publication Year :
- 2002
- Publisher :
- Elsevier BV, 2002.
-
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.
- Subjects :
- DNA, Complementary
Xenopus
Molecular Sequence Data
Receptors, Cell Surface
Ligands
Biochemistry
Receptors, G-Protein-Coupled
GTP-Binding Proteins
Complementary DNA
Animals
Drosophila Proteins
Amino Acid Sequence
Cloning, Molecular
Receptor
Molecular Biology
Chromatography, High Pressure Liquid
In Situ Hybridization
Phylogeny
G protein-coupled receptor
Orphan receptor
Reverse pharmacology
Base Sequence
Sequence Homology, Amino Acid
biology
Edman degradation
Reverse Transcriptase Polymerase Chain Reaction
Somatostatin receptor
Chromosome Mapping
Cell Biology
biology.organism_classification
Immunohistochemistry
Molecular biology
Protein Structure, Tertiary
Cell biology
Drosophila melanogaster
Oocytes
Peptides
Protein Binding
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 277
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....936245aa29e0ebc0e89b3f887b024c95