Identification in **Drosophila melanogaster** of the invertebrate G protein-coupled FMRFamide receptor
Faculty of Sciences. Biology
Faculty of Sciences. Chemistry
Faculty of Medicine and Health Sciences
Engineering sciences. Technology
Proceedings of the National Academy of Sciences of the United States of America. - Washington, D.C.
, p. 15363-15368
We here describe the cloning and characterization of the functionally active Drosophila melanogaster (Drm) FMRFamide receptor, which we designated as DrmFMRFa-R. The full-length ORF of a D. melanogaster orphan receptor, CG 2114 (Berkeley Drosophila Genome Project), was cloned from genomic DNA. This receptor is distantly related to mammalian thyroid-stimulating hormone-releasing hormone receptors and to a set of Caenorhabditis elegans orphan receptors. An extract of 5,000 central nervous systems from the related but bigger flesh fly, Neobellieria bullata (Neb), was used to screen cells expressing the orphan receptor. Successive purification steps, followed by MS, revealed the sequence of two previously uncharacterized endogenous peptides, APPQPSDNFIRFamide (Neb-FIRFamide) and pQPSQDFMRFamide (Neb-FMRFamide). These are reminiscent of other insect FMRFamide peptides, having neurohormonal as well as neurotransmitter functions. Nanomolar concentrations of the Drm FMRFamides (DPKQDFMRFamide, TPAEDFMRFamide, SDNFMRFamide, SPKQDFMRFamide, and PDNFMRFamide) activated the cognate receptor in a dose-dependent manner. To our knowledge, the cloned DrmFMRFa-R is the first functionally active FMRFamide G protein-coupled receptor described in invertebrates to date. The publication of the complete euchromatic portion of the Drosophila melanogaster (Drm) genome by Adams et al. (1) enabled the analysis of the full repertoire of Drm G protein-coupled receptors (GPCRs) for the first time. The Drm genome encodes about 160 GPCR genes, including at least 21 GPCRs for classic neurotransmitters and neuromodulators and between 39 and 45 peptide receptors (2, 3). To date, only five Drm peptide GPCRs have been fully characterized functionally (48). The others are orphan receptors, meaning that neither their function nor their ligand is known. Based on sequence similarities revealed by blast analysis and phylogenetic tree construction, the orphan receptor CG2114 was classified within the thyroid-stimulating hormone (TSH)-releasing hormone (TRH) receptors (3). This similarity is of particular interest, because the presence of TRH or thyroid hormones has as yet not been demonstrated in insects or invertebrates in general. Vertebrate thyroid hormones T3 and T4 could play a role in the reproduction of arthropods, because they have effects similar to juvenile hormone on the follicle cells during vitellogenesis (9). Because we found that this orphan receptor was not efficiently activated by bovine TRH, the natural ligand in Drm was searched for by means of a reverse physiology approach (10). The functionally expressed orphan Drm receptor was screened against peptide fractions purified from a CNS tissue extract from the related but much bigger flesh fly, Neobellieria bullata (Neb). In the present paper, we show that FMRFamides are the cognate ligands for this orphan receptor, which we annotated as the Drm FMRFamide receptor or DrmFMRFa-R.