Identification of Drosophila neuropeptide receptors by G protein-coupled receptors-beta-arrestin2 interactions.
Activation of G protein-coupled receptors (GPCR) leads to the recruitment of beta-arrestins. By tagging the beta-arrestin molecule with a green fluorescent protein, we can visualize the activation of GPCRs in living cells. We have used this approach to de-orphan and study 11 GPCRs for neuropeptide receptors in Drosophila melanogaster. Here we verify the identities of ligands for several recently de-orphaned receptors, including the receptors for the Drosophila neuropeptides proctolin (CG6986), neuropeptide F (CG1147), corazonin (CG10698), dFMRF-amide (CG2114), and allatostatin C (CG7285 and CG13702). We also de-orphan CG6515 and CG7887 by showing these two suspected tachykinin receptor family members respond specifically to a Drosophila tachykinin neuropeptide. Additionally, the translocation assay was used to de-orphan three Drosophila receptors. We show that CG14484, encoding a receptor related to vertebrate bombesin receptors, responds specifically to allatostatin B. Furthermore, the pair of paralogous receptors CG8985 and CG13803 responds specifically to the FMRF-amide-related peptide dromyosuppressin. To corroborate the findings on orphan receptors obtained by the translocation assay, we show that dromyosuppressin also stimulated GTPgammaS binding and inhibited cAMP by CG8985 and CG13803. Together these observations demonstrate the beta-arrestin-green fluorescent protein translocation assay is an important tool in the repertoire of strategies for ligand identification of novel G protein-coupled receptors.
Johnson
Erik C
EC
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Bohn
Laura M
LM
Barak
Larry S
LS
Birse
Ryan T
RT
Nässel
Dick R
DR
Caron
Marc G
MG
Taghert
Paul H
PH
D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D029721:Drosophila Proteins / Q000378:metabolism; D019835:FMRFamide / Q000378:metabolism; D049452:Green Fluorescent Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007301:Insect Hormones / Q000378:metabolism; D019476:Insect Proteins; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D009479:Neuropeptides / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D018013:Receptors, Neuropeptide / Q000378:metabolism*; D018000:Receptors, Peptide / Q000378:metabolism; D018039:Receptors, Tachykinin / Q000378:metabolism; D014162:Transfection; D000071557:beta-Arrestins
D019390:Arrestins; C489221:Crz protein, Drosophila; D029721:Drosophila Proteins; D007301:Insect Hormones; D019476:Insect Proteins; D008024:Ligands; D008164:Luminescent Proteins; C478106:MsR1 protein, Drosophila; C478107:MsR2 protein, Drosophila; D009479:Neuropeptides; D009842:Oligopeptides; D010455:Peptides; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D018000:Receptors, Peptide; D018039:Receptors, Tachykinin; D000071557:beta-Arrestins; C076523:allatostatin; C060602:corazonin protein, insect; C076101:neuropeptide F; C077905:neomyosuppressin; D049452:Green Fluorescent Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C011536:proctolin; D019835:FMRFamide; D000242:Cyclic AMP