Identification of Drosophila Neuropeptide Receptors by G Protein-coupled Receptors-β-Arrestin2 Interactions
2003; Elsevier BV; Volume: 278; Issue: 52 Linguagem: Inglês
10.1074/jbc.m306756200
ISSN1083-351X
AutoresErik C. Johnson, Laura Bohn, Larry S. Barak, Ryan T. Birse, Dick R. Nässel, Marc G. Caron, Paul H. Taghert,
Tópico(s)Computational Drug Discovery Methods
ResumoActivation of G protein-coupled receptors (GPCR) leads to the recruitment of β-arrestins. By tagging the β-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 GTPγS binding and inhibited cAMP by CG8985 and CG13803. Together these observations demonstrate the β-arrestin-green fluorescent protein translocation assay is an important tool in the repertoire of strategies for ligand identification of novel G protein-coupled receptors. Activation of G protein-coupled receptors (GPCR) leads to the recruitment of β-arrestins. By tagging the β-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 GTPγS binding and inhibited cAMP by CG8985 and CG13803. Together these observations demonstrate the β-arrestin-green fluorescent protein translocation assay is an important tool in the repertoire of strategies for ligand identification of novel G protein-coupled receptors. G protein-coupled receptors form a family of structurally similar transmembrane signaling proteins. The recent sequencing of the Drosophila melanogaster genome (1Adams M.D. Celniker S.E. Holt R.A. Evans C.A. Gocayne J.D. et al.Science. 2000; 287: 2185-2195Crossref PubMed Scopus (4854) Google Scholar) has enabled the identification of the entire complement of ∼160 fly GPCRs 1The abbreviations used are: GPCRG protein-coupled receptorsGFPgreen fluorescent proteinDMSdromyosuppressinPDFpigment dispersing factorNPFneuropeptide FGRKsGPCR kinasesβarr2β-arrestin2GTPγSguanosine 5′-3-O-(thio)triphosphateDTKDrosophila tachykininAstallatostatin. (2Brody T. Cravchik A. J. Cell Biol. 2000; 150: F83-F88Crossref PubMed Scopus (223) Google Scholar). Of these, 44 encode GPCRs for peptide ligands (3Hewes R. Taghert P.H. Genome Res. 2001; 11: 1126-1142Crossref PubMed Scopus (460) Google Scholar) of which approximately half have been functionally characterized. The number of genes encoding neuropeptides is currently estimated to be at least 32 (4Vanden Broeck J. Peptides (Elmsford). 2001; 22: 241-254Crossref PubMed Scopus (274) Google Scholar, 5Taghert P.H. Veenstra J.A. Adv. Genet. 2003; 49: 1-65Crossref PubMed Scopus (85) Google Scholar). G protein-coupled receptors green fluorescent protein dromyosuppressin pigment dispersing factor neuropeptide F GPCR kinases β-arrestin2 guanosine 5′-3-O-(thio)triphosphate Drosophila tachykinin allatostatin. Many Drosophila GPCRs have been de-orphaned through binding or signaling assays tailored to individual receptor subtypes. De-orphaning the remaining ∼20 fly peptide GPCRs would involve the development of several different signaling assays, which are dependent upon the specific G protein signaling pathway that is utilized by each specific receptor. A more general approach of ligand identification would rely on measurement of a universally shared GPCR property. For example, most GPCRs share a common mechanism by which signaling is terminated. In particular, outside the visual system mammalian GPCRs are desensitized by the actions of five GPCR kinases (GRKs) and the two β-arrestins (6Kohout T.A. Lefkowitz R.J. Mol. Pharmacol. 2003; 63: 9-18Crossref PubMed Scopus (364) Google Scholar). In Drosophila, GPCRs may be desensitized by a similar GRK/arrestin-mediated mechanism; two GRKs (7Cassill J.A. Whitney M. Joazeiro C.A. Becker A. Zuker C.S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11067-11070Crossref PubMed Scopus (73) Google Scholar) and one non-visual arrestin (8Roman G. He J. Davis R.L. Genetics. 2000; 155: 1281-1295Crossref PubMed Google Scholar) have been identified. We have reported previously (9Kim K.M. Valenzano K.J. Robinson S.R. Yao W.D. Barak L.S. Caron M.G. J. Biol. Chem. 2001; 276: 37409-37414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 10Barak L.S. Warabi K. Feng X. Caron M.G. Kwatra M.M. J. Biol. Chem. 1999; 274: 7565-7569Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 11Walker J.K. Premont R.T. Barak L.S. Caron M.G. Shetzline M.A. J. Biol. Chem. 1999; 274: 31515-31523Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) the use of β-arrestin2-green fluorescent protein (βarr2-GFP) chimeras to assess desensitization of a variety of known diverse mammalian GPCRs. Unstimulated receptor-expressing cells display a diffuse GFP fluorescence that fills the cytoplasm. Within minutes of exposure to specific ligands, the majority of the fluorescence translocates to become associated with the membrane. On that basis, we have suggested its use as a generalized method for GPCR de-orphaning (12Barak L.S. Ferguson S.S. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). In this report we demonstrate the utility of the βarr2-GFP assay to study 11 Drosophila peptide GPCRs that are evolutionarily diverse and that couple to multiple second messengers. We first confirm the specific activities of five peptides for six receptors that were previously de-orphaned: proctolin (PROC, CG6986), neuropeptide F (NPF, CG1147), corazonin (COR, CG10698), dFMRF-amide (CG2114), and allatostatin C (AstC, CG7285 and CG13702). Then we extend the observations to confirm the activity of Drosophila tachykinin peptides for two putative Drosophila tachykinin receptors (DTKs, CG6515 and CG7887). Finally, we employ the assay to deorphan three receptors. These include CG14484, which responds specifically to allatostatin B (AstB) neuropeptide, and a pair of receptor paralogues (CG8985 and CG13803) that are sensitive to the FMRF-amide-related neuropeptide dromyosupressin (DMS). Molecular Cloning—We generated full-length receptors constructs for the following genes using methods described in Johnson et al. (13Johnson E.C. Garczynski S.F. Park D. Crim J.W. Nässel D.R. Taghert P.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6198-6203Crossref PubMed Scopus (69) Google Scholar): CG1147, CG2114, CG6515, CG6986, CG7285, CG7887, CG8985, CG10698, CG13702, CG13803, and CG14484. Rapid amplification of cDNA ends PCR was performed using ESTs or fly head cDNA as templates. Primers flanking the predicted open reading frames incorporated restriction sites to facilitate directional cloning into the pcDNA5/FRT vector (Invitrogen) and a 5′ Kozak sequence to facilitate expression in mammalian cells. The sequences of primers used for all reactions are provided in the Supplemental Material. All receptor constructs were fully sequenced. The Supplemental Material lists sequence information for the following receptor cDNAs: CG7285, CG8985, CG13702, CG13803, and CG14484. Those receptors that had been previously published, and for which we found no differences, are not listed (CG1147, CG2114, CG6515, CG7887, and CG10698). The sequence of CG6986 was published previously (13Johnson E.C. Garczynski S.F. Park D. Crim J.W. Nässel D.R. Taghert P.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6198-6203Crossref PubMed Scopus (69) Google Scholar). Transfections and Cell Culture—HEK-293 cells were transiently transfected using electroporation or LipofectAMINE using 10 μg of DNA per 4 × 106 cells. Cells were transfected with a 5:1 ratio of GPCR DNA and β-arrestin2-GFP (βarr2-GFP) DNAs. The dominant negative dynamin mutant (K44A) was included in some transfections, at a ratio of 5:1:1 (GPCR:βarr2-GFP:K44A) (14Zhang J. Barak L.S. Winkler K.E. Caron M.G. Ferguson S.S. J. Biol. Chem. 1997; 272: 27005-27014Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Some experiments included overexpression of a G protein receptor kinase (GRK2). This was achieved using HEK-293 cells that had GRK2 stably integrated into the genome (12Barak L.S. Ferguson S.S. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar), or by including GRK2 in transfections at a ratio of 5:1:1 (GPCR:βarr2-GFP:GRK2). To study calcium responses, stable lines expressing different receptors were generated and selected for resistance to hygromycin B (13Johnson E.C. Garczynski S.F. Park D. Crim J.W. Nässel D.R. Taghert P.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6198-6203Crossref PubMed Scopus (69) Google Scholar). Cells were maintained in a humidified incubator under 5% CO2 atmosphere and at 37 °C and split every 3 days 1:5. Growth medium was Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics. Confocal Microscopy—HEK-293 cells were transfected as described above and plated onto 35-mm dishes with a centered glass coverslip to facilitate imaging. Growth media were removed and replaced with serum-free minimum essential media without phenol red 30 min prior to assays. Peptides were dissolved in the same media and added at room temperature without mixing in 100 μl (equal to 1:10 the volume of culture medium). Images were collected using 488 nm excitation and a 505-nm long-pass filter on a Zeiss laser-scanning microscope or on an Olympus laser scanning microscope. Images were imported into Adobe Photoshop and adjusted for contrast. Peptides—Dromyosuppressin (DMS), adipokinetic hormone, crustacean cardioactive peptide, ecdysis triggering hormone, and pigment dispersing factor (PDF) were purchased from Multiple Peptide Systems, San Diego. The Drosophila tachykinin (DTK1) was purchased from MedProbe, Oslo, Norway. Allatostatin A (AstA-1), allatostatin C (AstC), and DPKQDFMRF-amide were purchased from Bachem. Proctolin and corazonin were purchased from Sigma. Diuretic hormones 31 (DH31) and 44 (DH44) were generously donated by Julian Dow; allatostatin B (AstB-1) and IF-amide were donated by Jan Veenstra; NPF was donated by Joe Crim; and sex peptide was donated by Erik Kubli. Cellular Assays—Intracellular calcium was monitored as described (13Johnson E.C. Garczynski S.F. Park D. Crim J.W. Nässel D.R. Taghert P.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6198-6203Crossref PubMed Scopus (69) Google Scholar). Briefly, HEK-293 cells stably expressing receptor DNA were loaded with 5 μm FLUO3-AM dye (Molecular Probes, Eugene, OR) dissolved in a 20% Pluronic F-127/Me2SO solution in an HBSS solution containing 20 mm HEPES and 2.5 mm probenecid. Cells were measured for changes in fluorescence dependent upon ligand application. To monitor changes in intracellular cAMP levels, HEK-293 cells were transiently transfected with receptor cDNA and a multimerized CRE-luciferase reporter gene (15Hearn M.G. Ren Y. McBride E.W. Reveillaud I. Beinborn M. Kopin A.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14554-14559Crossref PubMed Scopus (158) Google Scholar) and assayed 24 h post-transfection for luciferase activity with a LucLite Kit (PerkinElmer Life Sciences). Peptides were dissolved in serum-free media and incubated for 4 h; forskolin (Sigma) was added at a 1 μm concentration to test for Gi activity. Luminescence was measured on a Victor Wallac 2 Plate reader (PerkinElmer Life Sciences). EC50 values were calculated from concentration-response curves using computerized nonlinear curve fitting (PRISM 3.0, GraphPad, San Diego, CA). [35S]GTPγS Binding Assay—HEK-293 cells were transiently transfected by electroporation using 10 μgof CG13803 per 4 × 106 cells. Cells plated in 10-cm dishes were washed twice in phosphate-buffered saline and collected by cell scraper in ice-cold homogenization buffer (20 mm Tris-HCl (pH 7.4), 100 mm NaCl, 1 mm EDTA, 10 μm GDP (Sigma), 1 mm phenylmethylsulfonyl fluoride, 1 tablet of protease inhibitor mixture (Roche Applied Science) per 10 ml) and were disrupted by a Dounce homogenizer. Crude membranes were prepared by centrifugation at 30,000 × g for 30 min at 4 °C. Membranes were resuspended in assay buffer (50 mm Tris-HCl (pH 7.4), 100 mm NaCl, 5 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol, 10 μm GDP). Membranes (20 μg of protein per assay tube) were incubated in the presence or absence of DMS and 100 pm [35S]GTPγS (1250 Ci/mmol, PerkinElmer Life Sciences) for 1 h at 30 °C as described previously (16Wilbanks A.M. Laporte S.A. Bohn L.M. Barak L.S. Caron M.G. Biochemistry. 2002; 41: 11981-11989Crossref PubMed Scopus (72) Google Scholar). Binding was terminated by rapid filtration over GF/B filters using a Brandel cell harvester (Brandel, Gaithersburg, MD). Filters were washed 3 times with ice-cold distilled H2O and counted by a liquid scintillation counter. The data consist of three separate transfections assayed in quadruplicate. Statistical Analysis—Two-tailed single sample t tests (Microsoft EXCEL) were used to examine basal versus stimulated receptor activity for the measurements of CRE-luciferase activity. p values < 0.05 were considered significant. The translocation of βarr2-GFP chimeras has been used to assess desensitization of a variety of known diverse mammalian GPCRs (9Kim K.M. Valenzano K.J. Robinson S.R. Yao W.D. Barak L.S. Caron M.G. J. Biol. Chem. 2001; 276: 37409-37414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 10Barak L.S. Warabi K. Feng X. Caron M.G. Kwatra M.M. J. Biol. Chem. 1999; 274: 7565-7569Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 11Walker J.K. Premont R.T. Barak L.S. Caron M.G. Shetzline M.A. J. Biol. Chem. 1999; 274: 31515-31523Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). To evaluate the efficacy of the translocation assay to study Drosophila GPCRs, we first tested known Drosophila peptide GPCRs that were demonstrated previously to signal through disparate pathways with a panel of synthetic Drosophila peptides. HEK-293 cells transiently expressing the proctolin receptor encoded by CG6986 (13Johnson E.C. Garczynski S.F. Park D. Crim J.W. Nässel D.R. Taghert P.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6198-6203Crossref PubMed Scopus (69) Google Scholar) displayed clear translocation of the βarr2-GFP to the membrane within 10 min of exposure to 1 μm proctolin (Fig. 1, top) but not to any of the other 16 Drosophila neuropeptides (Fig. 1, middle; Table I). Translocated GFP had the appearance of discrete puncta ranging to a continuous halo. To quantify the robust nature of the response, 100 GFP-positive cells in each of three independent transfections were scored for translocation 20 min after exposure to proctolin. In these cases, we found 89, 91, and 95 cells, respectively, displaying GFP translocation. A typical field of GFP-positive cells is shown in Fig. 1 (bottom panels). In these and subsequent experiments we scored cells within 20 min of exposure to test ligands.Table ISummary of βarr2-GFP translocation following exposure of Drosophila GPCRs to candidate peptide ligands The 11 different Drosophila receptors were transiently expressed in HEK-293 cells, and different peptides were added, all at 1 μm concentrations. Yes indicates translocation, and No indicates translocation was not evident. A dash indicates that this peptide was not tested on the indicated receptor. Every receptor studied was examined following at least two independent transfections. For each transfection at least two dishes were examined, and for each dish at least 25 cells were examined for agonist-stimulated responses. Responses were scored positive when at least one-half of all cells examined produced visible and robust translocation.CG6986CG1147CG10698CG2114CG7285aResponse required additional GRK2CG13702aResponse required additional GRK2CG6515CG7887CG14484CG8985CG13803AstA-1NoNoNoNoNoNoNoNoNoAstB-1NoNoNoNoNoNoNoNoYesNoNoAstCNoNoNoNoYesYesNoNoNoNoNoAKHNoNoNoNo––NoNoNoNoNoCORNoNoYesNoNoNoNoNoNoNoNoCCAPNoNoNoNoNoNoNoNoNoNoNoDMSNoNoNoYesNoNoNoNoNoYesbDynamin K44A included in transfectionYesDH31NoNoNo–NoNoNoNoNoNoNoDH44NoNoNoNoNoNoNoNoNoNoNoETHNoNoNo–––NoNoNoNoNodFMRFa-1NoNoNoYesNoNoNoNoNoNoYesbDynamin K44A included in transfectionIFaNoNoNoNoNoNoNoNoNoNoNoNPFNoYesNoNo––NoNoNoNoNoPDFNoNoNo–NoNoNoNoNoNoNoPROCYesNoNoNo–NoNoNoNoNoSPNoNoNoNoNoNoNoNoNoNoNoDTK(1)NoNoNo–––YesYesNoNoNoa Response required additional GRK2b Dynamin K44A included in transfection Open table in a new tab The receptor for neuropeptide F (CG1147) belongs to the family of NPY-like receptors and signals through Gi-mediated pathways (17Garczynski S.F. Brown M.R. Shen P. Murray T.F. Crim J.W. Peptides (Elmsford). 2002; 23: 773-780Crossref PubMed Scopus (139) Google Scholar). Cells expressing this receptor displayed βarr2-GFP translocation in response to its cognate ligand, NPF, at micromolar concentrations (Fig. 2). These cells did not respond to any of the other peptides tested (Table I). Cells expressing the corazonin receptor encoded by CG10698 (18Cazzamali G. Saxild N. Grimmelikhuijzen C. Biochem. Biophys. Res. Commun. 2002; 298: 31-36Crossref PubMed Scopus (89) Google Scholar, 19Park Y. Kim Y.J. Adams M.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11423-11428Crossref PubMed Scopus (307) Google Scholar), which is related to the vasopressin/oxytocin receptor family, displayed translocation of βarr2-GFP to the membrane specifically in response to 1 μm corazonin (Fig. 2). Cells expressing the dFMRF-amide receptor encoded by CG2114 (20Cazzamali G. Grimmelikhuijzen C.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12073-12078Crossref PubMed Scopus (104) Google Scholar, 21Meeusen T. Mertens I. Clynen E. Baggerman G. Nichols R. Nachman R.J. Huybrechts R. De Loof A. Schoofs L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15363-15368Crossref PubMed Scopus (85) Google Scholar), which is related to the neurotensin/thyrotropin-releasing factor receptor family, displayed βarr2-GFP translocation in response to the dFMRF-amide peptide DPKQDFMRF-amide (Fig. 2) and to the related peptide DMS (data not shown), at micromolar concentrations, but not to any other peptide tested (Table I). Two somatostatin-like receptors (CG7285 and CG13702) in Drosophila were demonstrated recently to respond to Ast-C by using Xenopus oocytes (22Kreienkamp H.J. Larusson H.J. Witte I. Roeder T. Birgul N. Honck H.H. Harder S. Ellinghausen G. Buck F. Richter D. J. Biol. Chem. 2002; 277: 39937-39943Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Exposure of HEK-293 cells expressing CG7285 or CG13702 receptors to Ast-C peptide at micromolar concentrations did not produce detectable GFP translocation (data not shown). Likewise, these receptors did not display any changes in intracellular calcium levels in response to Ast-C application, with or without a co-expressed promiscuous Gα protein subunit. 2E. Johnson, unpublished data. The C termini of these receptors have multiple clusters of serine/threonine residues that are potential targets for GRK phosphorylation (23Barak L.S. Oakley R.H. Laporte S.A. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 93-98Crossref PubMed Scopus (202) Google Scholar). We reasoned that this lack of a detectable response to Ast-C might be due to intrinsic phosphorylation by the endogenous complement of GRKs. In mammalian GPCRs, certain receptors are constitutively phosphorylated and associated with β-arrestin which, at steady state, results in their trafficking to endocytic vesicles and a loss of signaling function (16Wilbanks A.M. Laporte S.A. Bohn L.M. Barak L.S. Caron M.G. Biochemistry. 2002; 41: 11981-11989Crossref PubMed Scopus (72) Google Scholar, 23Barak L.S. Oakley R.H. Laporte S.A. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 93-98Crossref PubMed Scopus (202) Google Scholar). With these receptors, inhibiting clathrin-mediated endocytosis reverses their intracellular localization phenotype. It also re-establishes, at least in part, their signaling function (23Barak L.S. Oakley R.H. Laporte S.A. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 93-98Crossref PubMed Scopus (202) Google Scholar). To address that possibility, we co-transfected CG7285 and CG13702 each with the dynamin K44A mutant to inhibit internalization, and potentially redistribute the receptors to the plasma membrane (14Zhang J. Barak L.S. Winkler K.E. Caron M.G. Ferguson S.S. J. Biol. Chem. 1997; 272: 27005-27014Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). As shown in Fig. 3, now in the presence of dynamin K44A, the association of the CG13702 receptor with βarr2-GFP becomes apparent in the unstimulated state and is further enhanced upon addition of Ast-C (Fig. 3). Cells expressing these receptors (with dynamin K44A) did not respond to any of the other peptides tested (Table I). Co-expression of dynamin K44A with the CG6986 receptor did not alter the βarr2-GFP translocation responses of those cells to proctolin or trigger translocation in response to peptide not previously active (data not shown). Thus, blocking endocytosis can interfere with receptor trafficking but does not change the pharmacological specificity of ligand interactions. CG6515 and CG7887 are predicted to encode paralogous receptors that are related to the tachykinin family of receptors. Both receptors have been shown to respond to heterologous tachykinin peptide (24Li X.J. Wolfgang W. Wu Y.N. North R.A. Forte M. EMBO J. 1991; 10: 3221-3229Crossref PubMed Scopus (136) Google Scholar, 25Monnier D. Colas J.F. Rosay P. Hen R. Borrelli E. Maroteaux L. J. Biol. Chem. 1995; 267: 1298-1302Abstract Full Text PDF Google Scholar, 26Torfs H. Oonk H.B. Broeck J.V. Poels J. Van Poyer W. De Loof A. Guerrero F. Meloen R.H. Akerman K. Nachman R.J. Arch. Insect. Biochem. Physiol. 2001; 48: 39-49Crossref PubMed Scopus (25) Google Scholar). However, neither Drosophila receptor has yet been shown to respond to native Drosophila tachykinins, and thus both remain essentially orphans. Following exposure to a putative Drosophila tachykinin peptide (27Siviter R.J. Coast G.M. Winther A.M. Nachman R.J. Taylor C.A. Shirras A.D. Coates D. Isaac R.E. Nässel D.R. J. Biol. Chem. 2000; 275: 23273-23280Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), we observed translocation of βarr2-GFP in cells expressing either CG6515 or CG7887 (Fig. 4). Cells expressing either receptor did not respond to any of the other peptides tested (Table I). We made several observations that indicated the possible identities of certain orphan GPCRs. CG13803 is predicted to encode a receptor related to the neurotensin/thyrotropin-releasing factor receptor family (3Hewes R. Taghert P.H. Genome Res. 2001; 11: 1126-1142Crossref PubMed Scopus (460) Google Scholar). Cells expressing CG13803 displayed translocation of βarr2-GFP to the membrane following exposure to the neuropeptide DMS at 1 μm (Fig. 5) and at 100 nm concentrations (data not shown). CG13803 cells did not respond to any other peptide tested (Table I). CG14484 is predicted to encode a receptor related to the bombesin receptor family. We observed translocation of βarr2-GFP by these cells in response to 1 μm Ast-B-1 neuropeptide (Fig. 5) but not to any other peptide tested (Table I). We extended our observations on the CG13803 orphan receptor by considering the potentiating effects of co-expressing GRK. With mammalian GPCRs, co-expression of GRKs can accelerate the kinetics and the extent of βarr2-GFP translocation. It is thought that certain receptors may require more GRK to be effectively phosphorylated. CG8985 encodes a member of the neurotensin/thyrotropin-releasing factor receptor family and is paralogous to CG13803 (3Hewes R. Taghert P.H. Genome Res. 2001; 11: 1126-1142Crossref PubMed Scopus (460) Google Scholar). Cells expressing CG8985 and overexpressing GRK2 also responded to 1 μm DMS with βarr2-GFP translocation (Fig. 6) but not to 1 μm DPKQDFMRF-amide or any other peptide tested, under any condition (Table I). Although cells expressing CG13803 responded to DMS independently of additional GRK2 expression (Fig. 5), this manipulation did cause a change in the response profile (Fig. 6); CG13803 cells co-expressing GRK2 now also responded to DPKQDFMRF-amide at both 1 μm and 100 nm but not to any other peptide tested (Table I). Co-expression of GRK2 with the CG6986, CG1147, CG10698, CG6515, CG7887, or CG14484 receptors did not alter the profiles of βarr2-GFP translocation responses of those cells to a broad range of test peptides (data not shown). To extend the observations established with the βarr2-GFP translocation assay, we evaluated the sensitivity and selectivity of CG13803 and CG8985 for DMS and DPKQDFMRF-amide by two additional measures of GPCR activation. CG13803 expressing cells displayed significantly higher [35S]GTPγS binding at doses as low as 10 nm of the DMS peptide (Fig. 7A). We also assayed for changes in intracellular calcium and (indirectly) for cAMP levels in response to CG13803 or CG8985 activation. No significant increases in intracellular calcium or cAMP were elicited by exposure of either receptor to DMS or to DPKQDFMRF-amide (data not shown). With cells that were exposed to forskolin, both DMS and DPKQDFMRF-amide produced significant decreases in cAMP levels (Fig. 7B) suggesting that these receptors are coupled to inhibitory G proteins. Estimated EC50 values for CG13803 were 0.17 nm (r2 = 0.95) for DMS and 4.2 nm (r2 = 0.95) for DPKQDFMRF-amide. For CG8985 cells, estimated EC50 values were = 1.8 nm (r2 = 0.89) for DMS and 13 nm (r2 = 0.92) for DPKQDFMRF-amide. Again DMS was significantly more potent than DPKQDFMRF-amide, and that feature recapitulated results obtained with the βarr2-GFP translocation. For mammalian GPCRs, it has been shown that the pattern of βarr2-GFP translocation falls into two categories. Class A receptors maintain translocated βarr2-GFP at the plasma membrane. Class B receptors, which have C-terminal clusters of serine and threonine residues and thus higher affinity for β-arrestins, internalize the translocated βarr2-GFP into endocytic vesicles (28Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2001; 276: 19452-19460Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). This receptor internalization (14Zhang J. Barak L.S. Winkler K.E. Caron M.G. Ferguson S.S. J. Biol. Chem. 1997; 272: 27005-27014Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar) is visible within 10 min post-treatment as the formation of round fluorescent vesicles that often have non-fluorescent centers. The translocation responses of cells expressing the Drosophila peptide GPCRs tended to show a similar categorization (Fig. 8). Specifically, cells expressing CG2114, CG6515, CG6986, CG7285, CG8985, CG13702, CG13803, and CG14484 typically displayed class A type characteristics. Cells expressing CG1147, CG7887, and CG10698 typically displayed class B type characteristics. The βarr2-GFP translocation assay has been used effectively and extensively to study diverse GPCRs that are sensitive to different peptides and amines (9Kim K.M. Valenzano K.J. Robinson S.R. Yao W.D. Barak L.S. Caron M.G. J. Biol. Chem. 2001; 276: 37409-37414Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 10Barak L.S. Warabi K. Feng X. Caron M.G. Kwatra M.M. J. Biol. Chem. 1999; 274: 7565-7569Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 11Walker J.K. Premont R.T. Barak L.S. Caron M.G. Shetzline M.A. J. Biol. Chem. 1999; 274: 31515-31523Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 12Barak L.S. Ferguson S.S. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). It is broadly applicable for GPCR de-orphaning because mammalian receptors that couple to different signaling pathways (12Barak L.S. Ferguson S.S. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar) desensitize by using a common set of GRKs and arrestin proteins. Here we demonstrate that the application of βarr2-GFP translocation can be used to detect specific ligands for a very
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