Identification of Drosophila Neuropeptide Receptors by G Protein-coupled Receptors-β-Arrestin2 Interactions*

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 (1) has en-abled the identification of the entire complement of ϳ160 fly GPCRs 1 (2). Of these, 44 encode GPCRs for peptide ligands (3) of which approximately half have been functionally characterized. The number of genes encoding neuropeptides is currently estimated to be at least 32 (4,5).
Many Drosophila GPCRs have been de-orphaned through binding or signaling assays tailored to individual receptor subtypes. De-orphaning the remaining ϳ20 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 ␤-arrestins (6). In Drosophila, GPCRs may be desensitized by a similar GRK/arrestinmediated mechanism; two GRKs (7) and one non-visual arrestin (8) have been identified. We have reported previously (9 -11) the use of ␤-arrestin2-green fluorescent protein (␤arr2-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 (12).
In this report we demonstrate the utility of the ␤arr2-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).

EXPERIMENTAL PROCEDURES
Molecular Cloning-We generated full-length receptors constructs for the following genes using methods described in Johnson et al. (13): 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 5Ј 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 (13).
Transfections and Cell Culture-HEK-293 cells were transiently transfected using electroporation or LipofectAMINE using 10 g of DNA per 4 ϫ 10 6 cells. Cells were transfected with a 5:1 ratio of GPCR DNA and ␤-arrestin2-GFP (␤arr2-GFP) DNAs. The dominant negative dynamin mutant (K44A) was included in some transfections, at a ratio of 5:1:1 (GPCR:␤arr2-GFP:K44A) (14). 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 (12), or by including GRK2 in transfections at a ratio of 5:1:1 (GPCR:␤arr2-GFP:GRK2). To study calcium responses, stable lines expressing different receptors were generated and selected for resistance to hygromycin B (13). Cells were maintained in a humidified incubator under 5% CO 2 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 l (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.
Cellular Assays-Intracellular calcium was monitored as described (13). Briefly, HEK-293 cells stably expressing receptor DNA were loaded with 5 M FLUO3-AM dye (Molecular Probes, Eugene, OR) dissolved in a 20% Pluronic F-127/Me 2 SO 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 (15) 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 M concentration to test for G i activity. Luminescence was measured on a Victor Wallac 2 Plate reader (PerkinElmer Life Sciences). EC 50 values were calculated from concentration-response curves using computerized nonlinear curve fitting (PRISM 3.0, GraphPad, San Diego, CA).
[ 35 S]GTP␥S Binding Assay-HEK-293 cells were transiently transfected by electroporation using 10 g of CG13803 per 4 ϫ 10 6 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 M 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 ϫ 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 MgCl 2 , 1 mM EDTA, 1 mM dithiothreitol, 10 M GDP). Membranes (20 g of protein per assay tube) were incubated in the presence or absence of DMS and 100 pM [ 35 S]GTP␥S (1250 Ci/mmol, PerkinElmer Life Sciences) for 1 h at 30°C as described previously (16). 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 H 2 O 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 EX-CEL) were used to examine basal versus stimulated receptor activity for the measurements of CRE-luciferase activity. p values Ͻ 0.05 were considered significant.

RESULTS
The translocation of ␤arr2-GFP chimeras has been used to assess desensitization of a variety of known diverse mammalian GPCRs (9 -11). 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 (13) 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.
The receptor for neuropeptide F (CG1147) belongs to the family of NPY-like receptors and signals through G i -mediated pathways (17). Cells expressing this receptor displayed ␤arr2-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 (18,19), which is related to the vasopressin/oxytocin receptor family, displayed translocation of ␤arr2-GFP to the membrane specifically in response to 1 M corazonin (Fig. 2). Cells expressing the dFMRF-amide receptor encoded by CG2114 (20,21), which is related to the neurotensin/thyrotropin-releasing factor receptor family, displayed ␤arr2-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 (22). 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 G␣ protein subunit. 2 The C termini of these receptors have multiple clusters of serine/threonine residues that are potential targets for GRK phosphorylation (23). 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 ␤-arrestin which, at steady state, results in their trafficking to endocytic vesicles and a loss of signaling function (16,23). With these receptors, inhibiting clathrin-mediated endocytosis reverses their intracellular localization phenotype. It also re-establishes, at least in part, their signaling function (23). 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 (14). As shown in Fig. 3, now in the presence of dynamin K44A, the association of the CG13702 receptor with ␤arr2-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 ␤arr2-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 (24 -26). 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 (27), we observed translocation of ␤arr2-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-re- The numbers in each panel refer to minutes following peptide application. The variation in intensity to PDF was due to changes in imaging plane as the cell moved during the observation period. No such changes were consistently seen in response to PDF. Bottom, wide field view of multiple cells transfected with CG6986 at 0 and 20 min after proctolin application (numbers in panels refer to minutes). Asterisks indicate cells giving a positive ␤arr2-GFP translocation; the minus symbol indicates a cell that did not respond. leasing factor receptor family (3). Cells expressing CG13803 displayed translocation of ␤arr2-GFP to the membrane following exposure to the neuropeptide DMS at 1 M (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 ␤arr2-GFP by these cells in response to 1 M 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 ␤arr2-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 (3). Cells expressing CG8985 and overexpressing GRK2 also responded to 1 M DMS with ␤arr2-GFP translocation (Fig. 6) but not to 1 M DPKQDFMRFamide 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 DP-KQDFMRF-amide at both 1 M 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 ␤arr2-GFP translocation responses of those cells to a broad range of test peptides (data not shown).
To extend the observations established with the ␤arr2-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 [ 35 S]GTP␥S 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 EC 50 values for CG13803 were 0.17 nM (r 2 ϭ 0.95) for DMS and 4.2 nM (r 2 ϭ 0.95) for DPKQDFMRF-amide. For CG8985 cells, estimated EC 50 values were ϭ 1.8 nM (r 2 ϭ 0.89) for DMS and 13 nM (r 2 ϭ 0.92) for DPKQDFMRF-amide. Again DMS was significantly more potent than DPKQDFMRF-amide, and that feature recapitulated results obtained with the ␤arr2-GFP translocation.
For mammalian GPCRs, it has been shown that the pattern of ␤arr2-GFP translocation falls into two categories. Class A receptors maintain translocated ␤arr2-GFP at the plasma membrane. Class B receptors, which have C-terminal clusters of serine and threonine residues and thus higher affinity for ␤-arrestins, internalize the translocated ␤arr2-GFP into endocytic vesicles (28). This receptor internalization (14) 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.

DISCUSSION
The ␤arr2-GFP translocation assay has been used effectively and extensively to study diverse GPCRs that are sensitive to different peptides and amines (9 -12). It is broadly applicable for GPCR de-orphaning because mammalian receptors that couple to different signaling pathways (12) desensitize by using a common set of GRKs and arrestin proteins. Here we demonstrate that the application of ␤arr2-GFP translocation can be used to detect specific ligands for a very wide variety of Drosophila peptide GPCRs. We found that each of 11 different peptide GPCRs, representing six distinct families of Drosophila peptide GPCRs, could be analyzed by this method. The 11 receptors include members of the vasopressin, somatostatin, tachykinin, bombesin, thyrotropin-releasing factor, and neuropeptide Y receptor ancestral groups (3). By using this approach, we have identified Ast-B as a potential ligand for CG14484 and DMS as a potential ligand for CG13803 and CG8985, and we demonstrated responsiveness of two purported tachykinin receptors (CG7887 and CG6515) to Drosophila tachykinin peptides. Furthermore, we have confirmed the identity of native peptide ligands for six recently characterized Drosophila receptors. Additionally, many of the distinct features previously observed for mammalian GPCRs, including GRK sensitivity, differential ␤-arrestin affinities, and consti-tutive association with ␤-arrestin, were found in the case of functionally expressed Drosophila receptors.
The ␤arr2-GFP translocation response following exposure to cognate ligands is known to be dependent on the high level of receptor expression that is typically found in transfected cell lines (29). The translocation assay produced robust responses in the case of the Drosophila proctolin receptor CG6986. We found that the majority of GFP-expressing cells responded to desensitizing doses of ligand and displayed clear GFP membrane association within minutes. Thus, at least in the case of this Drosophila receptor, the strength of the response was not limited by transfection efficiency or by levels of receptor expression in a heterologous system. Furthermore, the specificity of the proctolin response by CG6986 was supported by the absence of any response to 16 other peptides tested. Although we did not quantify responses for each of the other 10 receptors tested to the same extent, the results were similar in most cases in that positive responses (when observed) were displayed by a majority of GFP-expressing cells (Fig. 1). In this regard, the ␤arr2-GFP translocation assay can be a robust measure of interactions by orphan Drosophila receptors with their cognate ligands.
We used the ␤arr-2-GFP translocation assay to de-orphan the CG13803 and CG8985 GPCRs as receptors for the neuropeptide DMS. Although these results were specific and clear, unequivocal receptor characterization still requires multiple independent assays. We therefore turned to GTP␥S incorporation and cAMP level measurements. Those experiments strongly support the hypothesis that CG13803 and CG8985 are bona fide DMS receptors. Likewise, the ␤arr2-GFP translocation assay can be used to re-evaluate a putative receptor (as we have analyzed the proctolin receptor CG6986 and five other "identified" receptors) and to supply independent confirmation of receptor identity. Thus, we note that for each of 10 receptors tested here (all except CG14484), we have now provided additional and independent lines of evidence to argue for their specific identities.
CG13803 and CG8985 displayed robust responses to both DMS and dFMRF-amide peptides. We favor the hypothesis that the CG13803 and CG8985 receptors are more sensitive to DMS for three reasons. First, cells expressing CG13803 were ϳ10-fold more sensitive to DMS over DPKQDFMRF-amide in decreasing levels of cAMP. Second, cells expressing CG13803 responded to DMS as indicated by the translocation of ␤arr-2 but did not respond to DPKQDFMRF-amide, unless co-transfected with GRK2. This suggests that whereas both peptides are effective agonists, DPKQDFMRF-amide either has much less affinity or it places the signaling receptor in a different conformation. Finally, CG8985 never responded to DPKQD-FMRF-amide in the translocation assay, and it gave a smaller maximal response in the cAMP assay to that peptide. In contrast, CG8985 responded strongly to DMS in the cAMP assay and, with GRK2 co-expression, responded robustly in the translocation assay. We note that DMS displays inhibitory physiological activity in vivo (e.g. Ref. 30), and this is consistent with the finding that DMS activation of CG13803 and CG8985 causes a decrease in cyclic nucleotide levels, presumably by coupling via G i/o . Additional second messenger studies and binding studies will be useful to refine further the profile of receptor sensitivity to these related peptides.
Many neuropeptides are co-synthesized with structurally related peptides as parts of larger precursors. For example, the dFMRF-amide precursor contains at least six structurally related, and biologically active, FMRF-amide-like peptides (31). Many of these peptides signal through the CG2114 receptor at very low doses (20,21). We found that one of six co-synthesized FIG. 6. Co-expression of GRK2 helps define the response profiles of the paralogous receptors CG8985/CG13803. CG8985 only displays ␤arr2-GFP in response to DMS with additional GRK2 (top). Its paralogue CG13803 does not require additional GRK2 to respond to DMS (middle) but does require additional GRK2 to exhibit translocation in response to the related peptide dFMRF-amide (bottom).
DTK peptides can trigger ␤arr2-GFP translocation in cells expressing either CG6515 or CG7887; those observations support the hypothesis that these receptors represent DTK receptors. The question remains whether DTK-1 or the other five DTKs, or all, are endogenous ligands for these receptors in vivo. When used in a qualitative "all-or-none" fashion as we have done in this work with saturating ligand concentrations, ␤arr2-GFP translocation is of limited value to evaluate systematically the rank order potencies of "related" ligands, such as the case of the six related DTK peptides. We note that the assay can also be used quantitatively (with available confocal microscopy platforms) to establish potency orders as effectively as other signaling or receptor binding assays (29,32). Quantitative assays should now be employed to rank the potential interactions of multiple and related DTK peptides with the CG6515 and CG7887 receptors.
We found that both DPKQDFMRF-amide and DMS triggered a ␤arr2-GFP translocation in cells expressing CG2114. Two groups have reported recently (21,22) that the GPCR encoded by CG2114 is a receptor for several of the peptides derived from the dFMRF-amide precursor CG2346. DMS (TD-VDHVFLRFa) is an FMRF-amide-related peptide that is derived from a separate neuropeptide gene precursor CG6440 (33). In the previous studies, the CG2114 receptor responded to DMS but was more sensitive to dFMRF-amide peptides (like DPKQDFMRF-amide) by ϳ2 orders of magnitude. The pharmacological overlap between dFMRF-amide and DMS neuropeptides was also evident in our observations of cells expressing the paralogous receptors encoded by CG8985 and CG13803. All three receptors (CG2114, CG13803, and CG8985) belong to the same neurotensin/thyrotropin-releasing factor receptor family (3). At present, there are no indications that the Drosophila genome contains sequences that encode peptides related to authentic neurotensin or thyrotropin-releasing factor (or related peptides). Thus, akin to the situation with other Drosophila peptides GPCRs (13, 19, 22, 34 -36), the evolutionary origins of the peptide receptors are presently better described than that of their cognate ligands. During the review process for this paper, an independent group published data (37) using a promiscuous G␣ protein to indicate that the receptors encoded by CG13803 and CG8985 respond specifically to DMS.
We also found that cells expressing the orphan receptor CG14484 displayed a specific and robust ␤arr-2 translocation response to the Ast-B neuropeptide. The CG14484 receptor is related to the bombesin family of peptide GPCRs. There are three families of allatostatic neuropeptides so far identified in different insect groups (5); these factors can inhibit the production of the sesquiterpinoid juvenile hormone. Ast-B peptides are structurally related to mammalian galanin peptides (W(X 6 )W-amide), and to date they have not been associated with any specific physiological response in Drosophila. The identification of a candidate receptor for the Ast-B peptide(s) will likely facilitate the definition of their functional roles. Drosophila contains a paralogous receptor CG14593, which we have not yet tested in these assays.
Recent studies of arrestin-mediated GPCR regulation have identified agonists that fail to recruit ␤-arrestin efficiently. The primary example of this is morphine binding at the -opioid receptor (38 -40). Such ligands signal well but are very poor at inducing phosphorylation of the activated receptor. In order to study such receptors in the ␤arr2-GFP translocation assay, it has proven useful to overexpress a GRK to enhance receptor phosphorylation, thereby facilitating arrestin recruitment (12,38). In the case of the Drosophila receptors we have studied, we observed that most did not require additional GRK activity to display specific responses. However, that same strategy did prove useful to help de-orphan CG13803 and CG8985. Specifically, GRK overexpression revealed sensitivity in the case of CG13803 cells to a 100 nM dose of DPKQDFMRF-amide and sensitivity in the case of the paralogous receptor CG8985 to 1 M DMS. The apparent requirement for additional GRK activity may reflect the fact that our studies took place within a heterologous expression system, mammalian HEK-293 cells. However, mammalian GPCRs also display differing responsiveness to distinct GRK subtypes. These differences are reduced by GRK overexpression that guarantees through mass action that even receptors that are poor GRK substrates become maximally phosphorylated. We presume that these particular Drosophila receptors are poor substrates for mammalian GRKs. The general issue of GPCR responsiveness in a heterologous environment will be best resolved by re-evaluating these properties in insect cells. Together these data suggest the utility of continued use of GRK overexpression when a sluggish or absent ␤arr2-GFP response is observed following ligand addition.
Mammalian GPCRs usually internalize in response to agonist activation. However, certain GPCRs, such as constitutively active and "loss of function" mutant receptors, are efficient GRK substrates even in the absence of agonist activation (16,23). This property leads to the constitutive trafficking of these receptors to endocytic vesicles and thus effectively removes them from the plasma membrane and inhibits their signaling function. Trapping them at the plasma membrane using internalization inhibitors such as dynamin K44A permits the visualization of these arrestin-receptor complexes (16,23). We found that overexpression of dynamin K44A did not alter the normal ␤arr-2 translocation response profile of the proctolin receptor CG6986. However, this refinement was useful for study of Drosophila GPCRs CG7285 and CG13702 to establish robust responses to their cognate ligand, the Ast-C peptide. Therefore, similar to the use of GRK2 overexpression, our results clearly suggest the use of dynamin K44A overexpression may be a useful adjunct when applying the ␤arr-2 translocation assay to de-orphan novel receptors.
Two classes of mammalian GPCRs have been observed that differ in arrestin association during endocytosis (28). Class A receptors (e.g. ␤ 2 -adrenergic receptors) translocate arrestins to the plasma membrane but dissociate from them in an early phase of endocytosis. Class B receptors (e.g. angiotensin 1A receptors) translocate and remain associated with arrestins throughout their internalization. For the Drosophila receptors examined here in mammalian cells, CG6986, CG2114, CG7285, CG13702, CG6515, CG14484, CG13803, and CG8985 behaved as class A receptors, whereas CG1147, CG7887, and CG10698 behaved as class B receptors. With mammalian GPCRs, these different internalization patterns contribute to the kinetics of resensitization and the ability to signal through the mitogenactivated protein kinase signaling pathway (28,41). The significance of these internalization patterns will have to be reevaluated for each of the Drosophila receptors in a native environment.
In summary, by using two independent assays (Fig. 7), we have confirmed the DMS sensitivity of CG13803 and CG8985 that was indicated by the ␤arr2-GFP translocation assay. Those findings, together with the de-orphaning of the CG14484, CG7887, and CG6515 receptors, and our positive results studying the other six (previously identified) receptors, support the conclusion that the ␤arr2-GFP translocation assay is an effective method with which to de-orphan Drosophila peptide GPCRs. Because GPCR/␤-arrestin interactions are independent of the specific signaling pathways engaged, this approach should represent a universal tool for the identification of orphan GPCRs and their ligands.