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Proc Natl Acad Sci U S A. Oct 29, 2002; 99(22): 14554–14559.
Published online Oct 21, 2002. doi:  10.1073/pnas.202498299
PMCID: PMC137921
Pharmacology

A Drosophila dopamine 2-like receptor: Molecular characterization and identification of multiple alternatively spliced variants

Abstract

Dopamine is an important neurotransmitter in the central nervous system of both Drosophila and mammals. Despite the evolutionary distance, functional parallels exist between the fly and mammalian dopaminergic systems, with both playing roles in modulating locomotor activity, sexual function, and the response to drugs of abuse. In mammals, dopamine exerts its effects through either dopamine 1-like (D1-like) or D2-like G protein-coupled receptors. Although pharmacologic data suggest the presence of both receptor subtypes in insects, only cDNAs encoding D1-like proteins have been isolated previously. Here we report the cloning and characterization of a newly discovered Drosophila dopamine receptor. Sequence analysis reveals that this putative protein shares highest homology with known mammalian dopamine 2-like receptors. Eight isoforms of the Drosophila D2-like receptor (DD2R) transcript have been identified, each the result of alternative splicing. The encoded heptahelical receptors range in size from 461 to 606 aa, with variability in the length and sequence of the third intracellular loop. Pharmacologic assessment of three DD2R isoforms, DD2R-606, DD2R-506, and DD2R-461, revealed that among the endogenous biogenic amines, dopamine is most potent at each receptor. As established for mammalian D2-like receptors, stimulation of the Drosophila homologs with dopamine triggers pertussis toxin-sensitive Gi/o-mediated signaling. The D2-like receptor agonist, bromocriptine, has nanomolar potency at DD2R-606, -506, and -461, whereas multiple D2-like receptor antagonists (as established with mammalian receptors) have markedly reduced if any affinity when assessed at the fly receptor isoforms. The isolation of cDNAs encoding Drosophila D2-like receptors extends the range of apparent parallels between the dopaminergic system in flies and mammals. Pharmacologic and genetic manipulation of the DD2Rs will provide the opportunity to better define the physiologic role of these proteins in vivo and further explore the utility of invertebrates as a model system for understanding dopaminergic function in higher organisms.

Dopamine is an essential catecholamine in the central nervous system of both mammals (1) and Drosophila (2). In mammals, dopamine modulates neurologic, cardiovascular, endocrine, and renal functions. In addition, this neurotransmitter regulates motor activity, sexual behavior, and the response to drugs of abuse (1, 3–6). In Drosophila, known dopamine-mediated functions overlap with those of mammals. Experimental evidence suggests that in flies dopamine modulates locomotor activity, sexual function and the response to cocaine, nicotine, and alcohol (7–11).

Dopaminergic signaling is mediated through seven transmembrane domain (TM) G protein-coupled receptors that fall within the class A or rhodopsin family based on amino acid homology and conservation of amino acid signature motifs. The dopamine receptor family is divided into two major subfamilies: the D1-like receptors (D1 and D5) and D2-like receptors (D2, D3, and D4). D1-like and D2-like receptor cDNAs have been isolated from many species. A subset of these proteins has been expressed in heterologous cell lines and characterized pharmacologically (1, 5, 12, 13).

Dopamine receptors have been classified as D1-like or D2-like based on amino acid sequence homology, signal transduction pathways, and sensitivity to class-specific agonist and antagonist drugs. D1-like receptors are coupled to stimulatory G proteins (Gαs) thereby triggering activation of adenylate cyclase. D2-like receptors transduce second-messenger signaling via inhibitory G proteins (Gαi/o), which lead to inhibition of adenylate cyclase and modulation of ion channels (1, 5, 12, 13).

Due to the physiologic significance of D1-like and D2-like receptors, there has been considerable interest in developing subtype-selective dopamine agonists and antagonists. These functionally diverse drugs include both established therapeutics as well as promising candidates for the treatment of neuropsychiatric illness, migraine, and cardiovascular disease (1, 13). D2 receptor agonists (e.g., bromocriptine) have proven efficacious in Parkinson's disease, whereas antagonists at this receptor (e.g., haloperidol and chlorpromazine) are used commonly in the treatment of psychiatric illness and hold promise as a treatment for acute migraine.

Selected dopaminergic drugs have also been shown to have effects in insects. Physiologic studies in cockroach, larval tobacco hornworm, and Drosophila indicate that neuronal signaling, hormone biosynthesis, and motor function are modulated by dopaminergic signaling (7, 14, 15). The subtype-selective ligands used in these studies suggest the presence of both D1-like and D2-like receptors in invertebrates. In vitro binding experiments with isolated membranes and autoradiographic analysis of honey-bee brain provide additional evidence that both D1- and D2-like receptors are expressed in insects (16, 17). Studies with selective radioligands revealed localization of respective receptor subtypes in distinct brain regions. Despite the pharmacological evidence suggesting the presence of both D1- and D2-like receptors in insects, to date only D1-like receptors have been cloned and characterized. Two Drosophila (DmDop1 and DopR99B) and one honey-bee (AmDop1) D1-like receptor cDNAs have been isolated (18–22).

The present study describes the cloning and characterization of a newly discovered Drosophila dopamine receptor. As a result of alternative splicing, at least eight variants of this receptor transcript are found in fruit flies. We have expressed and pharmacologically assessed three of these receptor isoforms: Drosophila D2-like receptor (DD2R)-606, DD2R-506, and DD2R-461. The amino acid sequence, G protein coupling, and pharmacologic profile support the classification of these proteins as insect D2-like receptors.

Materials and Methods

Materials.

The receptor ligands used in this study were purchased from Sigma; a complete listing of chemical names is included in Supporting Materials and Methods, which is published as supporting information on the PNAS web site, www.pnas.org. Receptor-mediated signaling was assessed using luciferase reporter gene constructs (23–25) comprised of either (i) five tandem repeats of the serum response element (SRE5x) ligated upstream from a reporter gene encoding firefly luciferase, or (ii) a multimerized cAMP response element (CRE6x) ligated upstream from the reporter gene.

Cloning of cDNAs Encoding Full-Length DD2Rs.

A highly conserved region of the human dopamine 2 receptor, amino acids 350–399 (GenBank accession no. P14416) was used to identify homologous sequence (GenBank accession no. AE002611) within the Drosophila genome (26) using a tblastn BLAST search (www.ncbi.nlm.nih.gov). Using PCR, overlapping DD2R cDNA fragments were generated (detailed in Supporting Materials and Methods) and ligated into the expression vector pcDNA1.1. The protein-coding region of each DD2R cDNA was sequenced using an ABI 37X DNA sequencer.

Sequence Analysis.

Amino acid alignments of the Drosophila receptor with homologous proteins were performed using ALIGN X with the blosum62mt2 scoring matrix (Vector NTI Suite, version 5.5, InforMax, North Bethesda, MD).

Identification of Multiple Alternatively Spliced DD2R Transcripts.

The region of the DD2R transcript encoding TM V, intracellular loop 3, TM VI, and TM VII was PCR-amplified from cDNA template using three independent sets of primers S3/A3, S5/A5, and S6/A6 (sequences are shown in Table 2, which is published as supporting information on the PNAS web site). PCR products were isolated, subcloned, and sequenced.

Assessment of DD2R Expression.

Total RNA was extracted from Drosophila Canton S larva, pupa, adult head, and adult body using the RNAqueous-4PCR kit (Ambion, Austin, TX). The presence of the DD2R transcript in each RNA sample was assessed by PCR using a 5′-directed pair of oligonucleotides (S4/A4) designed to amplify an invariant (i.e., outside the segment that is subject to alternative splicing) 565-bp region of the cDNA. Primer sequences and PCR conditions are detailed in Supporting Materials and Methods.

Quantitative assessment of the DD2R transcript used poly(A)-selected RNA isolated with the PolyATtract mRNA-isolation system III (Promega). First-strand cDNA was synthesized from equal amounts of larva, pupa, adult head, or adult body poly(A) RNA using the TaqMan reverse transcription kit (Applied Biosystems). Quantitative PCR was performed using the TaqMan PCR core reagent kit (Applied Biosystems). Two DD2R-specific probes (CT17758 corresponding to the 5′ end of the cDNA, CT37739 to the 3′ end), each double-labeled with the fluorescent dye 6-carboxyfluorescein and the quencher dye 6-carboxy-N,N,N′,N′-tetramethylrhodamine, were obtained from Applied Biosystems. For each labeled probe, two flanking PCR primers were selected to amplify a region of the DD2R cDNA that includes an intron/exon junction, thus minimizing the likelihood of generating a signal from residual genomic DNA (primer sequences are specified in supporting information, Table 2). The 5′ DD2R primers (TS1/TA1 and CT17758) target a 176-bp region of the DD2R cDNA, which includes portions of exons 1 and 2. The 3′ DD2R primers (TS2/TA2 and CT37739) amplify a 181-bp fragment corresponding to a segment of exons 8 and 9. Both sets of DD2R PCR primers (5′ and 3′) localize to invariant regions of the DD2R cDNA. 6-Carboxyfluorescein-labeled probes for the quantitative amplification of DmDop1 (CT27288) and actin 5C (CT13368) transcripts were obtained from Applied Biosystems. The sequences of the corresponding flanking PCR primers are provided in supporting information, Table 2. Quantitative PCR was performed using the following incubation conditions: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles: 95°C for 15 sec/60°C for either 15 sec (DD2R and actin 5C) or 20 sec (DmDop1). Amplification was performed using the Prism 7700 sequence-detection system (Applied Biosystems). Data recording and analysis were done with SEQUENCE DETECTOR software (version 1.7a).

Luciferase Assay.

Human embryonic kidney (HEK)293 cells were plated (5,000–7,000 per well) onto 96-well Primaria plates (BD Biosciences, Bedford, MA). Cells were transiently transfected with cDNAs encoding DD2R-606, DD2R-506, or DD2R-461 together with the appropriate luciferase reporter gene and/or Gq5i using Lipofectamine reagent (Invitrogen). Twenty-four hours posttransfection, cells were exposed to ligand for 3 h in serum-free medium. Cells then were lysed, and luciferase activity was quantified using LucLite reagents (Packard). Synthetic agonists were assessed at 1 μM concentration. Ligand potencies were determined by stimulating transfected cells with increasing concentrations of agonists. EC50 values were calculated from concentration response curves using computerized nonlinear curve fitting PRISM 3.0, GraphPad, San Diego).

Statistical Analysis.

Two-tailed single sample t tests were used to examine basal vs. stimulated receptor activity. When multiple conditions were compared for a given receptor, analysis was performed using one-way ANOVA. Post tests were done by Tukey–Kramer multiple comparisons analysis (INSTAT, GraphPad).

Results

Identification of Genomic Sequence Encoding a Putative Drosophila Dopamine Receptor.

Database mining with a limited TM VI region of the human dopamine 2 receptor (hD2R) revealed a high degree of amino acid homology with the translated Drosophila genomic fragment, GenBank accession no. AE002611, which includes both sequences, CG17004 and CG9569. CG17004 encodes a protein homologous with hD2R TMs III–VII, whereas the adjacent sequence (CG9569) encodes the TM I/II domain.

Cloning of Multiple Drosophila D2-Like Receptor Isoforms.

Primers (see supporting information, Table 2; and Fig. 5, which is published as supporting information on the PNAS web site) were designed to amplify the ORF as well as demonstrate that the putative receptor transcript was formed as a product of coding sequence from two adjacent annotated genes, CG17004 and CG9569. Overlapping clones were joined using a unique ClaI site, which is found within sequence encoding TM III. The full-length DD2R cDNA (Fig. (Fig.1)1) was subcloned into the expression vector pcDNA1.1.

Fig 1.
Alternative splicing of the DD2R gene generates multiple receptor variants. (A) The protein-coding exons are depicted as boxes (E1–E9). The size of each intron (number of bases) is shown in parentheses. The portion of the receptor encoded by each ...

Expression of the DD2R Transcript.

PCR analysis suggests that the DD2R transcript is expressed during development (in larva and pupa) as well as in the adult fly (Fig. (Fig.22A). To assess the relative abundance of the transcripts, quantitative PCR was carried out with two independent sets of primers (TS1/TA1 and TS2/TA2), which were used in combination with respective TaqMan 6-carboxyfluorescein-labeled oligonucleotides (Applied Biosystems). Analysis with each set of primers confirmed that the DD2R transcript is most abundant in mRNA isolated from Drosophila adult head. The data generated using the TS1/TA1 primer pair are shown in Fig. Fig.22B. The expression profile obtained for the DD2R transcript parallels that of another Drosophila dopamine receptor (the D1-like receptor, DmDop1) as assessed by quantitative PCR (Fig. (Fig.22C). Of note, actin 5C transcripts were present at comparable levels in each of the mRNA samples which were analyzed (Fig. (Fig.22D).

Fig 2.
The DD2R transcript is expressed at multiple developmental stages. Reverse transcription (RT) using random hexamers was performed to generate first-strand cDNA (+) from either total RNA (A) or poly(A) mRNA (BD). In addition, negative ...

Identification of Additional Alternatively Spliced Transcripts.

Using primer sets (S3/A3, S5/A5, and S6/A6; see supporting information, Fig. 5) that span the third intracellular loop, a series of DD2R splice variants was PCR-amplified from adult head cDNA (Fig. (Fig.1).1). The size of the PCR products obtained was highly sensitive to the salt conditions during amplification. Each of eight DD2R variants (i.e., DD2R-461, DD2R-506, DD2R-581, DD2R-589, DD2R-597, DD2R-598, DD2R-605, and DD2R-606) (i) was obtained using at least two different pairs of oligonucleotide primers, (ii) encoded an isoform with a distinct third intracellular loop size, and (iii) arose from alternatively spliced exons in the DD2R gene.

DD2R Gene Structure.

The exon/intron junctions of the DD2R gene (Fig. (Fig.1)1) were determined by comparison of the cDNAs and the genomic sequence obtained from the Drosophila genome project (26). Comparison of each alternatively spliced transcript with corresponding genomic sequence revealed the presence of consensus GT and AG dinucleotides at the termini of each putative intron (ref. 27; shown in supporting information, Fig. 5).

Sequence Analysis of the DD2Rs.

Sequence comparison of DD2R with the GenBank database revealed that these proteins have the highest degree of homology with other known D2-like receptors. As with other receptors in this subfamily, the DD2Rs are class A G protein-coupled receptors with corresponding amino acid signature motifs in the first (GN), third (DRY), sixth (CWLP), and seventh (NPXXY) TMs (28). The classification of the DD2Rs as biogenic amine receptors is supported also by the presence of a number of conserved amino acid residues that are established affinity and/or efficacy determinants for endogenous amines (29, 30). These residues include D156 in TM III, serine residues S237, S238, and S241 in TM V, and aromatic residues F242 in TM V, as well as W543, F546, and F547 in TM VI (numbers corresponding to position in DD2R-606; Fig. Fig.11).

Sequence comparison of the Drosophila receptor with known D1-like and D2-like receptors suggests that DD2R falls within the D2 subfamily. Paired alignments (not shown) with D2-like receptors (D2, D3, and D4) revealed amino acid identity ranging from 29% to 32%. In contrast, when compared with D1-like receptors (D1 and D5), identity ranged from 17 to 21%. The DD2Rs share additional structural features with D2-like vs. D1-like receptors including a relatively long third intracellular loop and a short carboxyl terminus.

Dopamine Stimulation of the DD2Rs Results in Gαi/o-Mediated Signaling.

It has generally been found that Drosophila receptor signaling observed in mammalian cells or Xenopus oocytes reflects results in a native Drosophila microenvironment (e.g., Schneider cells; refs. 21 and 31). Functional assessment of the Drosophila D2-like receptors in HEK293 cells revealed that, similar to the corresponding mammalian homologs, these proteins are coupled to inhibitory G proteins. DD2R-606, -506, and -461 were transiently expressed in HEK293 cells together with the reporter construct, CRE6x-luciferase, which is a sensitive indicator of intracellular cAMP levels (23, 24). Forskolin stimulation of these cells results in an increase in cAMP, reflected by an elevation in luciferase activity. The addition of dopamine led to a marked decrease in forskolin-induced cAMP levels, reflected by a concomitant reduction in luciferase activity (Fig. (Fig.3).3). The dopamine-induced effect can be inhibited by pretreatment of cells with pertussis toxin, an established selective inhibitor of Gi/o-mediated signaling (32).

Fig 3.
DD2R-mediated inhibition of adenylate cyclase is pertussis toxin (PTX)-sensitive. HEK293 cells were cotransfected with receptor cDNA encoding either a Drosophila or human dopamine receptor and a CRE6x-luciferase reporter gene construct. Cells were preincubated ...

A complementary approach that was used to confirm Gi/o-linked DD2R signaling relied on coexpression of the chimeric G protein, Gq5i (33), and the SRE5x-luciferase reporter gene construct. The 5 aa at the carboxyl-terminus of Gq5i are sufficient to enable interaction with Gi-coupled receptors (34, 35). Other domains of Gq5i direct receptor-mediated signaling to the Gq pathway, which in turn can be detected using the SRE5x-luciferase reporter gene construct. Ligand stimulation of a Gi-coupled receptor thus is expected to trigger the following cascade: Gq5i → phospholipase C → → protein kinase C → → SRE → luciferase transcript → luciferase activity → light emission. The significant elevation in light emission observed with this assay after dopamine stimulation of the DD2R (Fig. (Fig.4)4) further confirms Gi/o-mediated second-messenger signaling.

Fig 4.
Comparison of dopamine and synthetic agonists at the DD2R. HEK293 cells were cotransfected with receptor [DD2R or human D2-long receptor (hD2L)] cDNA in addition to a SRE5x-luciferase reporter construct and a plasmid encoding the chimeric ...

Dopamine Is the Highest Potency DD2R Endogenous Ligand.

It is not unusual for biogenic amine receptors (e.g., the Drosophila tyramine, octopamine, and dopamine-1 receptors) to recognize and signal in response to amines that share structural similarity with the principal endogenous agonist (18–21, 31, 36). In the current study, DD2R-606, -506, and -461 were therefore assessed with multiple aminergic ligands. At the concentration of 10 μM, dopamine, norepinephrine, epinephrine, serotonin, and tyramine significantly stimulated each of the DD2R variants, whereas histamine and octopamine failed to result in detectable receptor activation (not shown). Among the active biogenic amines, the potency for dopamine was highest at each of the DD2R isoforms (Table (Table1;1; and Table 3 and Fig. 6, which are published as supporting information on the PNAS web site). Although many authors suggest that flies lack endogenous norepinephrine or epinephrine, it is of interest that these ligands still possess full agonist activity at the DD2Rs. Given the relatively high potency of the DD2R for serotonin, norepinephrine, epinephrine, and tyramine, it cannot be excluded that the DD2R may be a target for other biogenic amines in addition to dopamine.

Table 1.
Dopamine is the preferred endogenous agonist for the DD2Rs

D2-Like Receptor Synthetic Agonists Stimulate Second-Messenger Signaling by DD2Rs.

The Drosophila receptors were assessed further with a series of D2-like receptor agonists. Selectivity of the ligands was established previously using mammalian receptors. The high-affinity D2-like receptor agonist, bromocriptine (1, 5, 13), stimulated each of the Drosophila receptors with full efficacy (vs. dopamine) and nanomolar potency (Fig. (Fig.4).4). In addition, the D2-like receptor agonist R(−)-propylnorapomophine hydrochloride (NPA; ref. 5), as well as several less D2-selective agonists including (±)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (6,7-ADTN), apomorphine, and SKF82958 (5, 37, 38), each triggered significant levels of ligand-induced signaling (vs. basal). Other synthetic compounds, including the D2-like receptor agonists quinpirole and 7-OH-(±)-2-dipropylamino-7-hydroxy1,2,3,4-tetrahydronaphthalene hydrobromide (7-OH-DPAT; ref. 5), failed to significantly stimulate DD2R-mediated signaling.

Antagonist Inhibition of Dopamine-Mediated Signaling.

A series of known dopamine receptor antagonists (raclopride, eticlopride, spiperone, haloperidol, butaclamol, and flupenthixol) was assessed at DD2R-606, -506, and -461 as inhibitors of 1 μM dopamine-induced signaling. In contrast to the marked effects of the tested antagonists at the human D2R, only butaclamol (10 μM) and flupenthixol (1 μM) showed detectable, albeit weak, activity at the fly DD2Rs (see Fig. 7, which is published as supporting information on the PNAS web site).

Discussion

In Drosophila, dopamine mediates a spectrum of physiological functions including locomotor activity (7), response to drugs of abuse (10, 11), fertility (39, 40), sexual behavior (8), learning, and memory (41, 42). Using drugs with established subtype selectivity in vertebrates, prior studies of dopamine-mediated function in Drosophila suggested that both D1-like and D2-like receptors are expressed. However, despite the pharmacologic evidence for receptor heterogeneity (7, 14–16), all previously characterized insect dopamine receptor cDNAs including two Drosophila homologs fall into the D1-like subclass.

In this study, we have cloned and functionally analyzed splice variants of a newly discovered dopamine receptor. We postulate that the characterized isoforms of the Drosophila protein are classified most appropriately as insect D2-like receptors. This conclusion is supported by the following findings: (i) the DD2R amino acid sequences share highest homology with mammalian D2-like receptors, (ii) the Drosophila receptors, similar to the corresponding mammalian D2-like homologs, are coupled to Gi/o, (iii) when evaluated with a series of endogenous biogenic amines, dopamine is the most potent agonist, and (iv) the DD2Rs can be activated fully by nanomolar concentrations of the synthetic D2-like receptor drug, bromocriptine.

Since the initial classification of dopamine receptors, effects on adenylate cyclase were considered one of the key pharmacological features to distinguish D1-like from D2-like proteins (43). Studies on isolated rat pituitary homogenates demonstrated that activation of mammalian D2 receptors leads to inhibition of adenylate cyclase activity via coupling to Gi/o (44). This was supported further by studies on the corresponding recombinant proteins (45–47). Signaling through Gi/o is a hallmark feature common to all pharmacologically characterized species homologs of D2-like receptors (1). In contrast, D1-like proteins signal primarily through Gs-linked pathways. The mammalian D1-like (D1 and D5) as well as both previously cloned Drosophila dopamine receptors, DmDop1 and Dop99B, signal via Gs-mediated activation of adenylate cyclase (1, 18–21). This contrasts with Gi/o-mediated signaling by the DD2Rs, which strongly supports classification of the latter proteins as D2-like.

After defining the signal transduction pathway linked to the DD2Rs, we established that among the endogenous biogenic amines, dopamine was the most potent agonist. Further characterization of the Drosophila receptors used a wide spectrum of dopamine receptor synthetic agonist and antagonist drugs. In contrast to the highly conserved amino acids on G protein-coupled receptors, which act as affinity or efficacy determinants for a given endogenous ligand (e.g., dopamine), there is no evolutionary pressure to conserve binding sites for synthetic molecules. In light of this, when one examines compounds that have been selected based on their high affinity for a given subclass of mammalian receptor, it is not surprising that many of these ligands (e.g., quinpirole, raclopride, and eticlopride) do not bind and/or activate the Drosophila homologs.

Despite this consideration, DD2R-606, -506, and -461 each show features that are consistent with classification as a D2-like receptor. All three splice variants are fully activated, with nanomolar potency, by the D2-like receptor agonist bromocriptine (Fig. (Fig.4).4). In addition, several other agonists that induce mammalian D2-like receptor signaling including R(−)-propylnorapomophine hydrochloride (NPA; ref. 5), (±)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (6,7-ADTN), apomorphine, and SKF82958 also activate the DD2R isoforms (Fig. (Fig.4).4). Furthermore, the D1/D2 mixed antagonists (+)- butaclamol and flupenthixol (1, 13, 48) each inhibit agonist-induced DD2R signaling (see supporting information, Fig. 7).

In both Drosophila and mammals, multiple isoforms of the dopamine 2 receptor occur due to alternative splicing. In humans, a short, a long, and an extra long form of the D2 receptor have been identified and characterized (1, 12, 49). The long mammalian isoform includes an additional protein coding exon that extends the size of the third intracellular loop by 29 aa (1, 12). The extra-long form results from an alternative splice site leading to the inclusion of an additional 2 aa in the third intracellular loop (49). Despite the variability in the third intracellular loop, the mammalian receptors share similar pharmacologic profiles. In flies, alternative splicing results in the generation of an even more structurally diverse subfamily of D2-like receptors. Eight DD2R variants are described in this report. This variability among fly isoforms arises from alternative splicing of protein-coding exons 6 and 7. Similar to the corresponding mammalian homologs, the fly receptors differ in the size and composition of the third intracellular loop. Extended pharmacological characterization of the representative DD2R variants (DD2R-606, -506, and -461) revealed similar pharmacologic features.

It is well established that alternative splicing of G protein-coupled receptors may selectively affect the patterns of tissue- or cell-specific expression without altering receptor-mediated signaling. With the mammalian dopamine 2 receptors, the short isoform is expressed in presynaptic dopaminergic cell bodies and functions as an autoreceptor, thus controlling release of dopamine. In contrast, the mammalian D2 long form is localized to postsynaptic sites, where it mediates locomotor as well as other physiologic functions (50, 51). Whether Drosophila D2-like receptor splice variants will recapitulate the corresponding pre/postsynaptic localization with associated functions or will be distributed in a different tissue-specific pattern remains to be investigated.

In earlier studies, several behaviors were attributed to signaling through a previously unidentified Drosophila dopamine D2 receptor. Utilizing decapitated Drosophila preparations, it was shown that direct administration of quinpirole induced hind-leg grooming and an increase in locomotor activity (7, 52). Parallel administration of the “D2-like antagonists” eticlopride and raclopride resulted in loss of righting reflex and unresponsiveness to mechanical provocation. In contrast to the observed in vivo effects, quinpirole was inactive in cell-based assays examining DD2R pharmacology. In addition, both eticlopride and raclopride had little if any capacity in vitro to block DD2R-606-, DD2R-506-, and DD2R-461-mediated signaling. It is of note that higher doses of drug were used for in vivo (5–10 mM) vs. in vitro (10 μM) testing. The maximum concentrations of compounds tested in cell-based assays were limited by HEK293 cell toxicity. Therefore, it cannot be excluded that the doses of drug administered during in vitro testing were insufficient to detect activity. Alternatively, the observed in vivo effects may have resulted from high concentrations of administered ligand acting at another receptor.

The exceptional genetic tools available to study Drosophila make it an attractive model system in which to further examine dopamine receptor-mediated function. With the molecular and pharmacologic tools now available to identify additional DD2R subtype-selective ligands, it will be possible to further refine future in vivo studies to more specifically target receptor-mediated physiology or behavior with high-potency/affinity drugs. With this information will hopefully emerge a more complete view of the role of Drosophila dopamine receptors in vivo. Once a behavior has been clearly linked to DD2Rs, genetic modifiers of receptor-mediated function can potentially be identified and characterized. The predominant expression of the DD2R gene in the adult Drosophila head suggests that this receptor likely is involved in functions mediated by the central nervous system, whereas its role in the periphery remains more speculative and awaits clearer definition of tissue-specific expression. The DD2R transcript is also found in pupa and larva, suggesting an additional role for this protein in the developing organism.

As established for the mammalian dopaminergic system, it is now apparent that both D1-like and D2-like receptors are expressed in Drosophila. It will be of great interest to determine whether the physiology linked to the DD2Rs parallels known dopaminergic functions (e.g., motor control, sexual behavior, and response to drugs of abuse) in mammals. With additional study, it should become evident to what extent flies can be used to enhance the understanding of the molecular mechanisms underlying dopamine receptor-mediated function, and pathophysiology in higher organisms.

Supplementary Material

Supporting Information:

Acknowledgments

Drosophila RNA was generously provided by Dr. F. Rob Jackson and Bikem Akten (Tufts University, Boston, MA). This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK46767 and GRASP Digestive Disease Center Grant P30 DK34928. A.S.K. is a Tufts–New England Medical Center Molecular Cardiology Research Institute Investigator.

Abbreviations

  • TM, transmembrane domain
  • DD2R, Drosophila D2-like receptor
  • SRE, serum response element
  • CRE, cAMP response element
  • HEK293, human embryonic kidney

Notes

Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos. AY150862 (DD2R-606), AY150863 (DD2R-506), and AY150864 (DD2R-461)].

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