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.

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 exon is illustrated, highlighting the number of amino acids (aa) per exon and the relative position of TMs I–VII (solid gray boxes). (B) The complete amino acid sequence of DD2R-606. Double and single underlining marks the sequence that corresponds to exons 6 and 7, respectively. DD2R-506 includes exon 7 but not exon 6 coding sequence. DD2R-461 lacks both exon-6 and -7 coding sequence. (C) Variable splicing of exon 6 results in DD2R splice variants DD2R-606, -605, -598, -597, -589, and -581. The carboxyl- and amino-terminal 5 aa of exons 5 and 7, respectively, are shown. Dashes represent amino acids that are absent in the corresponding transcript. Forty-eight amino acids that are shared among DD2R-606, -605, -598, -597, -589, and -581 are depicted by (48 aa). The putative splice sites are illustrated in supporting information, Fig. 5.

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 (B–D). In addition, negative controls for each RNA sample were carried out in the absence of reverse transcriptase (−). (A) PCR was performed using the primer pair S4/A4 (as detailed in Supporting Materials and Methods). The products were visualized on an ethidium bromide-stained 1.2% agarose gel. (B–D) The tissue/stage-specific levels of expression of DD2R (B), DmDop1 (C), and actin 5C (D) in adult head (○), adult body (•), pupa (▾), and larva (▴) were quantified by TaqMan analysis as detailed in Materials and Methods. The increase in fluorescence (ΔRn values) was plotted against cycle number (PRISM 3.0, GraphPad). The graphs shown are representative of multiple experiments. No detectable amplification was observed in negative controls (RT−), which were run in parallel for each tissue/transcript examined (data not shown).

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 CRE_6x-luciferase reporter gene construct. Cells were preincubated in the presence or absence of pertussis toxin (200 ng/ml) for 16 h. Forskolin (1 μM) was then added with or without dopamine (1 μM). After 3 h, luciferase activity was assayed as described in Materials and Methods. Light emission is expressed as a percentage of the respective forskolin-induced value (without dopamine, without pertussis toxin = 100%). The data represent the mean ± SEM from at least three independent experiments. Significance, presence vs. absence of dopamine (DA); **, P < 0.01. hD2L, human D2 long receptor; hD1, human dopamine 1 receptor.

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 SRE_5x-luciferase reporter construct and a plasmid encoding the chimeric G protein Gq5i. After a 3-h stimulation with ligand, luciferase activity was assessed. (A) The Drosophila DD2R and the hD2L have comparable potencies for dopamine and for the D2-selective agonist bromocriptine. Both dopamine and bromocriptine at maximally effective concentration induced a >10-fold increase in luciferase activity (vs. basal), which was defined as 100% for respective ligands. The graphs represent the mean ± SEM from at least three independent experiments, each performed in triplicate. Calculated EC₅₀ values (μM) for dopamine were 0.5 (DD2R-606, not shown), 0.4 (DD2R-506), 0.4 (DD2R-461, not shown) and 0.3 (hD2L). Corresponding EC₅₀ values (nM) for bromocriptine were 1.5 (DD2R-606, not shown), 1.4 (DD2R-506), 2.1 (DD2R-461, not shown), and 0.2 (hD2L). (B) Selected dopamine receptor ligands stimulate DD2R second-messenger signaling. Luciferase activity is expressed as a percentage of the dopamine (10 μM)-induced maximum for each receptor (100%, indicated by dashed line). The data represent the mean ± SEM from at least three independent experiments, each performed in triplicate. All agonist-stimulated values are significantly different (P < 0.05) from corresponding basal values except where indicated by ns (not significant).