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InsectBiochemistry

andMolecularBiology

0965-1748/$ - se

doi:10.1016/j.ib

�CorrespondE-mail addr

(A.R. Mercer).1Present addr2Present addr

University, Col

Insect Biochemistry and Molecular Biology 35 (2005) 873–882

www.elsevier.com/locate/ibmb

Characterization of a D2-like dopamine receptor (AmDOP3) inhoney bee, Apis mellifera

Kyle T. Beggs, Ingrid S. Hamilton, Peri T. Kurshan1, Julie A. Mustard2, Alison R. Mercer�

Department of Zoology, University of Otago, PO BOX 56, Dunedin, New Zealand

Received 23 December 2004; received in revised form 9 March 2005; accepted 21 March 2005

Abstract

Dopamine is an important neurotransmitter in vertebrate and invertebrate nervous systems and is widely distributed in the brain

of the honey bee, Apis mellifera. We report here the functional characterization and cellular localization of the putative dopamine

receptor gene, Amdop3, a cDNA clone isolated and identified in previous studies as AmBAR3 (Apis mellifera Biogenic Amine

Receptor 3). The Amdop3 cDNA encodes a 694 amino acid protein, AmDOP3. Comparison of AmDOP3 to Drosophila melanogaster

sequences indicates that it is orthologous to the D2-like dopamine receptor, DD2R. Using AmDOP3 receptors expressed in HEK293

cells we show that of the endogenous biogenic amines, dopamine is the most potent AmDOP3 agonist, and that activation of

AmDOP3 receptors results in down regulation of intracellular levels of cAMP, a property characteristic of D2-like dopamine

receptors. In situ hybridization reveals that Amdop3 is widely expressed in the brain but shows a pattern of expression that differs

from that of either Amdop1 or Amdop2, both of which encode D1-like dopamine receptors. Nonetheless, overlaps in the distribution

of cells expressing Amdop1, Amdop2 and Amdop3 mRNAs suggest the likelihood of D1:D2 receptor interactions in some cells,

including subpopulations of mushroom body neurons.

r 2005 Elsevier Ltd. All rights reserved.

Keywords: Apis mellifera; Dopamine receptor; G protein-coupled receptor; camp; Mushroom body

1. Introduction

There is compelling evidence that the catecholamine,dopamine (DA), plays multiple roles in insects. DA hasbeen strongly implicated in the regulation of locomotoractivity (Yellman et al., 1997; Cooper and Neckameyer,1999; Feany and Bender, 2000; Pendleton et al., 2002;Friggi-Grelin et al., 2003), sexual behaviour (Neck-ameyer, 1998a), development (Neckameyer, 1996; Neck-ameyer et al., 2001) and endocrine function (Granger etal., 1996, 2000), and it is believed to play a role also inbrain and behavioural plasticity (Mercer and Menzel,1982; Mercer and Erber, 1983; Temple et al., 1984;

e front matter r 2005 Elsevier Ltd. All rights reserved.

mb.2005.03.005

ing author. Tel.: +643 479 7961; fax: +64 3 479 7584.

ess: alison.mercer@stonebow.otago.ac.nz

ess: 120 Riverway ]7, Boston, MA 02215, USA.ess: 400D Aronoff Labs, 318 W 12th Ave, Ohio State

umbus, OH 43210, USA.

Macmillan and Mercer, 1987; Michelsen, 1988; Neck-ameyer, 1998b; Kirchhof and Mercer, 1997; Kirchhofet al., 1999; Schwaerzel et al., 2003). While many ofthese functions strongly parallel those seen in mamma-lian systems, knowledge of the physiology and pharma-cology of dopaminergic systems in insects is not yet asadvanced.Understanding the functional organization of dopa-

minergic systems requires knowledge of the receptorsubtypes that mediate the actions of DA, and thecellular localization of these receptor subtypes. Inmammals, DA receptors have been broadly classifiedinto two distinct groups: D1-like and D2-like receptors.One of the hallmark features that distinguishes D1-likefrom D2-like DA receptors is their coupling to cAMPsignaling pathways (for review see Missale et al., 1998).Activation of members of the D1 receptor family, whichin mammals includes D1 (D1A) and D5 (D1B)receptors, leads to an increase in intracellular cAMP

ARTICLE IN PRESSK.T. Beggs et al. / Insect Biochemistry and Molecular Biology 35 (2005) 873–882874

levels, whereas receptors from the D2 receptor sub-family, which includes the mammalian receptor sub-types D2, D3 and D4, either reduce cAMP levels or actvia different second messenger pathways.Based on sequence analysis and functional character-

ization of receptors encoded by cloned cDNA, two D1-like DA receptors have been identified in the honey bee,AmDOP1 (Blenau et al., 1998) and AmDOP2 (Humph-ries et al., 2003). The Amdop1 gene is most homologousto the fruit fly gene, Dmdop1/dDA1 (Gotzes et al., 1994;Sugamori et al., 1995), whereas Amdop2 is mosthomologous to the fruit fly gene DopR99B/DAMB(Feng et al., 1996; Han et al., 1996).Here, we examine the functional properties and

cellular distribution of the receptor encoded by Amdop3,a putative DA receptor referred to elsewhere asAmBAR3 (Apis mellifera Biogenic Amine Receptor 3;Ebert et al., 1998; Kokay et al., 1999; Humphries et al.,2003). We describe features of the AmDOP3 protein, thefunctional properties of AmDOP3 receptors and thedistribution of cells in the brain expressing Amdop3

mRNA. We also compare the pattern of expression ofAmdop3 with the expression patterns of the 2 D1-likeDA receptor genes in honey bee, Amdop1 and Amdop2.

2. Materials and methods

2.1. Sequence analysis

Genomic sequence data (assembly Amel 1.2) wereobtained from the Baylor College of Medicine HoneyBee Genome Project (http://www.hgsc.bcm.tmc.edu/pro-jects/honeybee/). Amino acid sequences were aligned usingClustalW software (version 1.82) using default settings(http://ww.ebi.ac.uk/clustalw/: Thompson et al., 1994).Prediction of the transmembrane-spanning protein do-mains was achieved using hydropathy index analysis (Kyteand Doolittle, 1982) and alignment of conserved residues.Boundary positions for the transmembrane domainswere estimated using the TMpred program (http://www.ch.embnet.org/software/TMPRED_form.html) andare indicative only. Protein motif recognition was per-formed using the eukaryotic linear motif (ELM) resourcefor functional sites in proteins (http://elm.eu.org/).

2.2. Heterologous expression of AmDOP3 receptors

For constitutive expression of AmDOP3 receptorsin human embryonic kidney (HEK293) cells, theAmdop3 coding region was inserted into the plasmidvector pcDNA3.1(+) (Invitrogen). The Amdop3 codingregion was PCR amplified from cDNA template(AmBAR3; Ebert et al., 1998) using a forward primercontaining a KpnI restriction site and consensus Kozaksequence (Kozak, 1987) immediately 50 of the trans-

lation initiation codon (GTCAGGTACCGCCAC-CATGGGGACGAAGACGGGCGAC) and a reverseprimer containing a XbaI restriction site immediately 30

of the stop codon (GACGTCTAGACTAGAAGCT-GACAAGTTTATGAAAGG). The amplification pro-duct was inserted into the pcDNA3.1(+) multiplecloning site using the described KpnI and XbaIrestriction sites to give the pcAmDOP3 construct. Theplasmid pcDNA3.1/His/LacZ (Invitrogen) was used toexpress the b-galactosidase (b-gal) protein as a controlfor receptor expression studies. Plasmid stocks wereprepared in parallel using a Qiagen EndoFree Maxi Kit.HEK293 cells were maintained as adherent mono-

layer cultures at 37 1C and 7% CO2 in high-glucoseDulbecco’s modified eagle medium (GibcoBRL) supple-mented with 10% fetal calf serum (GibcoBRL). Forreceptor expression, exponentially growing cells (2� 105

cells/well) were dispensed into 24-well plates (Nunc) andgrown for 24 h at 37 1C. Expression plasmid constructswere transfected into cells using FuGene6 reagent(Roche) and cells were grown for a further 48 h priorto analysis of receptor properties.

2.3. Functional properties of the AmDOP3 receptor

Measurements of intracellular cAMP were used tomonitor the effects of amines on HEK293 cellsexpressing AmDOP3 receptors. Mock-transfected cells(no plasmid DNA) and cells transfected with thepcDNA3.1/His/LacZ expression (for expression con-struct of the b-gal protein) were used as controls in allassays. Growth medium was removed and replaced withfresh serum-free growth medium containing 100 mM 3-isobutyl-1-methylxanthine (IBMX), 1 mM forskolin plusone of the following biogenic amines (Sigma): dopaminehydrochloride, (-)-norepinephrine bitartrate, tyraminehydrochloride, DL-octopamine hydrochloride, 5-hydro-xytryptamine creatinine sulphate, or histamine dihy-drochloride. Cells were exposed for 20min at 37 1C atthe range of concentrations indicated in the figurelegends.Intracellular cAMP levels were measured immediately

after amine treatment using a Biotrak competitiveenzyme immunoassay (Amersham Biosciences). Eachmeasurement was performed in duplicate and a mini-mum of three independent assays were carried out foreach compound and concentration tested. Statisticalanalysis of data was performed using Prism forMacintosh, version 3.0a (GraphPad Software, SanDiego, CA, USA).

2.4. Distribution of cells in the brain expressing Amdop3

mRNA

In situ hybridization was used to examine thedistribution of cells in the brain expressing Amdop3

ARTICLE IN PRESSK.T. Beggs et al. / Insect Biochemistry and Molecular Biology 35 (2005) 873–882 875

mRNA and to compare the pattern of Amdop3-expressing cells with the distribution of cells express-ing the D1-like DA receptor genes, Amdop1 andAmdop2. Brains of pupae midway through metamor-phic adult development (pupal stage 5) and of adultbees collected at the hive entrance were examined.Prior to dissection, the bees were cold anaesthetized.For bees at pupal stage 5 (P5) the entire head wasremoved and placed into fixative. For adults, the brainwas dissected from the head capsule under coldphosphate-buffered saline (PBS; 2mM NaH2PO4,5.8mM Na2HPO4, 154mM NaCl) prior to fixation.Isolated brains or whole heads were fixed in 4%paraformaldehyde in PBS at room temperature for 2 h(brains) or 4 h (heads). The tissue was then transferredto 18% sucrose/PBS for cryoprotection overnight. Thefollowing day, the tissue was embedded in freezingmedium (Cryomatrix, Shandon) and sectioned into16 mm slices, which were mounted onto polylysine-coated slides (Sigma).Hybridization to cryosections of honey bee brains was

performed with digoxigenin (DIG)-labelled riboprobesas described by Kurshan et al. (2003). For Amdop3,antisense and sense probes were transcribed from aplasmid containing an 890-bp Pst1/BglII fragment fromthe untranslated 50 region of the Amdop3 cDNA inpBluescript II (Stratagene). Antisense probe was synthe-sized using T7 RNA polymerase with an EcoRVlinearized plasmid, whereas sense probe was synthesizedusing T3 RNA polymerase with an XbaI linearizedplasmid.Antisense and sense probes for Amdop1 were tran-

scribed from a subclone containing an 870 bp ClaIfragment from the 30 untranslated region (bp1900–2770) of the Amdop1 cDNA (Blenau et al.,1998). Antisense probe was synthesized using T7 RNApolymerase with an XhoI linearized plasmid, whereassense probe was synthesized using T3 RNA polymerasewith an XbaI linearized plasmid. For Amdop2, antisenseand sense probes were synthesized from a 1214 bpfragment from the untranslated 30 end of the Amdop2

cDNA clone (Humphries et al., 2003). Antisense probewas synthesized using T7 RNA polymerase with a SalIlinearized plasmid, whereas sense probe was synthesizedusing T3 RNA polymerase with an ApaI linearizedplasmid. All DIG-labelled riboprobes were synthesizedusing a DIG RNA Labelling Kit (Roche) according tothe manufacturer’s instructions.In situ hybridization was performed as previously

described in Kurshan et al. (2003). Sections werephotographed with Fujichrome tungsten slide film(ASA 64) using an Olympus C-35AD-4 camera. Thephotographs (slides) were scanned (using AGFA Photo-look) and saved as Photoshop files. Minor contrast andbrightness adjustments were made to render the imagesmore clearly.

For description of cell populations in mushroombodies (MBs) of the brain we employ the terminologyused by Farris et al. (1999) to denote three main Kenyoncell groupings; inner compact cells (ICCs), noncompactcells (NCCs) and outer compact cells (OCCs). Furthersubdivisions of these three major groupings of peri-karya can be made based on the dendritic morpho-logy and projection patterns of the cells (Strausfeld,2002), but these have not been considered further inthis study.

3. Results

3.1. Sequence analysis

The Amdop3 cDNA sequence (GenBank accessionnumber AY921573) is 3256 bp in length and contains amajor open reading frame encoding a 694 amino acidprotein, AmDOP3. Hydropathy index analysis (Kyteand Doolittle, 1982) of the AmDOP3 sequence predictsthat the protein contains seven transmembrane-span-ning domains (Fig. 1B, TM I-VII), a highly character-istic feature of G protein-coupled receptors (Pierce et al.,2002). Comparison of AmDOP3 to described Drosophila

melanogaster proteins indicates that it is highly likely tobe the honey bee ortholog of the D2-like DA receptorDD2R (Hearn et al., 2002). A further database search ofknown arthropod protein sequences also revealed highhomology to a partial Anopheles gambia proteinsequence (GenBank accession no. XM_313744), whichappears truncated prior to the third TM domain.Alignment of the Amdop3 cDNA sequence to honey

bee genomic sequences indicates that the gene is encodedby eight exons (Fig. 1A). Comparison of this putativegene structure for Amdop3 to that described for theDD2R gene (Hearn et al., 2002) suggests that therelative positions of the intron-exon boundaries havebeen largely conserved. The DD2R gene has beendemonstrated to produce eight mRNA splice variants(Hearn et al., 2002). The Amdop3 sequence appears mostequivalent to DD2R-606, the longest of the splicevariants described by Hearn et al. (2002).Alignment of the amino acid sequences of AmDOP3

and DD2R-606 (Fig. 1B) demonstrates greatest se-quence divergence in regions considered to have thelowest functional constraints (Wess, 1998; Missale et al.,1998), namely the extracellular N-terminal domain andthe central sequences of the third intracellular loop.Both proteins have relatively short C-terminal tails andlong third intracellular loops, features that are char-acteristic of mammalian D2-like DA receptors (Missaleet al., 1998). A particular feature of AmDOP3 is acomparatively long N-terminal domain that containsseveral putative protein motifs for N-glycosylation anda putative N-terminal myristoylation signal.

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Fig. 1. Analysis of the Amdop3 nucleic acid and protein sequences. (A) Predicted exon-intron structure of the Amdop3 gene (not to scale) based on a

best-fit alignment of the Amdop3 cDNA sequence to the contig sequences of the Apis mellifera genome (release Amel 1.2). Intron sequences are

represented as dashed lines and exon sequences (numbers E1 to E8) as solid lines (non-coding) and boxes (coding). The relative positions of the

coding sections for the seven transmembrane loops (I–VII) are shown in grey. (B) Alignment of the AmDOP3 and DD2R-606 protein sequences

(Hearn et al., 2002), prediction of the protein structure, and identification of potential functional motifs. The protein structure is based on the

predicted locations of the seven transmembrane domains (shown as TM1-VII in grey boxes) typical of G protein-coupled receptors, and indicates the

position of the N- and C-terminal domains, and the three intracellular (il 1-3) and extracellular (el 1-3) loops. Residues implicated in ligand binding

(see text) are shown in bold. Putative sites of post-translational modification, based on sequence motif identification by the ELM resource, are

represented as follows: Asn-glycosylation sites are in bold italics; N-terminal myristoylation sites are underlined; PKA phosphorylation sites are in

bold and underlined; PKC phosphorylation sites are double underlined.

K.T. Beggs et al. / Insect Biochemistry and Molecular Biology 35 (2005) 873–882876

3.2. Functional characterization of the AmDOP3

receptor

Expression of AmDOP3 receptors in HEK293 cellsresulted in a small, but significant rise in the basal levelsof intracellular cAMP (Fig. 2A). AmDOP3-expressingcells treated with forskolin (1 mM) also exhibitedsignificantly higher levels of cAMP than forskolin-treated controls (mock-transfected cells, and cellsexpressing b-gal; Fig. 2B). Exposing AmDOP3-expres-sing cells to DA, however, resulted in a significantreduction in intracellular levels of cAMP (Fig. 2C, 3),whereas in control (mock-transfected, or b-gal -expres-sing) cells, no such reduction in cAMP was observed(Fig. 2C). These results suggest that DA activation ofAmDOP3 receptors is responsible for down regulationof cAMP in AmDOP3-expressing cells. The effect of DAon these cells was dose dependent (Fig. 3).To confirm that DA was the most potent of the

biogenic amines at activating AmDOP3 receptors,forskolin-stimulated cells were exposed to a rangeof biogenic amines, all at a concentration of 10 mM(Fig. 2C). Serotonin, octopamine and histamine had nosignificant effect on intracellular levels of cAMP in cells

expressing AmDOP3 receptors. Tyramine, like DA,lowered cAMP levels in these cells (Fig. 2C), but asignificant reduction in intracellular cAMP was detectedonly if tyramine was applied at concentrations greaterthan 10 mM (Fig. 3).Norepinephrine greatly increased cAMP levels both

in AmDOP3-expressing cells and in control cells(Fig. 2C) suggesting, as shown elsewhere (Gerhardtet al., 1997; Ohta et al., 2003; Grohmann et al., 2003),that HEK293 cells can express endogenous adrenergicreceptors. Our results also suggest that DA activates anendogenous receptor population, as exposing mock-transfected cells (not shown) or b-gal-expressing cells toDA resulted in a small, but significant rise in intracel-lular cAMP levels in these cells (see Fig. 2C). In cellsexpressing AmDOP3 receptors, however, these actionsof DA were completely masked by DA-activation ofAmDOP3 (Figs. 2C, 3).Interestingly, bromocryptine (10 mM), a potent ago-

nist of the Drosophila DD2R receptor (Hearn et al.,2002) caused a significant reduction in the levels ofintracellular cAMP in cells expressing AmDOP3(P ¼ 0:0324, Fig. 2C), but had no effect on cAMPlevels in control cells.

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Fig. 2. Relative levels of intracellular cAMP in control (mock-

transfected and b-gal-expressing) cells compared to cells expressingAmDOP3 receptors and the effect of potential receptor agonists. (A)

Basal levels of cAMP (measured in 100mM IBMX) expressed as a

percentage of the levels detected in control (mock-transfected) cells.

The mean concentration of cAMP determined for mock-transfected

cells was 0.43 pmol/well 70.21 SD. Expression of the AmDOP3

receptor in HEK293 cells increases the basal level of cAMP. Overall

statistical significance was determined by one-way ANOVA followed

by Tukey-Kramer tests (F ¼ 27:21, P ¼ 0:001). Letters above each baron the graph indicate whether or not differences between groups are

significant. Groups that share a letter are not significantly different.

Each bar represents the mean response+SD. (B) As for (A) except

cells were treated additionally with 1mM forskolin to induce adenylyl

cyclase activity and raise basal cAMP levels (F ¼ 16:62, P ¼ 0:0002).The mean concentration of cAMP determined for mock-transfected

cells treated with forskolin was 8.5 pmol/well 75.1 SD. (C) Effect ofpotential receptor agonists (10mM) on intracellular camp levels incontrol (b-gal-expressing) cells and in cells expressing AmDOP3

receptors. Mock-transfected (not shown), b-gal- and AmDOP3-

expressing cells were treated with IBMX, forskolin and 10mM of the

compounds indicated in the figure. For each treatment, data are

expressed as a percentage of the cAMP levels recorded in untreated

cells transfected with the same expression construct and treated with

forskolin and IBMX alone. Asterisks indicate treatments that induced

a significant reduction in cAMP levels compared to untreated controls.

The significance of differences between treated and untreated groups

was tested using Student’s two-tailed t-tests. A significance level of

po0:05 was accepted for all tests. The number of independent assaysfor each treatment is indicated in brackets. Each bar represents the

mean response7SD.

Fig. 3. Dose-dependent effect of dopamine (DA) and tyramine (TA)

on intracellular cAMP levels in cells expressing AmDOP3 receptors.

Data are normalized to cAMP levels recorded in cells treated with the

lowest ligand concentration applied (10�8M). Each data point was

measured in at least five or more independent assays. Error bars

represent the standard error of the mean.

K.T. Beggs et al. / Insect Biochemistry and Molecular Biology 35 (2005) 873–882 877

3.3. Amdop3 expression pattern

In situ hybridization analysis revealed the distribu-tion of cells in the brain expressing Amdop3 mRNA.The specificity of the probes used to detect Amdop3

transcript can be assessed by comparing stainingobtained using anti-sense probe (Figs. 4A, B and 5A)with sections from the same brain treated with senseprobe (control; Figs. 4C and 5B, respectively). Differ-ences in the patterns of expression of Amdop1, Amdop2

and Amdop3 (Figs. 4, 6; see below) provide additionalevidence of probe specificity.In the brains of stage 5 pupal bees, cells expressing

Amdop3 mRNA could be detected in somata located inthe protocerebrum, deutocerebrum and in tritocerebralregions of the brain (Figs. 4A, B). In the MBs of theprotocerebrum, staining for Amdop3 transcript at pupalstage 5 was most prominent within the NCCs that linethe inner margins of each calycal cup and spill out overthe developing neuropil of the MB ‘lip’ (Figs. 4A, B;large arrows). Staining within the OCCs of the MBswas weak (Fig. 4A; double-headed arrow), and therewas little if any staining of ICCs at this stage (Figs. 4A,B; small arrows). In adults, however, staining forAmdop3 mRNA within the MBs of the brain was nolonger restricted to NCCs. Amdop3 transcript could bedetected in all three major subpopulations of MBKenyon cells, the OCCs (Fig. 5A; double-headedarrow), ICCs (Fig. 5A; small arrow) and NCCs(Fig. 5A; large arrow). Both in adults and in pupalbees, staining with the Amdop3 probe could also bedetected in cells scattered around the antennal lobes(Figs. 4A, 5A; double-headed arrowheads) and inclusters of cells associated with the optic lobes of thebrain (Figs. 4A, 5A; arrowheads).

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Fig. 4. Distribution of cells in the brain at pupal stage 5 expressing

Amdop3 mRNA revealed using in situ hybridization. Staining using

antisense probe (A, B) can be compared with staining obtained using

sense (control) probe (C). (A) Frontal section of the anterior region of

the brain showing intense staining of cells in the protocerebrum and

deutocerebrum. In the mushroom bodies (MBs) of the protocerebrum,

staining is most intense in NCCs that line the inner margins of each

calycal cup and spill out over the developing ‘lip’ (large arrows). Weak

staining is apparent in outer compact cells (OCCs, double-headed

arrow), whereas little if any staining is apparent in inner compact cells

(ICCs, small arrow) at this stage. Intense staining is apparent also in

clusters of cells (arrowhead) that lie adjacent to developing optic lobes,

and in deutocerebral neurons located around the developing antennal

lobes (double-headed arrowheads). (B) Frontal section from the

posterior region of the brain showing intense staining of cells in the

protocerebrum and tritocerebrum. In the protocerebrum, staining is

most intense in NCCs of the MBs (large arrows) and in cells located

beneath the medial calyces (double-headed arrowheads). Intense

staining is also apparent in cells scattered throughout the tritocerebral

rind of somata (arrowheads). (C) Frontal section of the same brain as

shown in (A) and (B) stained with sense (control) probe. Scale

bar ¼ 200mm.

Fig. 5. Frontal sections of the adult brain show the distribution of cells

expressing Amdop3 mRNA revealed using in situ hybridization

techniques. (A) Staining obtained using the antisense probe. Intense

staining is apparent in ICCs (small arrow), NCCs (large arrow) and

OCCs (double-headed arrow) of the MBs. Staining is also apparent in

clusters of cells lying adjacent to the optic lobes (arrowheads) and in

neurons that surround the antennal lobes (e.g. double-headed arrow-

head). (B) Staining obtained using sense (control) probe. Scale

bar ¼ 200mm.

K.T. Beggs et al. / Insect Biochemistry and Molecular Biology 35 (2005) 873–882878

3.4. Comparison of Amdop3 expression pattern with that

of Amdop1 and of Amdop2

The distribution of cells in the brain expressingAmdop3 mRNA at pupal stage 5 (Fig. 4) was comparedto expression patterns obtained at the same develop-mental stage using probes for Amdop1 (Fig. 6A) orAmdop2 (Fig. 6B). Differences between the expres-sion patterns of these three DA-receptor genes wereparticularly striking within developing MB Kenyon cells(Table 1). At pupal stage 5, all three major subpopula-tions of Kenyon cells (OCCs, ICCs and NCCs) showedintense staining for Amdop1 mRNA (Fig. 6A). Incontrast, staining for Amdop2 mRNA was apparentonly in OCCs and ICCs (Fig. 6B), cells that at pupalstage 5 showed little or no staining for Amdop3 (Fig.4A). Amdop1 and Amdop2 transcripts were alsodetectable in prominent cell clusters located in thelateral protocerebrum adjacent to the developing opticlobes (Figs. 6Ai, Bi, respectively; arrowheads). WhileAmdop3 mRNA could not be detected in these cells atpupal stage 5 (Figs. 4A, B), cells lying adjacent to thedeutocerebrum (Fig. 4A; double-headed arrowheads)and tritocerebrum (Fig. 4B; arrowheads) stained moreintensely for Amdop3 transcript at this stage than foreither Amdop1 (Fig. 6Ai) or Amdop2 (Fig. 6Bi) mRNAs.

4. Discussion

Our study supports three main conclusions. First,Amdop3 (previously referred to as AmBAR3; Ebert etal., 1998; Kokay et al., 1999) encodes a DA receptor andits activation leads to a reduction in intracellular levels

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Fig. 6. Distribution of cells in the brain at pupal stage 5 expressing Amdop1 (Ai) and Amdop2 (Bi) mRNAs. (Ai, Bi) Frontal brain sections stained

with antisense probe. (Aii, Bii) Frontal sections taken from the same brains as shown in (Ai, Bi) respectively, stained with sense (control) probe. At

pupal stage 5, the patterns of expression of Amdop1 (Ai) and of Amdop2 (Bi) are markedly different from the expression pattern observed for Amdop3

(Fig. 4A, B). Differences are most striking in the MBs of the brain. At pupal stage 5, Amdop1 (see Ai) is expressed in OCCs (double-headed arrow),

NCCs (large arrows) and in ICCs (small arrow). Amdop2 (see Bi) is expressed in OCCs (double-headed arrow) and ICCs (small arrow), but not in

NCCs (large arrows). Cells in the lateral protocerebrum also stain intensely for Amdop1 (Ai) and Amdop2 (Bi) mRNAs at this stage. Scale

bar ¼ 200mm.

Table 1

Comparison of the expression patterns observed for the Amdop1,

Amdop2 and Amdop3 transcripts in mushroom body Kenyon cell

populations of stage 5 pupa

OCC ICC NCC

Amdop1 ++ ++ ++

Amdop2 ++ ++ —

Amdop3 + — ++

OCC outer compact cells; ICC inner compact cells; NCC noncompact

cells.

K.T. Beggs et al. / Insect Biochemistry and Molecular Biology 35 (2005) 873–882 879

of cAMP. Second, Amdop3 is widely expressed in thebrain, not only in adults, but also in pupal bees. Third,the pattern of expression of Amdop3 transcript in thebrain is markedly different from that of either Amdop1

or Amdop2, suggesting unique roles in the brain for eachof these DA receptor genes.Phylogenetic analysis of Amdop3 in previous studies

revealed that this gene shares highest homology to themammalian D2-like DA receptor genes (Ebert et al.,1998; Kokay et al., 1999; Humphries et al., 2003). Morerecently, a DA receptor gene isolated from Drosophila,DD2R, has been shown to encode a D2-like DAreceptor (Hearn et al., 2002). Amdop3 appears to bethe ortholog of DD2R (for review see Mustard et al.,2005) and here we show that AmDOP3 receptors, likeDD2R receptors, are D2-like in their functional proper-ties. Residues considered important for ligand bindingin mammalian biogenic amine receptors are seen incomparable positions in both DD2R and in AmDOP3

(Fig. 1B). These include an aspartate residue (D) in TMIII that interacts with the amine group, two serineresidues in TM V that interact with the benzyl hydroxylgroups, and a phenylalanine in TM VI that stabilizes thearomatic ring (Strader et al., 1995). An aspartate residuein TM II that has been shown to be important foractivation of the human D1 receptor (Tomic et al., 1993)is also conserved in both receptors.Significant differences between the proteins encoded

by Amdop3 and DD2R are also apparent. AmDOP3 hasa comparatively long extracellular N-terminal domain(Fig. 1B) with a putative myristoylation signal at its N-terminal. Post-translational addition of a hydrophobiclipid moiety to AmDOP3 might act to ‘anchor’ the N-terminus back to the membrane (Boutin, 1997).Receptor phosphorylation by the second messenger-dependent protein kinases, protein kinase A (PKA) andprotein kinase C (PKC), has been shown to be involvedin receptor desensitization of G protein-coupled recep-tors (Ferguson, 2001), and AmDOP3 contains severalpotential sites for both PKA and PKC phosphorylationin the third intracellular loop. Interestingly, many ofthese sites are not conserved between the AmDOP3 andDD2R proteins, with the notable exception of thosefound in the highly conserved C-terminal region of thethird intracellular loop (Fig. 1B). PKC phosphorylationof the same region of the rat D2L DA receptor hasrecently been implicated in receptor desensitization andinternalization responses (Namkung and Sibley, 2004).D2-like DA receptors characteristically function

through interaction with the Gi class of heterotrimeric

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G proteins to mediate repression of adenylyl cyclase,thereby lowering intracellular levels of cAMP (reviewedby Missale et al., 1998). Consistent with structuralevidence showing that AmDOP3 is most closely relatedto the D2-like DA receptors, DA-activation of Am-

DOP3 receptors resulted in significant down-regulationof intracellular cAMP. Intriguingly, however, basallevels of cAMP were higher in AmDOP3-expressingcells than in either, mock-transfected cells, or in controlcells expressing b-gal. While there is evidence to suggestthat the Gi class of G proteins can mediate activation ofcertain classes of adenylyl cyclase (Tang and Hurley,1998), upregulation of basal cAMP levels in AmDOP3-expressing cells might also suggest an interaction withthe Gs class of G proteins, which activate adenylylcyclase. There are many precedents for G protein-coupled receptors interacting with multiple G proteinpathways (Wess, 1998) and constitutive receptor activityhas been described for many G protein-coupledreceptors (Seifert and Wenzel-Seifert, 2002), includ-ing the honey bee D1-like DA receptor, AmDOP1(Mustard et al., 2003). One further possibility, however,is that the high levels of cAMP observed in this studyin cells expressing AmDOP3 are an artifact of high-level expression in a heterologous system. Furtherexperimentation is required to resolve this interestingobservation.Previous studies have revealed the highly dynamic

nature of DA receptor gene expression in the developingbrain of the honey bee (Kurshan et al., 2003). The D1-like DA receptor genes, Amdop1 and Amdop2, not onlyhave markedly different expression patterns in the brain,but also the patterns of expression change significantlyduring the course of development. The results of thisstudy suggest that the same is true of the D2-like DAreceptor gene, Amdop3. For example, while it was notpossible to detect Amdop3 transcript within ICCs of theMBs at pupal stage 5, strong staining for Amdop3

mRNA in this subpopulation of MB Kenyon cells wasdetectable in the adult brain. Although each of the threehoney bee DA receptor genes exhibits a unique patternof expression in the brain, our results show that there issignificant overlap in the distribution of cells expressingD1-like (Amdop1 and Amdop2) and D2-like (Amdop3)DA-receptor genes. In the brain of the adult worker bee,for example, ICCs of the MBs show intense staining forall 3 DA receptor gene mRNAs (Kurshan et al., 2003;present study). Studies in mammals have reportedcolocalization of D1- and D2-like DA receptors in thesame neuronal cells (Aizman et al., 2000; Lee et al.,2004). Recent work has also suggested that D1 and D2receptor proteins can form heteromers, and can actsynergistically to produce a novel phospholipase C-mediated calcium signal (Lee et al., 2004). In Caenor-

habditis elegans D1-like and D2-like receptors are bothexpressed in motor neurons of the ventral nerve cord

and, in a study on locomotion, appeared to actantagonistically (Chase et al., 2004). Our results suggestthat D1:D2 receptor interactions are likely to play aprominent role also in the brain of the bee.In the mouse, a number of DA receptor knockouts

have been analyzed including mutations of each of themammalian D2-like receptors, D2, D3 and D4 (Baik etal., 1995; Accili et al., 1996; Rubinstein et al., 1997). Themammalian D2 receptor, for example, has been shownto be intimately involved in motor function (Baik et al.,1995), and D2 knockout results in symptoms resemblingthose of Parkinson’s disease. DA has been stronglyimplicated in the regulation of locomotor activity in thefruit fly (Yellman et al., 1997; Cooper and Neckameyer,1999; Feany and Bender, 2000; Pendleton et al., 2002;Friggi-Grelin et al., 2003). The wide distribution ofAmdop3 mRNA in cells surrounding optic lobes andantennal lobes of the brain suggest that the D2-like DAreceptor in honey bees is likely to play a role also in theprocessing of information in sensory pathways of thebrain. Consistent with this hypothesis, recent studieshave shown that in honey bee antennal-lobe neurons,Ca2+-activated K+ currents are targets of DA modula-tion (Perk and Mercer, 2004). The identity of thereceptors responsible for mediating these effects has yetto be determined.While the cellular mechanisms through which DA

operates in the insect brain have yet to be fullyelucidated, the identification and characterization ofreceptors that mediate the actions of this importantamine provide important steps towards this goal. It willbe interesting in future studies to examine the role ofD1- and D2-like DA receptors in the honey bee, and inparticular, the functional significance of D1:D2 interac-tions in areas such as the MBs of the brain.

Acknowledgements

The authors wish to thank Paul Ebert of theUniversity of Queensland for supplying the AmBAR3cDNA clone, Ken Miller for assistance with the figuresand Kim Garrett for maintaining the honey beecolonies. This work was funded by grants from theRoyal Society of New Zealand Marsden Fund (UOO312), the Human Frontier Science Program (RG0014)and the University of Otago (UORG 200200484). Allexperiments described in this work comply with the lawsof New Zealand regulating scientific research.

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