functional expression of a locust tyramine receptor in murine erythroleukaemia cells

Insect Molecular Biology (2001)

10

(6), 541–548

© 2001 Blackwell Science Ltd

541

Blackwell Science Ltd

Functional expression of a locust tyramine receptor in murine erythroleukaemia cells

J. Poels

1

, M.-M. Suner

2

, M. Needham

3

, H. Torfs

1

, J. De Rijck

1

, A. De Loof

1

, S. J. Dunbar

2

and J. Vanden Broeck

1

1

Laboratory for Developmental Physiology and Molecular Biology, Zoological Institute, KULeuven, Leuven, Belgium;

2

Syngenta, Jealott’s Hill Research Station, Bracknell, UK;

3

AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, UK

Abstract

The LCR/MEL system (Locus Control Region/MurineErythroleukaemia cells) was employed to express andcharacterize the

Locusta migratoria

tyramine receptor(Tyr

Loc

), an insect G protein-coupled receptor. Func-tional agonist-dependent responses were recorded instable, tyramine receptor expressing cell clones (MEL-Tyr

Loc

). Tyramine elicited a dose-dependent increaseof cytosolic Ca

2+

-ions and an attenuation of forskolin-induced cyclic adenosine monophosphate (AMP) pro-duction. Octopamine was shown to be a weak agonistfor both responses. In addition, yohimbine proved tobe a potent tyramine receptor antagonist. This studyreports the first application of the LCR/MEL expressionsystem in functional assays for G protein-coupledreceptors and therefore expands the capabilities ofthis system by exploiting the functionality of the signaltransduction pathways.

Keywords: amine, calcium, cyclase, G protein, insect.

Introduction

Murine erythroleukaemia (MEL) cells are erythroid pro-genitor, robust, semiadherent cells with a short doubling timeof 10–16 h, which are derived from spleens of susceptiblemice infected with the Friend Virus Complex (Friend, 1957).Their differentiation is arrested at the proerythroblast stage,which allows for these cells to be maintained in tissue

culture indefinitely. Changes similar to normal red bloodcell maturation can be induced with a variety of chemicalagents, including polar–planar compounds such as di-methyl sulfoxide (DMSO). This terminal differentiation causesan induction of globin gene expression where

α

- and

β

-globins represent 25% of the total cellular protein content.The globin LCR enhancer is responsible for high levels oferythroid cell specific expression of globin proteins (Blomvon Assendelft

et al.

, 1989; Talbot

et al.

, 1989).The human globin LCR has been utilized in the LCR/

MEL system (Locus Control Region/Murine Erythro-leukaemia cells), alongside a human

β

-globin promoter

in cis

, to drive integration site independent expressionof cDNA and genomic sequences cloned in single-stepexpression vectors (Needham

et al.

, 1992, 1995). ThisLCR/MEL expression system has already been employedto express a variety of proteins. It is capable of producingfunctional secreted proteins (Needham

et al.

, 1992; Newton

et al.

, 1994). Stable expression of electrophysiologicallyfunctional mammalian homo- and hetero-multimeric ionchannel proteins has been obtained by means of theLCR/MEL system (Amar

et al.

, 1995; Garcia-Alonso,1997; Shelton

et al.

, 1993). It has also been shown to pro-duce very high levels of mammalian G protein-coupledreceptors (GPCRs) as a source for ligand binding experi-ments (Egerton

et al.

, 1995; Needham

et al.

, 1995). Withthis conventional LCR/MEL system, heterologous recep-tor expression was obtained after DMSO-induced dif-ferentiation of the cells into mature red blood cells.Unfortunately, G protein-coupled receptor over-expressionand terminal differentiation often led to a reduction or lossof the signalling events usually linked to these GPCRs.Therefore, this system seemed less suitable for deve-loping functional receptor assays, where the activity ofa ligand can be measured by a change in Ca

2+

or cyclicAMP concentrations.

In this paper, we report on the first functional analysis ofa GPCR by utilizing an uninduced LCR/MEL-C88L system.MEL-C88L, a recently identified subclone of MEL cells thatdisplays constitutive expression (i.e. in uninduced condi-tions) from the

β

-globin promoter, was employed to expressthe locust G protein-coupled tyramine receptor, Tyr

Loc

. Inaddition, because all proteins previously expressed in MELcells were of mammalian origin, this article describes the

Received 17 April 2001; accepted after revision 12 July 2001. Correspond-ence: Dr J. Vanden Broeck, Laboratory for Developmental Physiology andMolecular Biology, Zoological Institute, KULeuven, Naamsestraat 59, B-3000 Leuven, Belgium. Tel.: 32 16 323978; fax: 32 16 323902; e-mail:[email protected]

IMB_292.fm Page 541 Monday, November 19, 2001 8:41 AM

542

J. Poels

et al

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© 2001 Blackwell Science Ltd,

Insect Molecular Biology

,

10

, 541–548

first expression study of an insect protein in a murineerythroleukaemia cell line (MEL-C88L).

Results

Expression of Tyr

Loc

Individual pEV3Tyr

Loc

(Fig. 1) transfected, antibiotic-resistantcell clones were selected and analysed for

β

-globin andTyr

Loc

expression. A Northern blot was performed on RNA

samples (10

µ

g/ lane) extracted from induced (2% DMSO)and uninduced MEL-Tyr

Loc

clones. As a control, RNA frominduced, non-transfected cells (2

µ

g/ lane) was also loadedon the gel.

Uninduced MEL-Tyr

Loc

clones (Fig. 2, lanes 1–3) wereshown to contain a basal level of

β

-globin (Fig. 2A) andTyr

Loc

receptor (Fig. 2B) mRNA. The

β

-globin mRNAcontent of the MEL-Tyr

Loc

clones (Fig. 2A, lanes 6–8)strongly increased after DMSO treatment. Similarly, a largeincrease of Tyr

Loc

receptor mRNA levels was observedafter induction of MEL-Tyr

Loc

clones (Fig. 2B, lanes 6–8).The hybridization signal of the Tyr

Loc

mRNA was boostedapproximately forty times by DMSO induction, as mea-sured by densitometric analysis (ImageMaster, Amersham-Pharmacia Biotech). As expected, the receptor mRNAwas not detected in untransfected MEL-C88L cells (Fig. 2B,lanes 4 & 5). The observed difference in

β

-globin RNA bandintensity (Fig. 2A) between lanes 4–5 and lanes 6–8 cor-responds to the difference in RNA loaded on the gel (2

µ

g vs.10

µ

g), because the average ratio between both, as mea-sured by densitometric image analysis, was also about 1 : 5.

Ca

2+

responses

The different MEL-Tyr

Loc

clones were analysed in orderto test whether they produced a functional response totyramine. In all clones, a similar, tyramine-induced, tran-sient increase in cellular Ca

2+

was observed (Fig. 3).

Figure 1. The cDNA coding for the Locusta migratoria tyramine receptor (TyrLoc) was inserted into the multiple cloning site of pEV3, downstream of the human globin locus control region (LCR) between the promoter and the second exon of the β-globin gene. The human β-globin promoter and parts of the β-globin gene provide mRNA processing and maturation signals, give stability to the final mRNA and confer high expression levels in DMSO-induced cells.

Figure 2. Northern blot hybridization analysis of RNA samples derived from untransfected (MEL-C88L) or permanently transfected (MEL-TyrLoc) cell clones with β-globin (A) and TyrLoc (B) cDNA probes. Lanes 1–3: uninduced MEL-TyrLoc clones (10 µg RNA per lane); Lanes 4–5: DMSO-induced MEL-C88L cells (2 µg RNA per lane): positive control for β-globin mRNA (A), negative control for TyrLoc mRNA (B); Lanes 6–8: DMSO-induced MEL-TyrLoc clones (10 µg RNA per lane). The position of RNA molecular size standards (0.16–1.77 and 0.24–9.5 kb ladders were employed) is indicated on the right.

Figure 3. The tyramine-induced calcium response in MEL-TyrLoc cells is partially dependent on the Ca2+-concentration in the incubation medium. MEL-TyrLoc cells were treated with 1 µM tyramine (TA) in different buffer solutions (the ‘high Ca2+ solution’ contained 4 mM Ca2+, whereas in the ‘low Ca2+ solution’ no Ca2+ was added). Fura-2 fluorescence intensities were measured at an emission wavelength of 510 nm. The ratio of fluorescence intensities (F340/F380) measured at the excitation wavelengths of 340 nm (generated by Ca2+-bound fura-2) and 380 nm (corresponding to Ca2+-free fura-2) is indicated on the vertical axis. The arrow indicates the time of addition of the agonist.


Functional expression of locust tyramine receptor

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Therefore only one clone was used in further experiments.Untransfected MEL-C88L cells did not respond to tyramineat concentrations as high as 100

µ

M

.To investigate whether the response to tyramine was

depending on the presence of extracellular Ca

2+

-ions,experiments were performed in NCF buffers with differentCa

2+

concentrations. In ‘low Ca

2+

solution’ (no Ca

2+

wasadded to the NCF buffer), the cellular Ca

2+

-level dropped toits initial concentration after the agonist-induced increase,while the Ca

2+

-rise observed in the presence of ‘high Ca

2+

solution’ (4 m

M

Ca

2+

) lasted longer (Fig. 3). These resultsare consistent with the expectation that the initial cyto-plasmic Ca

2+

-increase was caused by the release fromintracellular stores, whereas a sustained Ca

2+

-levelwas achieved by an influx of Ca

2+

-ions from the outside ofthe cell.

Figure 4 shows the dose–response curves for theagonist-induced calcium rise in MEL-Tyr

Loc

cells. TheEC

50

value, calculated for the tyramine-dependent calciumresponses in these cells, was 4.7

×

10

–8

M

. A maximalresponse level was reached at 1

µ

M

tyramine. The effectsof other possible agonists were investigated. Octopamineproved to be a much weaker agonist for the Tyr

Loc

receptorthan tyramine (Fig. 4). At least 200 times higher concentra-tions of octopamine (EC

50

value

≥

10

–5

M) were neededto induce detectable calcium responses, showing the highspecificity of the receptor for tyramine. A small calciumrise could also be observed at very high concentrations(10

–4

M

) of dopamine. At this concentration, serotonin (5-hydroxytryptamine) did not produce any detectable response.

The

α

2

-adrenergic receptor antagonist yohimbine provedto be a potent antagonist of Tyr

Loc

-mediated responses.Yohimbine produced a concentration-dependent inhibitionof the calcium response to 0.5

µ

M

tyramine (Fig. 5). The

response to 0.5

µ

M

tyramine was completely blocked (i.e.no detectable Ca

2+

-increase or 100% inhibition) in thepresence of 2.5

µ

M

yohimbine.

Cyclic AMP responses

Tyramine had no significant effect on the cyclic AMPconcentration in untransfected MEL-C88L cells (negativecontrol). However, in MEL-Tyr

Loc

cells, addition of 1

µ

M

tyramine resulted in a decrease of cyclic AMP accumula-tion (Fig. 6A). As shown in Fig. 6(B), the agonist-induceddecrease in cyclic AMP production was much more pro-nounced in forskolin-treated MEL-Tyr

Loc

cells. This responsewas dose-dependent (Fig. 7). The EC

50

value, calculated forthe tyramine-dependent cyclic AMP decrease in forskolin-treated cells, was 4.0

×

10

–9

M

. A maximal response levelof approximately 84% inhibition of the forskolin-inducedcyclic AMP accumulation was obtained at a concentrationof 1

µ

M

tyramine. When comparing the effects of 1

µ

M

octopamine and 1

µ

M

tyramine, a 4-fold stronger decreasein cyclic AMP production was observed with 1

µ

M

tyramine,therefore indicating that tyramine is a more potent inhibitorof forskolin-induced cyclic AMP production in MEL-Tyr

Loc

cells (Fig. 6B). Challenging forskolin-treated MEL-C88Lcells with 1

µ

M

tyramine (negative control) did not result ina detectable change in cyclic AMP concentration. Forskolinis a potent stimulator of adenylyl cyclase activity.

Discussion

This paper describes the expression and functional ana-lysis of an insect G protein-coupled receptor in the uninducedLCR/MEL-C88L system. The present study shows thatthe locust tyramine receptor, Tyr

Loc

, when permanently

Figure 4. Dose–response relationship between tyramine (TA) and octopamine (OA) concentrations and Ca2+-increase in MEL-TyrLoc cells. A maximal response was obtained at 1 µM tyramine. Octopamine proved to be a much weaker agonist than tyramine, because it only elicited a Ca2+-response in a concentration range that was 2–3 orders of magnitude higher.

Figure 5. Dose-dependent effect of the receptor antagonist yohimbine on the intracellular Ca2+-rise induced by 0.5 µM tyramine (TA) in MEL-TyrLoc cells. In this figure, ‘0% inhibition’ corresponds to the Ca2+-rise that was observed in the absence of antagonist. At ‘100% inhibition’, there was no detectable Ca2+-elevation in the cells indicating that the antagonist completely blocked the response.


544

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, 10, 541–548

expressed in the mammalian pro-erythroid cell line, MEL-C88L, mediates agonist-induced changes in secondmessenger levels (i.e. increase of calcium ions and decreaseof cyclic AMP). The monophenolic amine tyramine proved

to be a more potent TyrLoc-agonist than octopamine. It activ-ated this receptor in a concentration range that was 2–3orders of magnitude lower than octopamine. In agreementwith the radio-ligand (3H-yohimbine) binding data obtainedin S2 cells (Vanden Broeck et al., 1995), the α2-adrenergicreceptor antagonist yohimbine proved to be a potent com-petitive antagonist of the TyrLoc receptor. Tyramine is thebiosynthetic precursor of octopamine and, since recently, itis also considered as an important neuro-active substancein insects. This idea is supported by the discovery of separ-ate activities, binding sites and uptake systems fortyramine and octopamine in the locust central nervoussystem (Downer et al., 1993; Hiripi et al., 1994; Roeder,1994; Vanden Broeck, 1996). Moreover, repeated tyramineinjections in Locusta migratoria and Schistocerca gregarialarvae led to an increased mortality and retardation of themoment of ecdysis (Torfs et al., 2000). When injected inadult female locusts, egg deposition was disturbed. Inter-estingly, these tyramine-induced effects were stronger thanthe ones observed with octopamine and could be antago-nized by yohimbine. Further evidence for a functional roleof tyramine in insects came from a study with a Drosophilamelanogaster tyramine receptor (Tyr/Oct-Dro, see alsobelow) mutant, designated as ‘hono’, that displayed adefective olfactory behaviour. In addition, the hono mut-ant was also impaired with responding to tyramine at

Figure 6. Tyramine (TA) reduces the accumulation of cyclic AMP in both uninduced (A) and forskolin-treated (B) MEL-TyrLoc cells. (A) Tyramine (TA, white bar) had a negative impact on the production of cyclic AMP in MEL-TyrLoc cells (compared to the basal cyclic AMP level, shaded bar). In non-transfected MEL-C88L cells, this inhibitory effect on adenylyl cyclase activity was not observed (negative control). Data are shown in percentage of the basal cyclic AMP level (‘100% cAMP’ corresponds to 1.1 pmol cyclic AMP per 106 cells). (B) In MEL-TyrLoc cells, tyramine (TA, white bar) and, to a much lesser extent, octopamine (OA, striped bar) clearly inhibited the forskolin-induced increase in cyclic AMP level. Data are shown in percentage of the average forskolin (10 µM) stimulated cyclic AMP level (‘100% cAMP’ corresponds to 12.1 pmol cyclic AMP per 106 cells). The significance of the tyramine and octopamine effects with respect to the cyclic AMP levels in the control condition is indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 7. The tyramine-induced attenuation of cyclic AMP accumulation in forskolin-treated MEL-TyrLoc cells is concentration-dependent. Compared to the calcium rise (Fig. 4), this effect occurs in a tenfold lower concentration range. A maximal inhibition of approximately 84% of the forskolin-induced cyclic AMP accumulation was obtained at a concentration of 1 µM tyramine. Data are displayed in percentage of the average forskolin (10 µM) stimulated cyclic AMP level (‘100% cAMP’ corresponds to 12.1 pmol cyclic AMP per 106 cells).


Functional expression of locust tyramine receptor 545

© 2001 Blackwell Science Ltd, Insect Molecular Biology, 10, 541–548

neuromuscular junctions, while it showed normal respon-siveness to octopamine (Kutsukake et al., 2000).

G protein-coupled receptors for phenolamines havebeen cloned from a few other insect species, such as Dro-sophila melanogaster (Arakawa et al., 1990; Saudou et al.,1990; Han et al., 1998), Heliothis virescens (Von Nickisch-Rosenegk et al., 1996) and Apis mellifera (Blenau et al.,2000), as well as from the mollusk, Lymnea stagnalis(Gerhardt et al., 1997a,b). The D. melanogaster OAMB,H. virescens K50Hel and L. stagnalis Lym-OA1 and Lym-OA2

receptors are preferentially activated by octopamine. Forthe honeybee Amtyr1, the fruitfly Tyr/Oct-Dro and the locustTyrLoc receptors, tyramine is a more potent agonistthan octopamine. Whether the octopamine- or tyramine-dependent activation results in a cyclic AMP and/or aCa2+ response not only depends on the receptor type (i.e.on receptor-specific G protein coupling), but also on the cellline which is utilized for heterologous expression (Blenau &Baumann, 2001). The specific supply of endogenous Gproteins and/or of other cellular signalling components mayinfluence the type of response(s) elicited upon activation ofa given receptor. In HEK293 cells, the OAMB receptorincreases cyclic AMP and Ca2+ concentrations, whilst in S2cells only a cyclic AMP rise can be detected (Han et al.,1998; Van Poyer et al., 2001). The Tyr/Oct-Dro receptor,which shows the highest degree of similarity to the TyrLocreceptor, initiates a cyclic AMP decrease when activatedin NIH3T3 (Saudou et al., 1990) or CHO cells (Robb et al.,1994), but not in COS7 cells (Saudou et al., 1990) or Xeno-pus oocytes (Reale et al., 1997). In addition, this fruitflyreceptor shows agonist-specific (tyramine vs. octopamine)coupling to different second messenger systems (cyclicAMP reduction vs. Ca2+-rise) when expressed in CHOcells (Robb et al., 1994). In MEL-TyrLoc cells, the tyramine-activated cyclic AMP decrease is much more pronouncedthan in S2-TyrLoc cells (Vanden Broeck et al., 1995). Also,in MEL-TyrLoc cells, an agonist-induced Ca2+ response (EC50

≈ 4.7 × 10–8 M) can be observed in a concentration rangethat is about tenfold higher than the one needed to elicit thecyclic AMP-decrease (EC50 ≈ 4.0 × 10–9 M). This calciumresponse appears to be relatively slow (Fig. 3) and itsmaximal effect is quite small (maximal change of the intra-cellular Ca2+-level ≈ 28 nM, Fig. 4). This calcium rise wasnot observed in S2-TyrLoc cells (our unpublished data). It hasbeen reported that the α2A-adrenergic receptor can initiatea Ca2+ response via Gi-associated βγ-subunits that activatephospholipase C (PLC) (Dorn et al., 1997; Musgrave &Seifert, 1995). This βγ-mediated coupling mechanism ispresent in multiple cell types, including human erythroleu-kaemia (HEL) cells. Whether this is also the case for MELcells expressing the insect α2-related receptor, TyrLoc,remains to be investigated. Nevertheless, the present studydemonstrates that the TyrLoc receptor mediates very pro-nounced effects on these second messenger systems in

MEL-TyrLoc cells. It clearly prefers tyramine to octopamineas an agonist for both types of responses (cyclic AMPinhibition and Ca2+ stimulation).

This paper presents the first application of the LCR/MELexpression system in functional assays for G protein-coupled receptors. The recently identified cell clone, MEL-C88L, allows for leaky, uninduced protein expression fromthe β-globin promoter. Uninduced cells retain nuclei thatare lost or otherwise functionally silenced on DMSO-induced terminal differentiation. Both the Ca2+ and cyclicAMP measurements demonstrate that, in uninduced MEL-TyrLoc cells, there is an efficient coupling of the expressedinsect receptor to the endogenous, mammalian signallingpathways. This observation indicates that the use of thisnovel LCR/MEL-C88L expression system should notnecessarily be restricted to the characterization and functionalanalysis of mammalian receptors. Moreover, the ability of theLCR/β-globin promoter combination to confer high levelsof expression, in a reproducible and position-independentmanner, is not lost in this MEL-C88L cell clone. Afterinduction, this clone also differentiates along the erythro-cyte pathway, loosing its nucleus and boosting the expres-sion levels of heterologous proteins. All these characteristicsshould make this LCR/MEL-C88L cell line an even moreflexible expression system: applicable in a variety of situa-tions, from functional (G protein-coupled receptor signallingcascade or ligand-gated ion channels) to radioligand bind-ing assays, for both mammalian and insect, secreted ortransmembrane proteins.

Experimental procedures

Generation of the pEV3TyrLoc expression vector

The coding region of the TyrLoc cDNA was amplified by poly-merase chain reaction from pVJ12 and pVJ12-IEGprom (VandenBroeck et al., 1995) using the following oligonucleotide primers:1 5′ PCR primer (TyrLoc-Fw): 5′-ttttaagcttgaattcagatctgccaccat-

gaacgggtcttcggctgc-3′2 3′ PCR primer (TyrLoc-Rev): 5′-ttttggatccgcggccgcgtcgactcat-

gtcttgaagtggagcagc-3′The 5′ primer contains the restriction sites HindIII, EcoRI and

BglII, and the consensus translation enhancing sequence(GCCACC) (Kozak, 1987). The 3′ primer contains the restrictionsites BamH I, NotI and Sal I. A PCR product was obtained with Pfupolymerase (Stratagene) utilizing the manufacturers protocol. Thefollowing PCR conditions were used: one cycle of denaturation at96 °C for 2 min followed by forty cycles of denaturation at 96 °C for1 min, annealing at 58 °C for 45 s and extension at 72 °C for 3 min,followed by a final extension reaction of 10 min at 72 °C. The result-ing PCR product was cloned into the pCR-Script (Amp SK +) vector(Stratagene) using the manufacturers protocol. Sequence analysisof a clone, confirmed the presence of a correctly edited insert. Thisinsert was released from the pCR-Script (Amp SK +) backgroundusing EcoRI and Sal I enzymes (Amersham-Pharmacia Biotech)and cloned into pEV3 (Fig. 1). The identity of the DNA utilized forMEL-C88L cell transformation was confirmed by restriction diges-tion and agarose gel electrophoresis as well as sequencing.

IMB_292.fm 45 Monday, November 19, 2001 8:41 AM

546 J. Poels et al.


Cell culture and cell transfections

Murine erythroleukaemia cells (Deisseroth & Hendrick, 1978)were cultured in Dulbecco’s modified Eagle’s medium (Gibco-BRL) supplemented with 10% foetal bovine serum and 2 mM

glutamine at 37 °C, under 10% CO2–90% air. Prior to transfection,50 µg of pEV3TyrLoc were linearized at the unique Asp 718 siteupstream of the neomycin cassette and downstream of the tyramineexpression cassette. Transfection into the cell line was performedby electroporation as described previously (Antoniou, 1991).

After transfection, cells were diluted in culture medium toconcentrations of about 104, 105 and 2.105 cells per ml and 1 mlaliquots were transferred to each well of a 24-well tissue cultureplate (NUNC, nunclon multidishes). Twenty-four hours after the trans-fection, G418 (Gibco-BRL) was added to a concentration of 1 mg/ml in order to select for stable transfectants. Individual clones werepicked 7–10 days after the addition of selective medium.

For RNA purification and functionality studies, cells were main-tained in exponential growth by passaging them for a period of4 days (cells should be growing from 2.105 cells/ml to 6–8.105

cells/ml in 24 h when in log phase). For the RNA purification, onehalf of the cells was induced using 2% DMSO and incubated for afurther period of 4 days.

RNA analysis

Approximately 1.107 cells were washed with phosphate-bufferedsaline and resuspended in 1 ml of RNAzol B (Biogenesis). RNAwas then purified according to the manufacturer’s protocol.

Electrophoresis of the RNA samples (10 µg per lane for theclones, 2 µg for the MEL-C88L controls) was performed in dupli-cate through agarose gels containing 2.2 M formaldehyde. TheRNA was then transferred to a nylon membrane (Hybond-N,Amersham-Pharmacia Biotech) in 20 × SSPE. After transfer, RNAwas covalently cross-linked to the membrane by short-wave ultra-violet irradiation using an UV StratalinkerTM 2400 (Stratagene).Each duplicate membrane was prehybridized and hybridized witheither 32P-labelled β-globin or TyrLoc cDNA probes.

Ca2+ measurements

Ca2+-ion concentrations were measured by using the acetoxyme-thyl (AM) ester of the fluorescent indicator fura-2 (Grynkiewiczet al., 1985). Cells were washed with NCF buffer (135 mM NaCl,5 mM KCl, 6 mM glucose, 0.62 mM MgCl2·6H2O, 10 mM HEPES,pH 7.4) containing 4 mM CaCl2 and resuspended at a concentra-tion of 2.106 cells/ml in NCF buffer containing 2 µM fura-2-AM(Molecular Probes). After 1 h incubation in the dark at 27 °C, thecell suspension was subjected to centrifugation at 300 g for5 min, resuspended in an equal volume of NCF buffer andincubated for an additional 30 min in the dark. Aliquots werecentrifuged as above and resuspended in 3 ml NCF buffer. Intra-cellular Ca2+ changes were analysed in an LS-50B LuminescenceSpectrophotometer (Perkin-Elmer) (ratiometric, dual excitationwavelength measurements, Fig. 3) or in a Hitachi F2000spectrofluorometer (single wavelength analysis, Figs 4 and 5) inthe presence or absence of the appropriate test chemical. Excita-tion wavelengths were 340 and/or 380 nm. The fluorescenceintensity was monitored at an emission wavelength of 510 nm. Noabsorbance or fluorescence artefacts were observed with any ofthe compounds used. For dose–response curves, the intracellularcalcium concentration was calculated from the fluorescence (F,

determined at a single excitation wavelength of 340 nm) data bymeans of the equation, [Ca2+]i = (F − Fmin) / (Fmax − F ) × Kd, in which theextracellular fura-2 fluorescence was subtracted from the F-values(Holmberg et al., 1998). The Kd of calcium binding to fura-2 wascalculated according to Shuttleworth & Thompson (1991). Dose–response curves were measured for the calcium effects that weregenerated in MEL-TyrLoc cells by tyramine (n = 8) and octopamine(n = 5). The dose-dependence of the antagonistic effect of yohim-bine (n = 3) on the agonist-evoked calcium rise in MEL-TyrLoccells was determined at a concentration of 0.5 µM tyramine. Datawere processed by SigmaPlot 4.0 (SPSS Inc.) software. Thisprogram was employed to analyse the results by means of a three-parameter sigmoidal curve-fitting algorithm, to plot dose–response curves and to calculate the EC50 values.

Cyclic AMP measurements

MEL-C88L and MEL-TyrLoc cells were seeded in six-well plates toa density of 8.105 cells/well and allowed to attach. The cell mediumwas then removed from the wells, and the wells were washed withNCF buffer. Incubations were performed in NCF buffer containing200 µM isobutylmethylxanthine (IBMX, to inhibit cyclic AMP phos-phodiesterase) with or without 10 µM forskolin (Fsk), and the addi-tional substances at their appropriate concentrations. In eachexperiment, three wells were incubated with the same solution at37 °C for exactly 30 min. In order to extract the cyclic AMP, 100%ice-cold ethanol was added to each well to a final concentration of65%. After 5 min incubation at room temperature, the solution wasremoved from the wells, which were then rinsed with 65% ethanol.The eluates from the same wells were then pooled together andevaporated. Cyclic AMP was measured by employing a ‘Scintilla-tion Proximity Assay’ (SPA kit, Amersham-Pharmacia Biotech),according to the manufacturer’s recommended procedure. Eachexperiment was done in triplicate. Data were processed by meansof the Excel (Microsoft) program and are shown as the average± SD (standard deviation) of n measurements. Statistical signific-ance was calculated with Student’s two-tailed t-test. Non-linearcurve fitting was performed with SigmaPlot 4.0 (SPSS Inc.). Thisprogram was employed to plot dose–response curves and tocalculate the EC50 values. Amines, yohimbine, IBMX and forskolinwere purchased from Sigma.

Sequence registration

Accession number of TyrLoc cDNA: X-69520.

Acknowledgements

The authors wish to thank Fergus Earley and Liz Hirst (Syn-genta) for technical advice and John Windass (Syngenta)for advocating the use of MEL cell expression studies withinsect receptors. Research was supported by IUAP/PAI(‘Interuniversity Poles of Attraction Programme’; BelgianState, Prime Minister’s Office – Federal Office for Scientific,Technical and Cultural Affairs). J.Vd.B. was a SeniorResearch Associate of the FWO-Flanders.

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Functional expression of locust tyramine receptor 547


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