Functional comparison of two evolutionary conserved insect neurokinin-like receptors

peptides 28 (2007) 103–108

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Functional comparison of two evolutionary conserved insect neurokinin-like receptors Jeroen Poels a, Heleen Verlinden a, Jakub Fichna b, Tom Van Loy a, Vanessa Franssens a, Kazimierz Studzian b, Anna Janecka b, Ronald J. Nachman c, Jozef Vanden Broeck a,* a

Laboratory for Developmental Physiology, Genomics and Proteomics, Catholic University Leuven, Naamsestraat 59, B-3000 Leuven, Belgium b Department of Medicinal Chemistry, Medical University of Lodz, Mazowiecka 6/8, 92-215 Lodz, Poland c Areawide Pest Management Research Unit, Southern Plains Agricultural Research Center, USDA, 2881F/B Road, College Station, TX 77845, USA

article info

abstract

Article history:

Tachykinins are multifunctional neuropeptides that have been identified in vertebrates as

Received 3 April 2006

well as invertebrates. The C-terminal FXGXRa-motif constitutes the consensus active core

Received in revised form

region of invertebrate tachykinins. In Drosophila, two putative G protein-coupled tachykinin

30 June 2006

receptors have been cloned: DTKR and NKD. This study focuses on the functional char-

Accepted 30 June 2006

acterization of DTKR, the Drosophila ortholog of the stable fly’s tachykinin receptor (STKR).

Published on line 4 December 2006

Tachykinins containing an alanine residue instead of the highly conserved glycine (FXAXRa) display partial agonism on STKR-mediated Ca2+-responses, but not on cAMP-responses.

Keywords:

STKR therefore seems to differentiate between a number of tachykinins. Gly- and Ala-

Agonist

containing tachykinins are both encoded in the Drosophila tachykinin precursor, thus raising

Antagonist

the question of whether DTKR can also distinguish between these two tachykinin types.

Drosophila

DTKR was activated by all Drosophila tachykinins and inhibited by tachykinin antagonists.

G protein

Ala-containing analogs did not produce the remarkable activation behavior previously

Neuropeptide

observed with STKR, suggesting different mechanisms of discerning ligands and/or activat-

Tachykinin

ing effector pathways for STKR and DTKR. # 2006 Elsevier Inc. All rights reserved.

Spantide

1.

Introduction

Neurokinins or tachykinins comprise a family of multifunctional brain/gut peptides that are present in both vertebrates and invertebrates. They play a role in a variety of physiological (cf. ‘‘tachykinin’’ refers to the ability to cause tachycardia) and even pathological situations [20,24]. The best-known vertebrate member is substance P (SP). In insects, immunoreactivity to vertebrate tachykinins has been demonstrated multiple times, but the first insectatachykinins (TKs, also designated as

tachykinin-related peptides or TRPs) were not identified until 1990 [22,23]. Since then, several insect TKs have been purified from insect tissue extracts and/or identified in silico from nucleotide sequence databases. The majority of insect TKs possess a C-terminal consensus sequence, FX1GX2Ra, while most vertebrate neurokinins own a FXGLMa motif. The functions of tachykinins in insects remain largely unknown. Despite the large intra- and interspecies diversity of TKs in invertebrates, only few corresponding receptors have been fully cloned. These receptors belong to the class of G protein-coupled

* Corresponding author. Tel.: +32 16 324260; fax: +32 16 323902. E-mail address: [email protected] (J.V. Broeck). 0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2006.06.014

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peptides 28 (2007) 103–108

receptors (GPCRs). The first cloned insect tachykinin receptor originated from Drosophila and was termed DTKR (Drosophila tachykinin receptor, Takr99D) [11]. When expressed in Xenopus oocytes, DTKR showed pertussis toxin sensitive responses to micromolar concentrations of SP and physalaemin (an amphibian tachykinin), but not to some other vertebrate tachykinins. However, the endogenous peptide ligands of the fruit fly had not yet been discovered at that time. Years later, a polypeptide precursor gene (Tk) encoding six putative TKs (designated DrmTKs) was identified in the Drosophila melanogaster genome data [25,30] and the occurrence of several Drm-TK peptides has recently been confirmed by mass spectrometry [1,33]. STKR, a neurokinin-like receptor cloned from the stable fly, Stomoxys calcitrans [7], shares 79% amino acid sequence identity with DTKR in the region flanked by the extracellular N- and intracellular C-terminal domains. When stably expressed in Drosophila Schneider 2 (S2) cells, this receptor was activated by insectatachykinin family members and stimulated second messenger production, showing both IP3/ Ca2+ and cAMP responses [29]. So far, only one tachykinin has been purified from the stable fly (Stomoxytachykinin, Stc-TK). Interestingly, Stc-TK contains an Ala-residue instead of the highly conserved Gly-residue that is present in most other invertebrate tachykinins (FX1AX2Ra) [15]. This apparently small change of Gly to Ala has some important functional consequences. Stc-TK is a potent, partial agonist for STKRmediated changes in cellular Ca2+-levels, but behaves as a full agonist for STKR-induced cAMP changes [17,18,27]. Substitution of the exceptional Ala-residue by Gly (Stc-TK-G) results in a full agonist for STKR-mediated Ca2+ responses, but is accompanied by a reduction in potency. The fact that STKR can differentiate between a number of tachykinins could prove to be a significant mechanism of physiological finetuning for this important class of peptides [18]. It could also form one explanation for the discrepancies that exist between the limited number of tachykinin receptors and the various alleged ligands that are usually encoded as multiple variants on a large precursor protein [9,12,14]. Indeed, in Drosophila, at least six tachykinins exist while only two putative receptors have been identified [11,13]. These results led us to investigate the functional activities of the different Drosophila tachykinins on DTKR, which was, more than 15 years after its initial discovery, never challenged with these supposed endogenous ligands. In addition, given the high degree of molecular similarity between the tachykinin receptors from two fly species, DTKR and STKR, we compared the functional properties of these receptors when expressed in an insect cell line (S2 cells), and evaluated the potencies and efficacies of two naturally occurring Ala-containing TKs; DrmTK-6 and Stc-TK, as well as of synthetic analogs that contained a Gly ! Ala switch. Furthermore, the efficacies of different antagonists for DTKR and STKR were evaluated.

2.

Materials and methods

2.1.

Molecular cloning and expression constructs

DTKR encoding cDNA was generated from a bacterial clone corresponding to EST GH10154 (Invitrogen; Carlsbad, USA) by

means of a PCR reaction including the following primers: 50 GCGGCCGCAGCCATGGAGAATCGCAGTGACTTCGAG-30 and 50 TCTAGAGGTTGTCAGGAGAGCAGTTGGGTGG-30 , containing NotI and XbaI restriction sites, respectively (shown in italics). ProofStart DNA polymerase was used according to the manufacturer’s protocol (Qiagen). The obtained PCR product was cloned into pCR4-TOPO (Invitrogen) and sequenced. A correct DTKR cDNA fragment was placed downstream of the Drosophila actin 5C promoter in pAc5.1/V5-His (Invitrogen) using appropriate restriction sites. The cloning and sequencing of the STKR cDNA has been described elsewhere, as well as the generation of the STKR expression construct [7,29]. Transcription of mitochondrially targeted apoaequorin is controlled by the constitutive actin promoter in the pAcaequorin vector, as described previously [28].

2.2.

Cell culture and transfections

Drosophila Schneider 2 cells [21] were cultured in Schneider’s Drosophila medium (Serva) supplemented with 0.6 g/l CaCl2, 0.4 g/l NaHCO3, 50 U/ml penicillin, and 50 mg/ml streptomycin (Invitrogen) and 10% heat-inactivated fetal calf serum (Invitrogen). Cells were grown in monolayers at 23 8C. Transfections were carried out in serum containing media with 45 ml TransFectin reagent (Biorad) and 15 mg DNA (7.5 mg pAc-aequorin and 7.5 mg receptor expression plasmid) per 75 cm2 culture flask. Cells were 60–90% confluent before transfection. Stable receptor expressing cells were obtained by co-transfection with pCoBlast (Invitrogen) and selection in blasticidin S containing media.

2.3.

Peptides

The insect tachykinin-related peptides, drosotachykinins 1-6 (Drm-TK-1-6), locustatachykinins 1-4 (Lom-TK-1-4), and stomoxytachykinin (Stc-TK), as well as Gly ! Ala substituted LomTK-3-A were synthesized by means of Fmoc polyamide chemistry. The purity (>95%) of these peptides was determined by MALDI-TOF-MS. Peptides were stored at 20 8C as freeze-dried stocks that were dissolved in basal Schneider’s medium (pH 6.45; supplemented with CaCl2 and NaHCO3) directly prior to use. The sequences of the natural and synthetic insectatachykinin analogs employed in this study are displayed in Table 1. Spantides I–III, potent substance P antagonists, were obtained from Bachem.

2.4.

Aequorin assay

Receptor and aequorin expressing cells were examined for viability and counted by trypan blue exclusion. Cells were spun down (6 min at 150 g) and resuspended in basal Schneider’s medium at a density of 1  107 cells per ml. ‘‘Coelenterazine h’’ (Invitrogen) was added to a concentration of 5 mM and the cells were incubated on a platform shaker in the dark at room temperature for 2–4 h. These coelenterazineloaded cells were diluted five times in basal Schneider’s medium immediately before starting the luminescence assay experiments. Test substances were dissolved in 100 ml basal Schneider’s medium and dispensed in triplicate into the wells

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peptides 28 (2007) 103–108

Table 1 – Amino acid sequences and EC50-values of tachykinin-like peptides on DTKR and STKR Peptide Drm-TK-1 Drm-TK-2 Drm-TK-3 Drm-TK-4 Drm-TK-5 Drm-TK-6 Lom-TK-1 Lom-TK-2 Lom-TK-3 Lom-TK-4 Lom-TK-3-A Stc-TK

Consensus: FXGXRa

EC50 (nM) on DTKR

APTSSFIGMRa APLAFVGLRa APTGFTGMRa APVNSFVGMRa APNGFLGMRa [pGlu]RFADFNSKFVAVRa GPSGFYGVRa APLSGFYGVRa APQAGFYGVRa APSLGFHGVRa APQAGFYAVRa APTGFFAVRa

6.4 46.0 8.5 10.4 6.5 10.7 836.7 744.0 8.4 10.0 10.1 8.3

EC50 (nM) on STKR 800.2 152.3 82.5 >1 mM 7.5 10.1 175.0 120.5 103.0 n.d. 0.6 8.2

Drm: Drosophila melanogaster, Lom: Locusta migratoria, Stc: Stomoxys calcitrans. The glycine-substituting alanine residue in Drm-TK-6, Lom-TK-3-A, and Stc-TK is underlined; n.d.: not determined.

of a white 96-well plate. Light emission was recorded (EG&G Microplate Luminometer LB96V, Berthold) for 30 s immediately after injection of 100 ml cell suspension into each well. Cells were then lysed by a second injection of 50 ml 0.5% Triton X-100, followed by a 8 s monitoring period. Results were calculated by means of Winglow software (Perkin-Elmer) as the fractional luminescence, i.e. the ratio of the agonist generated signal and the total luminescence (agonist + lysed cells), thereby correcting for potential well-towell variation in the number of injected cells [10]. The resulting data were then transferred to and processed by SigmaPlot (SSI) software.

3.

Results

3.1.

Activities of endogenous DTKR ligands

increases. DTKR shares a remarkable degree of amino acid sequence identity with STKR (79%), as opposed to 40% with the second Drosophila tachykinin-like receptor, NKD (Takr86C) [13]. While all six Drm-TKs perform as STKR agonists, Drm-TK-6, which contains the same Ala-replacement as Stc-TK, also proved to be a partial agonist for STKR-mediated intracellular Ca2+ changes [13]. Interestingly, this is not the case for DTKR, where Drm-TK-6 behaves as a full agonist (Fig. 1). In order to confirm this result, we also determined the efficacy and potency of several other insect tachykinins, such as stomoxytachykinin, Stc-TK, and locustatachykinins (Lom-TKs) (Fig. 2). Based on their potencies, the Lom-TKs can be divided in two groups; Lom-TKs 1 and 2 are two orders of magnitude less active on DTKR than Lom-TKs 3 and 4 (Table 1). As opposed to the effects on STKR [18], the Ala-substituted form of Lom-TK-3 (APQAGFYAVRa) does not possess a decreased efficacy nor an

We co-expressed DTKR and the Ca2+-sensitive photoprotein apoaequorin in Drosophila Schneider 2 (S2) cells. Receptor expressing, but not untransfected cells, showed dose-dependent Ca2+-responses to the six different Drosophila TKs (DrmTK-1-6). Except for Drm-TK-2, the corresponding EC50-values (Table 1) were similar for all Drm-TKs and in the nanomolar range. Substance P, which contains the vertebrate characteristic C-terminal FXGLMa-motif, was inactive at concentrations 1 mM (Fig. 1).

3.2.

Actions of nonendogenous tachykinins and analogs

The sites of TK release in the adult fruit fly brain and the expression pattern of DTKR correspond well, already providing a strong indication that DTKR can indeed be activated by the different endogenous TKs [2,25,33]. In addition to this, in a recent report, we proposed a model where the tachykinin receptor from the stable fly, STKR, possesses altered active states that can differentiate between a number of tachykinins [17,18]. The endogenous stable fly TK (Stc-TK) thereby has an Ala-residue instead of the highly conserved Gly-residue (FX1AX2Ra) present in most other invertebrate members of this peptide family [15]. This small modification causes Stc-TK to behave as a potent, partial agonist for STKR-mediated Ca2+responses, but as a full agonist for receptor-induced cAMP-

Fig. 1 – Dose-response curves for bioluminescent responses induced in S2-DTKR-aequorin cells by different Drosophila tachykinin-related peptides (Drm-TK-1-6) and substance P (SP). Data are the average WS.D. of three independent measurements done in triplicate and are given in percentage of the maximal response obtained with Drm-TK-4. The minimal response corresponds to treatment with buffer only. For clarity, the fitted curves for Drm-TK-3, -5, and -6 were omitted from the graph since they collide with the two leftmost curves on display.

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peptides 28 (2007) 103–108

Fig. 2 – Dose-response curves for bioluminescent responses induced in S2-DTKR-aequorin cells by different Locusta tachykinin-related peptides (Lom-TK-1-4) and Ala-containing tachykinins (Lom-TK-3-Ala and Stomoxys Stc-TK). Data are the average WS.D. of three measurements done in triplicate and are given in percentage of the maximal response obtained with Lom-TK-4. For clarity, one curve represents Lom-TK-3, -4, -3-Ala, and Stc-TK, while the right curve fits both Lom-TK-1 and -2.

augmented potency for DTKR-mediated Ca2+-level changes when compared to the activity of the Gly-containing analog. In addition, Stc-TK performs as a full agonist on DTKR.

3.3. I–III

Effects of tachykinin receptor antagonists; spantides

Spantide I was originally developed as a D-amino acid containing substance P analog ([D-Arg1, D-Trp7,9, Leu11]SP) that behaved as an antagonist on the mammalian neurokinin receptor, NK1. The high neurotoxicity and limited antagonistic potency of spantide I directed the development of spantide II ([D-NicLys1, Pal3, D-Cl2Phe5, Asn6, D-Trp7,9, Nle11]SP) and spantide III ([D-NicLys1, Pal3, D-Cl2Phe5, Asn6, D-Trp7, D-Pal9, Nle11]SP) [4–6]. The three spantides display some pronounced preferences for the different neurokinin receptors (NK1–3), whereby NK3-activity could not be inhibited by any type of spantide [8]. On STKR, the rank order of antagonistic potencies was: spantide III > spantide II > spantide I [28]. The activity of Lom-TK-2 on the second Drosophila tachykinin-like receptor, NKD, could be blocked by spantide I, but no dose-response relationship was established, nor were there any other antagonists examined [13]. So far, no spantides had been tested on DTKR. The rank order of inhibitory effectiveness of spantides on this receptor was: spantide II > spantide III  spantide I (Fig. 3A–C).

4.

Discussion

We have confirmed that DTKR, a Drosophila GPCR with sequence similarities to vertebrate tachykinin receptors, can indeed be activated by the putative endogenous ligands, DrmTK-1-6, and that this receptor is coupled to the Ca2+-pathway,

Fig. 3 – A, B, and C: Antagonistic effect of spantides I–III on Drm-TK-1 evoked bioluminescent Ca2+-responses in S2DTKR-aequorin cells. All three spantides behaved as competitive inhibitors of DTKR, as indicated by the rightward shift of dose-response curves with increasing antagonist concentrations. Data are the average WS.D. of three independent measurements done in triplicate and are given in percentage of the maximal response that was generated using 1 mM Drm-TK-1 without antagonist.

peptides 28 (2007) 103–108

as has been established for other members of the tachykinin receptor family. While this report was in preparation, another paper was published confirming the activities generated by Drm-TKs in HEK-293 cells expressing DTKR [2]. When we expressed DTKR in CHO cells, the different Drm-TKs also proved to be agonists, but their EC50-values were much higher than in S2 cells (data not shown). In any case, Drosophila cells as an expression system for insect receptors provide the benefit of mimicking the in vivo situation by coupling to homologous G proteins and cellular effectors, whereas in mammalian-based expression systems the coupling between insect receptor and mammalian G protein(s) is by definition artificial, and thus might lead to sub-optimal functioning. Five of the six Drm-TKs display quite similar EC50-values for the DTKR-mediated calcium response, pointing to an almost equipotent function for these agonists at this receptor. DrmTKs are encoded by a single transcript that does not seem to possess alternative splicing, hence giving rise to a sole precursor protein and thus, most likely, equimolar concentrations of all six TKs. Whether the small potency differences of the six Drosophila tachykinins for DTKR are also reflected in in vivo circumstances remains unknown. However, the generation of multiple TKs processed from a single precursor protein is a common theme in invertebrate species, where so far only a limited number of receptors have been identified [3,16,19,26,31]. In contrast to the related stable fly tachykinin-like receptor (STKR), no partial agonism was detected with Ala-containing TKs on DTKR-mediated Ca2+-responses in S2 cells. Also, the rank order of antagonistic potencies for the members of the spantide family differs between the two receptors. These observations suggest a notable difference in how these GPCRs make a distinction between their respective (ant)agonists. It thus appears that DTKR does not possess the additional mechanism for distinguishing Ala-containing tachykinin analogs, at least not in S2 or CHO cells. Concerning the functions of DTKR, the expression patterns of the peptide ligands, as well as the receptor, suggest multiple functional neuromodulatory roles in the adult fly. Very recently, utilizing the RNAi technique, Winther et al. [32] established a role for Drm-TKs in locomotor activity in adult flies and odor perception in both larvae and adults. Further experiments are needed to reveal whether these activities are mediated via DTKR and/or NKD. It will thus be of interest to determine the rank order of Drm-TK agonism, as well as a detailed distribution pattern for the second Drosophila tachykinin-like receptor, NKD. Also, it should be verified whether both Drosophila tachykinin receptors have the same order of spantide antagonistic properties. This could prove useful for in vitro as well as in vivo assays where the activities of endogenous Drosophila tachykinins are examined and one wishes to distinguish NKD and DTKR related effects.

Acknowledgements The authors thank Sofie Van Soest and Luc Vanden Bosch for excellent technical assistance. This work was supported by a grant for Bilateral Scientific Cooperation between Flanders

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and Poland (BIL05/17). The authors also acknowledge the Belgian Interuniversity Attraction Poles Programme (IUAP/PAI P5/30, Belgian Science Policy) and the FWO-Vlaanderen for financial support. J. Poels obtained a postdoctoral fellowship from the FWO-Vlaanderen. H. Verlinden, T. Van Loy, and V. Franssens obtained a Ph.D. fellowship from the IWT.

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