In insects, molting and metamorphosis are strictly regulated by ecdysteroids. Ecdysteroid synthesis is positively or negatively controlled by several neuropeptides. The prothoracicostatic peptide (PTSP) BmPTSP (Bombyx mori prothoracicostatic peptide), isolated from the larval brain of B. mori, has been demonstrated to inhibit ecdysteroid synthesis in the prothoracic glands (PGs) [Hua et al. (1999) J. Biol. Chem. 274, 31169–31173]. More recently, the newly recognized B. mori receptor for Drosophila melanogaster sex peptide (DmSP) has been identified as a receptor for BmPTSP. However, details on the signalling pathways and physiological functions of this receptor have remained elusive. In the present paper, we report the functional characterization of the BmPTSP receptor (BmPTSPR)/sex peptide (SP) receptor (SPR) using both mammalian and insect cells. Synthetic DmSP shows the potential to inhibit forskolin (FSK) or adipokinetic hormone (AKH)-induced cAMP-response element (CRE)-driven luciferase (Luc) activity in a manner comparable with synthetic BmPTSP1. However, DmSP displayed a much lower activity in triggering Ca2+ mobilization and internalization than did BmPTSP1. Additionally, 6-carboxy-fluorescein fluorophore (FAM)-labelled DmSP and BmPTSP3 were found to bind specifically to BmPTSPR/SPR. The binding of FAM–DmSP was displaced by unlabelled DmSP, but not by unlabelled BmPTSP1 and, vice versa, the binding of FAM–BmPTSP3 was blocked by unlabelled BmPTSP3, but not by unlabelled DmSP. Moreover, internalization assays demonstrated that BmPTSP1, but not DmSP, evoked recruitment of the Bombyx non-visual arrestin, Kurtz, to the activated BmPTSPR/SPR in the plasma membrane. This was followed by induction of internalization. This suggests that BmPTSP1 is probably an endogenous ligand specific for BmPTSPR/SPR. We therefore designate this receptor BmPTSPR. In contrast, DmSP is an allosteric agonist that is biased towards Gαi/o-dependent cAMP production and away from Ca2+ mobilization and arrestin recruitment.

INTRODUCTION

All insect species undergo several transformations prior to reaching adulthood. Insect metamorphosis is strictly regulated by molting hormones known as ecdysteroids [1]. The biosynthesis of ecdysteroids (also referred as ecdysteroidogenesis) is now known to occur predominately in the prothoracic gland (PG) and is stimulated by the tropic factor prothoracicotropic hormone (PTTH) [2,3]. PTTH is synthesized in the brain's neurosecretory cells and released to the haemolymph at specific times and at particular developmental stages. In addition to this tropic effect, some inhibitory factors to ecdysteroid biosynthesis in the PGs of Bombyx mori have also been identified. These include prothoracicostatic peptide (PTSP) [4,5], bommo–myosuppressin (BMS) [6] and bommo–FMRFamides (Phe-Met-Arg-Phe-NH2) (BRFas) [7]. The varied timings of synthesis and release of the above factors generate a finely tuned fluctuation of ecdysteroid titre in the haemolymph during the various stages of insect development.

PTSP is a multi-functional neuropeptide that has been detected in numerous cells throughout the central nervous system and in the peripheral neurosecretory cells in various insects species [8,9]. This peptide belongs to the WX6Wamide neuropeptide family that shares the conserved C-terminal motif -WX6Wamide. The B. mori prothoracicostatic peptide (BmPTSP) was first isolated from the larval brain of B. mori and was shown to exert a strong inhibitory effect on PTTH-stimulated ecdysteroid synthesis in the PG at both the spinning and the feeding stages [4]. In B. mori, eight copies of PTSPs or PTSP-related peptides are encoded by a single PTSP peptide precursor gene which is based on the potential endoproteolytic cleavage sites and C-terminal amidation sites. Most of these BmPTSPs showed prothoracicostatic activity at concentrations of 10−7 M and 10−6 M, except in the case of PTSP-VIII [5].

A Bombyx neuropeptide G-protein coupled-receptor (GPCR), mainly expressed in the PG, has recently been identified as a functional receptor for PTSP and the Drosophila sex peptide (DmSP) [5]. Sex peptide (SP) is produced in the male Drosophila's accessory gland and is transported to the female genital tract in mating. This results in an increase in egg-laying and a reduction in receptivity to courting males in female Drosophila [10,11]. This post-mating switch in female D. melanogaster is common to most insect species [12,13]. Using Ca2+ imaging analysis in a heterologous expression system, human embryonic kidney (HEK)293 cells expressing this receptor respond to BmPTSPs in a dose-dependent manner but fail to respond to other neuropeptides [5]. The receptor was first identified as a functional receptor for DmSP using Chinese hamster ovary (CHO) cells co-expressing the receptor together with the Ca2+ reporter aequorin and chimeric G-proteins (Gαqi or Gαqo) [14]. It was thus named as BmPTSP receptor/SP receptor (BmPTSPR/SPR). This functional characterization has demonstrated that the BmPTSPR/SPR is activated via a Gαi/o-coupled signalling pathway to regulate cAMP formation and Ca2+ mobilization in response to DmSP. The analysis of receptor expression during development showed that BmPTSPR/SPR is highly expressed during each head capsule slippage period and on the last day of the spinning stage (i.e., 1 day before each ecdysis). However, it is only weakly expressed during each intermolt period. The expression pattern of BmPTSPR/SPR is closely related to its function in the regulation of ecdysteroidogenesis. However, detailed information on the signalling cascades of BmPTSPR/SPR remains largely unknown.

In the present study, BmPTSPR/SPR was cloned from the brain of the silkworm, B. mori and functionally characterized using synthetic BmPTSP1 and DmSP in HEK293 cells and Spodoptera frugiperda 21 (sf21) cells. Our data, derived from functional assays and binding assays using fluorophore-tagged DmSP and BmPTSP3 demonstrates that BmPTSPR/SPR is differentially activated in its binding, signalling and internalization by BmPTSP1 and DmSP.

MATERIALS AND METHODS

Materials

The larvae of the B. mori silkworm, strain (p50), were kindly provided by Dr Liangen Shi (College of Animal Science, Zhejiang University). The DmSP, BmPTSP1 and FAM-labelled DmSP (FAM–DmSP) were synthesized by GL Biochem. The cell culture media and FBS were purchased from Hyclone. Lipofectamine 2000, G418 and OPTI®-MEMI reduced serum media were purchased from Invitrogen. The pCMV–FLAG vector, forskolin (FSK) and pertussis toxin (PTX) were purchased from Sigma and the pEGFP–N1 vector were from Clontech Laboratories Inc. Primary antibodies for Western blotting were purchased from Cell Signaling and Beyotime.

Cloning of BmPTSPR/SPR cDNA and construction of mammalian expression vectors and insect expression vectors

The cDNA of the brain of larval silkworms was prepared using a PrimeScript™ 1st Strand cDNA Synthesis Kit according to the manufacturer's instructions. The cDNA-fragment encoding BmPTSPR/SPR (GenBank accession number: NM_001114874) was generated by means of a PCR reaction using brain cDNA as a template and included the following primers: 5′-ATGGCG GTCACC ATAGAC AATT-3′ and 5′-TTAAAG CACAGT TTCGTT TGTAC-3′. The obtained PCR product was directionally cloned into a pCMV–FLAG vector and a pEGFP–N1 vector using the following primers: 5′-AAGCTT ATGGCG GTCACC ATAGAC AATT-3′ and 5′-GGATCC TTAAAG CACAGT TTCGTT TGTAC-3′ for pCMV–FLAG; 5′-CTCGAG GCCACC ATGGCG GTCACC ATAGAC-3′ and 5′-GGTACC GTAAGC ACAGTT TCGTTT GTAC-3′ for pEGFP–N1. The two vectors were named as BmPTSPR/SPR and BmPTSPR/SPR–EGFP respectively. In order to permit expression in insect cells, BmPTSPR/SPR encoding fragments from BmPTSPR/SPR and BmPTSPR/SPR–EGFP were cloned downstream of the whispovirus ie1 promoter. All constructs were sequenced to verify the correctness of their sequences and orientations.

Culture and transfection

Sf21cells were maintained in insect cell culture media TC100 from Applichem supplemented with 10% FBS at 28°C and seeded on to a 6-well tissue culture plate 2 h prior to transfection. The BmPTSPR/SPR cDNA plasmid constructs were transfected into sf21 cells using a SuperFectinTM II DNA Transfection Reagent (Pufei) according to the manufacturer's instruction. The HEK293 cells were maintained in Dulbecco's Modified Eagles Medium (DMEM; Hyclone) supplemented with 10% heat-inactivated FBS (Pufei) and were incubated at 37°C in a humidified atmosphere with 5% CO2/95% air. The BmPTSPR/SPR cDNA plasmid constructs were transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. 24 h after transfection, stably expressing cells were selected by the addition of 800 mg/l G418.

cAMP accumulation measurement

A reporter gene assay was performed to investigate changes in the intracellular levels of cAMP. In the present study, the pCRE (cAMP-response element)–Luc (luciferase) or pBmCRE–Luc reporter gene systems, consisting of the firefly Luc coding region under the control of a minimal promoter containing CREs, were used to measuring the intracellular cAMP levels. HEK293 and sf21 cells expressing FLAG–PTSPR/SPR and its corresponding reporter gene were seeded into 96-well plates. Twenty-four hours later, the cells were stimulated with different concentrations of BmPTSP1 or DmSP in serum-free DMEM or TC100 in the presence of adipokinetic hormone 1 (AKH1) or FSK and then incubated for 4 h. Luc activity was detected using a firefly Luc assay kit (Ken-real). When required, cells were pre-treated overnight with PTX (100 ng/ml) or CTX (300 ng/ml) in serum-free medium before the start of the experiment. The cAMP concentration was assessed using a commercially available cAMP detection kit (R&D).

Intracellular calcium measurement

The fluorescent Ca2+ indicator fura-2-am (fura-2-acetoxymethyl ester) was employed to monitor changes in intracellular calcium. Calcium mobilization was performed as described previously with slight modifications [15]. The stable FLAG–PTSPR/SPR-expressing HEK293 cells were harvested using Cell Stripper (Mediatech), washed twice with PBS and resuspended at 5×106 cells/ml in Hanks’ balanced salt solution containing 0.025% BSA. The cells were then loaded with 3 μM fura-2-am (Molecular Probes) for 30 min at 37°C. For sf21 cells, the experiment was performed at 28°C in HBM [Hepes-buffered medium: 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.2 mM Na2HPO4, 5 mM NaHCO3, 10 mM glucose and 20 mM HEPES–NaOH, CaCl2 (1 mM), pH 6.2] instead of Hank's solution. The calcium flux was measured using excitation wavelengths of 340 and 380 nm in a fluorescence spectrometer (LS55, PerkinElmer Life Sciences).

Immunoblot analysis

HEK293 cells stably expressing FLAG–PTSPR/SPR were seeded in 6-well plates and were starved by growth in serum-free media for an hour. After stimulation with BmPTSP1/DmSP, cells were lysed. Equal amounts of total cell lysates were size-fractionated using Tris/glycine SDS/PAGE (10% gel) and transferred to a PVDF membrane (Millipore). Membranes were blocked in blocking buffer Tris-Buffered Saline with 0.1% Tween 20 (TBST) containing 5% non-fat dry milk for 1 h at room temperature (RT) and then probed with rabbit monoclonal anti-p-extracellular signal-regulated kinase 1/2 (ERK1/2) antibody (Cell Signaling). Following this, they were probed with anti-rabbit horseradish peroxidase (HRP)-conjugated second antibody (Chemicon) according to manufacturer's protocol. As a loading control, total ERK1/2 (Cell Signaling) was assessed after p-ERK1/2 chemiluminescence detection using HRP-substrate purchased from Cell Signaling.

Internalization assay and fluorescence microscopy

For the internalization assay, HEK293 cells stably expressing PTSPR/SPR–EGFP were seeded in cover glass-bottomed 6-well plates. After treatment with PTSP1/SP peptides at 37°C for 60 min, HEK293 cells were stained with the membrane probe DiI (Beyotime) at 37°C for 5–10 min, fixed with 3.7% paraformaldehyde for 15 min and finally incubated with Hoechst 33258 (Beyotime) for cell nuclei staining for 10 min. The cells were then mounted in a mounting reagent (DTT/PBS/glycerol). Fluorescence microscopy was performed using a Zeiss LSM510 laser scanning confocal microscope attached to a Zeiss Axiovert 200 microscope using a Zeiss Plan-Apo 63×1.40 numerical aperture (NA) oil immersion lens.

Ligand competition binding assay

We used the same buffers for the ligand-binding assay as for the calcium response assay. HEK293 cells stably expressing the FLAG–PTSPR/SPR were detached using non-enzyme cell dissociation buffer and were washed twice with 1 ml of ice cold PBS. Labelled and unlabelled peptides were made up in PBS at a concentration of four times the final concentration specified. We added 100 μl of either labelled ligand with 100 μl of buffer or unlabelled ligand per well. Cells were incubated at 22°C for 90 min and then put on ice. Cells were then washed three times with 500 μl of ice cold standard solution with 0.1% BSA. Binding was determined by measuring the fluorescence intensity with a FlowCytometer (Beckman Coulter). We used the fluorescence-labelled BmPTSP3 or DmSP (100 nM), in the absence of the unlabelled ligand, to determine total binding. Increasing concentrations of synthetic unlabelled BmPTSP1 and DmSP were used for displacement binding analyses. Binding is presented as the percentage of total binding. The binding displacement curves were analysed and the Ki values were determined using GraphPad Prism.

RESULTS

Expression and localization of BmPTSPR/SPR in HEK293 and sf21 cells

BmPTSPR/SPR, a typical seven-transmembrane receptor, was first identified as a functional receptor for the DmSP [14]. To confirm its correct expression and localization in exogenous expression systems, two chimaeras of BmPTSPR/SPR, fused with EGFP in the C-terminal end and with an N-terminal FLAG-tag, were constructed and stably or transiently expressed in HEK293 cells and insect sf21 cells. Confocal microscopy revealed that in HEK293 cells in the absence of the ligand, BmPTSPR/SPR–EGFP was mainly expressed and localized to the plasma membrane but with a certain amount of intracellular accumulation (Figure 1A). Meanwhile, in sf21 cells, BmPTSPR/SPR–EGFP was predominantly distributed in the plasma membrane (Figure 1C). Whole cell ELISA analysis confirmed the significant cell surface expression of BmPTSPR/SPR (Figures 1B and 1D). These data suggests that the fusion expression with FLAG-tag and EGFP has no effect on BmPTSPR/SPR expression and localization in HEK293 and sf21 cells.

Expression of BmPTSPR/SPR in HEK293 and sf21 cells

Figure 1
Expression of BmPTSPR/SPR in HEK293 and sf21 cells

(A and C) HEK293 and sf21 cells expressing BmPTSPR/SPR–EGFP were stained with a membrane plasma probe (Dil) and a nuclei probe (DAPI). The cells stably expressing BmPTSPR/SPR–EGFP were seeded on glass bottom 6-well plates overnight, incubated with DiI (5 μM) and DAPI and examined by confocal microscopy as described in the ‘Materials and Methods’. All images present are representative of at least three independent experiments. (B and D) ELISA analysis of BmPTSPR/SPR expression. HEK293 (B) and sf21 cells (D) were transiently transfected with FLAG-epitope-tagged receptor (right column) or without FLAG-epitope-tagged receptor (left column) and subsequently detected by the M2 anti-FLAG monoclonal antibody via ELISA assay. Values represent the mean ± S.D. (n=3). Statistical significance was determined by using the Student's ttest. ***P<0.001. All experiments were repeated independently three times with similar results.

Figure 1
Expression of BmPTSPR/SPR in HEK293 and sf21 cells

(A and C) HEK293 and sf21 cells expressing BmPTSPR/SPR–EGFP were stained with a membrane plasma probe (Dil) and a nuclei probe (DAPI). The cells stably expressing BmPTSPR/SPR–EGFP were seeded on glass bottom 6-well plates overnight, incubated with DiI (5 μM) and DAPI and examined by confocal microscopy as described in the ‘Materials and Methods’. All images present are representative of at least three independent experiments. (B and D) ELISA analysis of BmPTSPR/SPR expression. HEK293 (B) and sf21 cells (D) were transiently transfected with FLAG-epitope-tagged receptor (right column) or without FLAG-epitope-tagged receptor (left column) and subsequently detected by the M2 anti-FLAG monoclonal antibody via ELISA assay. Values represent the mean ± S.D. (n=3). Statistical significance was determined by using the Student's ttest. ***P<0.001. All experiments were repeated independently three times with similar results.

DmSP directly bound to BmPTSPR/SPR at a different site to BmPTSP

To assess whether BmPTSP1 or DmSP directly binds to BmPTSPR/SPR, a competitive binding assay, using an FAM fluorophore-tagged peptide, was developed. Two N-terminally FAM fluorophore-labelled peptides, FAM–BmPTSP3 and FAM–DmSP, were synthesized. Analysis using a CRE-driven Luc assay demonstrated that FAM–BmPTSP3 and FAM–DmSP could activate BmPTSPR/SPR, leading to inhibition of the FSK-induced Luc activity in HEK293 cells. Our data derived from the functional assay indicated that FAM–BmPTSP3 and FAM–DmSP could activate BmPTSPR/SPR with IC50 values of 234.3 nM and 1.5 nM respectively in the HEK293 cells (Figure 2A). Competitive binding analysis using FAM–BmPTSP3 and FAM–DmSP were performed in HEK293 cells expressing BmPTSPR/SPR. As shown in Figure 2(B), the binding of FAM–BmPTSP3 to BmPTSPR/SPR was competitively displaced by unlabelled BmPTSP1 with an observed inhibition constant (Ki) value of 266.7 nM, but not by unlabelled DmSP and vice versa, FAM–DmSP binding was significantly blocked by DmSP with a Ki value of 1.8 μM, but not by BmPTSP1 (Figure 2C). Confocal microscopy observation also revealed that the binding of FAM–BmPTSP3 to BmPTSPR/SPR was specifically blocked by BmPTSP1, whereas FAM–DmSP binding was significantly blocked by unlabelled DmSP, but not by unlabelled BmPTSP1 (Figures 2D and 2E). This suggests that BmPTSP1 directly binds to BmPTSPR/SPR, but at a different site to that of DmSP binding.

Direct interaction of BmPTSPR/SPR with BmPTSP1 and DmSP

Figure 2
Direct interaction of BmPTSPR/SPR with BmPTSP1 and DmSP

(A) Ligand dosage inhibition of FSK-induced cAMP accumulation through BmPTSPR/SPR. Increasing concentrations of DmSP and FAM–DmSP were added to BmPTSPR/SPR and CRE–Luc expressing HEK293 cells. Results are the mean ± S.D. (n=3). (B and C) Competitive binding analysis. Binding of FAM–BmPTSP3 (B) and FAM–DmSP (C) to BmPTSPR/SPR expressed in HEK293 cells was measured in the absence of unlabelled peptides (total binding) or in the presence of different concentrations of unlabelled BmPTSP1 and DmSP. The extent of binding was determined by fluorescence intensity and is presented as a percentage of the total binding. Values are mean ± S.D. (n=3). The binding displacement curves were analysed and the Ki values were determined using GraphPad Prism software. (D and E) The competition binding of FAM–BmPTSP3 and FAM–DmSP to BmPTSPR/SPR by BmPTSP1 and DmSP were visualized by confocal microscopy. Cells expressing BmPTSPR/SPR were incubated with 100 nM FAM–PTSP3 or FAM–DmSP in the presence of 1 μM unlabelled BmPTSP1 or DmSP for 90 min on ice, followed by wash with ice cold PBS and then detected by confocal microscopy. All experiments were repeated independently at least three times with similar results.

Figure 2
Direct interaction of BmPTSPR/SPR with BmPTSP1 and DmSP

(A) Ligand dosage inhibition of FSK-induced cAMP accumulation through BmPTSPR/SPR. Increasing concentrations of DmSP and FAM–DmSP were added to BmPTSPR/SPR and CRE–Luc expressing HEK293 cells. Results are the mean ± S.D. (n=3). (B and C) Competitive binding analysis. Binding of FAM–BmPTSP3 (B) and FAM–DmSP (C) to BmPTSPR/SPR expressed in HEK293 cells was measured in the absence of unlabelled peptides (total binding) or in the presence of different concentrations of unlabelled BmPTSP1 and DmSP. The extent of binding was determined by fluorescence intensity and is presented as a percentage of the total binding. Values are mean ± S.D. (n=3). The binding displacement curves were analysed and the Ki values were determined using GraphPad Prism software. (D and E) The competition binding of FAM–BmPTSP3 and FAM–DmSP to BmPTSPR/SPR by BmPTSP1 and DmSP were visualized by confocal microscopy. Cells expressing BmPTSPR/SPR were incubated with 100 nM FAM–PTSP3 or FAM–DmSP in the presence of 1 μM unlabelled BmPTSP1 or DmSP for 90 min on ice, followed by wash with ice cold PBS and then detected by confocal microscopy. All experiments were repeated independently at least three times with similar results.

BmPTSPR/SPR was specifically activated by BmPTSP1 and DmSP via PTX-sensitive Gαi-protein

We next sought to ascertain the functionality of BmPTSPR/SPR by assaying cAMP accumulation. HEK293 cells were stably co-transfected with BmPTSPR/SPR and pCRE-Luc and intracellular cAMP levels were determined by assaying Luc activity. As shown in Figures 3(A) and 3(B), upon stimulation with BmPTSP1 or DmSP, BmPTSPR/SPR was activated to induce a significant inhibition of FSK-stimulated Luc activity with IC50 values of 1.5 nM and 3.5 nM respectively. As a control, no change in the Luc activity was detected in mock-transfected HEK293 cells. The agonist-induced inhibition of the FSK-stimulated cAMP accumulation was completely blocked by pre-treatment with 100 ng/ml PTX, an inhibitor of Gαi-protein, in HEK293 cells (Figure 3C). To confirm these results obtained by the CRE-driven Luc assay system, a direct cAMP detection assay by ELISA was used. As illustrated in Figure 3(D), both BmPTSP1 and DmSP stimulation resulted in a significant inhibition of FSK-induced cAMP production in BmPTSPR/SPR-expressing HEK293 cells. Similarly, in sf21 cells co-transfected with the BmPTSPR/SPR and a Gαs-coupled Bombyx AKH receptor (AKHR), BmPTSP1 or DmSP were found to induce a significant inhibition of AKH-stimulated Luc activity (Figure 3E). Taken together, these results demonstrate that activated BmPTSPR/SPR couples to the PTX-sensitive Gαi-protein leading to inhibition of adenylate cyclase activity. BmPTSP1 and DmSP showed comparable potentials in the inhibition of both FSK- and AKH-induced cAMP production.

BmPTSP1- and DmSP-mediated inhibition of FSK-induced cAMP accumulation in BmPTSPR/SPR expressing cells

Figure 3
BmPTSP1- and DmSP-mediated inhibition of FSK-induced cAMP accumulation in BmPTSPR/SPR expressing cells

(A) cAMP accumulation in HEK293 cells transiently co-transfected CRE–Luc with BmPTSPR/SPR or with vehicle vector was determined in response to BmPTSP1 and DmSP stimulation (100 nM). (B) Ligand dosage curve of FSK-induced cAMP accumulation in BmPTSPR/SPR expressing HEK293 cells. cAMP accumulation in HEK293 cells stably expressing BmPTSPR/SPR and pCRE–Luc was determined in response to FSK and ligand. (C) Effects of PTX on peptide-mediated inhibition of cAMP accumulation. HEK293 cells expressing BmPTSPR/SPR and CRE–Luc were pre-treated with PTX (100 ng/ml) overnight prior to incubation with BmPTSP1 (100 nM) and DmSP (100 nM) for 4 h. (D) ELISA analysis of cAMP accumulation in BmPTSPR/SPR expressing cells. cAMP levels in BmPTSPR/SPR expressing HEK293 cells stimulated with DMEM, FSK (10 μM), FSK (10 μM) with BmPTSP1 (100 nM) and FSK (10 μM) with DmSP (100 nM) for 15 min were determined using a commercially available cAMP detection kit. (E) Effects of BmPTSP1 (1 μM) and DmSP (1 μM) on CRE-driven Luc expression induced by AKH1 (1 nM) in sf21 cells transiently co-transfected with pBmFLAG–AKHR, pBmFLAG–PTSPR and pBmCRE–Luc. Values are mean ± S.D. (n=3). All data, except those in (B), were analysed by using the Student's ttest. **P<0.01; ***P<0.001. All experiments were repeated independently at least three times with similar results.

Figure 3
BmPTSP1- and DmSP-mediated inhibition of FSK-induced cAMP accumulation in BmPTSPR/SPR expressing cells

(A) cAMP accumulation in HEK293 cells transiently co-transfected CRE–Luc with BmPTSPR/SPR or with vehicle vector was determined in response to BmPTSP1 and DmSP stimulation (100 nM). (B) Ligand dosage curve of FSK-induced cAMP accumulation in BmPTSPR/SPR expressing HEK293 cells. cAMP accumulation in HEK293 cells stably expressing BmPTSPR/SPR and pCRE–Luc was determined in response to FSK and ligand. (C) Effects of PTX on peptide-mediated inhibition of cAMP accumulation. HEK293 cells expressing BmPTSPR/SPR and CRE–Luc were pre-treated with PTX (100 ng/ml) overnight prior to incubation with BmPTSP1 (100 nM) and DmSP (100 nM) for 4 h. (D) ELISA analysis of cAMP accumulation in BmPTSPR/SPR expressing cells. cAMP levels in BmPTSPR/SPR expressing HEK293 cells stimulated with DMEM, FSK (10 μM), FSK (10 μM) with BmPTSP1 (100 nM) and FSK (10 μM) with DmSP (100 nM) for 15 min were determined using a commercially available cAMP detection kit. (E) Effects of BmPTSP1 (1 μM) and DmSP (1 μM) on CRE-driven Luc expression induced by AKH1 (1 nM) in sf21 cells transiently co-transfected with pBmFLAG–AKHR, pBmFLAG–PTSPR and pBmCRE–Luc. Values are mean ± S.D. (n=3). All data, except those in (B), were analysed by using the Student's ttest. **P<0.01; ***P<0.001. All experiments were repeated independently at least three times with similar results.

BmPTSP exhibited more potential activity in triggering ERK1/2 phosphorylation and Ca2+ mobilization than did DmSP through BmPTSPR/SPR

It is well known that activated GPCRs signal to the mitogen-activated protein kinase (MAPK) cascades via distinct Gαi-, Gαs-, and Gαq-dependent signalling pathways. This then leads to the phosphorylation of ERK1/2. We next investigated whether the activated BmPTSPR/SPR in HEK293 cells signal to the ERK1/2 pathway. HEK293 cells stably expressing BmPTSPR/SPR were seeded in 24-well plates overnight and were treated with the agonist BmPTSP1 or DmSP for the indicated time or with the indicated concentration. Then the cell lysates were assayed using a phospho-specific antibody against the phosphorylated ERK1/2 kinases. As indicated in Figure 4(A), BmPTSP1 and DmSP treatment elicited transient phosphorylation of ERK1/2 with maximal phosphorylation evident at 5 min in BmPTSPR/SPR transfected cells. In contrast, no obvious effects on ERK1/2 were observed in the mock-transfected HEK293 cells (Figure 4C). Analysis of the concentration–response curve showed that BmPTSP1 treatment induced a concentration-dependent activation of ERK1/2 with an EC50 of 6.3 nM, whereas DmSP stimulation triggered a much weaker response in ERK1/2 phosphorylation with an EC50 value of 68 nM (Figure 4B). Furthermore, we examined the effects of both BmPTSP1 and DmSP peptides on the intracellular Ca2+ change in the BmPTSPR/SPR-expressing cells using the calcium probe fura-2-am. As shown in Figure 4(D), stimulation with BmPTSP1 elicited a rapid and transient increase in intracellular Ca2+ mobilization. However, upon exposure to DmSP, a much weaker response in evoking intracellular Ca2+ mobilization was detected. Taken together, these results suggest that BmPTSP1 appears as an agonist with a higher potency in the induction of ERK1/2 activation and intracellular Ca2+ mobilization than does DmSP.

BmPTSP1- and DmSP-mediated ERK1/2 phorsphorylation and Ca2+ mobilization in BmPTSPR/SPR expressing cells

Figure 4
BmPTSP1- and DmSP-mediated ERK1/2 phorsphorylation and Ca2+ mobilization in BmPTSPR/SPR expressing cells

Dose dependence (A) and time course (B) of BmPTSP1- and DmSP-stimulated phosphorylation of ERK1/2 in stable BmPTSPR/SPR expressing HEK293 cells were further tested. (C) ERK1/2 phorsphorylation of HEK293 cells transiently transfected with BmPTSPR/SPR and vehicle vector were treated with 100 nM BmPTSP1 and DmSP for 5 min. The pERK was normalized to a loading control (total-ERK). Data from three independent experiments were analysed and the mean percentage densities ± S.E.M. (n=3 replicates) are shown. In (C), Student's t test was used to compare the basal group and agonist-treated groups. **P<0.01. HEK293 (D) and sf21 (E) cells transfected with BmPTSPR/SPR were measured in response to different peptides using the fluorescent Ca2+ indicator fura-2-am. The Figures are representative of more than three independent experiments.

Figure 4
BmPTSP1- and DmSP-mediated ERK1/2 phorsphorylation and Ca2+ mobilization in BmPTSPR/SPR expressing cells

Dose dependence (A) and time course (B) of BmPTSP1- and DmSP-stimulated phosphorylation of ERK1/2 in stable BmPTSPR/SPR expressing HEK293 cells were further tested. (C) ERK1/2 phorsphorylation of HEK293 cells transiently transfected with BmPTSPR/SPR and vehicle vector were treated with 100 nM BmPTSP1 and DmSP for 5 min. The pERK was normalized to a loading control (total-ERK). Data from three independent experiments were analysed and the mean percentage densities ± S.E.M. (n=3 replicates) are shown. In (C), Student's t test was used to compare the basal group and agonist-treated groups. **P<0.01. HEK293 (D) and sf21 (E) cells transfected with BmPTSPR/SPR were measured in response to different peptides using the fluorescent Ca2+ indicator fura-2-am. The Figures are representative of more than three independent experiments.

BmPTSPR/SPR underwent internalization upon activation by BmPTSP1

Upon agonist binding, GPCRs are rapidly internalized from the cell surface to the cytoplasm. This is a key process for regulating the strength and duration of receptor-induced signalling. To visualize the internalization and trafficking of BmPTSPR/SPR, an expression vector was constructed to express a chimaeric protein in which EGFP was fused to the C-terminal end of BmPTSPR/SPR and stably or transiently transfected into HEK293 and sf21 cells. Functional assays demonstrated that BmPTSPR/SPR–EGFP was shown to inhibit the FSK-stimulated Luc activity, as compared with the wild-type BmPTSPR/SPR, in response to BmPTSP1. This suggests that BmPTSPR/SPR–EGFP functions as normal as wild-type BmPTSPR/SPR (Figure 5A).

Internalization of BmPTSPR/SPR in response to BmPTSP1 and DmSP in HEK293 cells

Figure 5
Internalization of BmPTSPR/SPR in response to BmPTSP1 and DmSP in HEK293 cells

(A) The dose-dependent inhibition of FSK-induced cAMP accumulation in wild-type BmPTSPR/SPR and BmPTSPR/SPR–EGFP expressing cells. Values are mean ± S.D. (n=3). (B) HEK293 cells transiently transfected with FLAG–BmPTSPR/SPR and BmKurtz were stimulated with 1 μM BmPTSP1 and DmSP for the indicated time periods and the internalization of BmPTSPR/SPR was determined by ELISA. Values are given as mean ± S.D. (n=4) and are analysed by using Student's t test. (C) Confocal microscopy analysis of the internalization of BmPTSPR/SPR–EGFP initiated by BmPTSP1 and DmSP. HEK293 cells transiently transfected with BmPTSPR/SPR–EGFP and BmKurtz were stimulated with 1 μM BmPTSP1 and DmSP for 45 min. All pictures shown are representative of at least three independent experiments. All experiments were repeated at least three times on different days using different batches of cells.

Figure 5
Internalization of BmPTSPR/SPR in response to BmPTSP1 and DmSP in HEK293 cells

(A) The dose-dependent inhibition of FSK-induced cAMP accumulation in wild-type BmPTSPR/SPR and BmPTSPR/SPR–EGFP expressing cells. Values are mean ± S.D. (n=3). (B) HEK293 cells transiently transfected with FLAG–BmPTSPR/SPR and BmKurtz were stimulated with 1 μM BmPTSP1 and DmSP for the indicated time periods and the internalization of BmPTSPR/SPR was determined by ELISA. Values are given as mean ± S.D. (n=4) and are analysed by using Student's t test. (C) Confocal microscopy analysis of the internalization of BmPTSPR/SPR–EGFP initiated by BmPTSP1 and DmSP. HEK293 cells transiently transfected with BmPTSPR/SPR–EGFP and BmKurtz were stimulated with 1 μM BmPTSP1 and DmSP for 45 min. All pictures shown are representative of at least three independent experiments. All experiments were repeated at least three times on different days using different batches of cells.

In HEK293 cells expressing BmPTSPR/SPR–EGFP, observations using confocal microscopy revealed that the fluorescence of BmPTSPR/SPR–EGFP was primarily localized in the plasma membrane with certain intracellular accumulation, independent of the ligand. BmPTSP1 (1 μM) could induce significant internalization of BmPTSPR/SPR–EGFP but hardly any internalization was observed upon DmSP stimulation at high concentrations (Figure 5C). The quantitative ELISA data were highly consistent with the confocal microscopy observations (Figure 5B). Similar results were obtained in sf21 cells (Figure 6A). Furthermore, Kurtz, a novel non-visual arrestin identified in insects, was cloned from B. mori and a fusion expression of BmKurtz with EGFP at the C-terminal end was constructed. Sf21 and HEK293 cells co-transfected with BmPTSPR/SPR and BmKurtz–EGFP or β-arrestin1/2–EGFP were then exposed to BmPTSP1 and examined using confocal microscopy. As shown in Figure 6(B), in the presence of BmPTSP1, BmKurtz–EGFP was significantly translocated to the plasma membrane in sf21 cells, whereas no BmKurtz–EGFP was recruited to the plasma membrane upon stimulation of DmSP. No β-arrestin1–EGFP and β-arrestin2–EGFP were recruited to the plasma membrane upon stimulation of BmPTSP1 and DmSP in HEK293 cells (result not show). The sf21 cells co-transfected with BmPTSPR/SPR–EGFP and BmKurtz were treated with different concentrations of BmPTSP1 for 45 min or with 1 μM BmPTSP1 and for different periods. The BmPTSPR/SPR–EGFP receptor was internalized in a dose- and time-dependent manner upon stimulation with BmPTSP1 (Figures 6C and 6D). These results suggest that BmPTSP1 appears to be an endogenous agonist to potently induce the recruitment of Kurtz to the activated BmPTSPR/SPR, leading to its internalization.

Internalization and recruitment of BmKurtz in BmPTSPR/SPR expressing sf21 cells

Figure 6
Internalization and recruitment of BmKurtz in BmPTSPR/SPR expressing sf21 cells

(A) sf21 cells transiently transfected with BmPTSPR/SPR–EGFP and BmKurtz were stimulated with 1 μM BmPTSP1 and DmSP for 45 min and the internalization of BmPTSPR/SPR–EGFP was observed. (B) The BmPTSP1- and DmSP-mediated recruitment of BmKurtz in BmPTSPR/SPR expressing cells was determined by confocal microscopy. Sf21 cells were transiently expressed with BmKurtz–EGFP and BmPTSPR/SPR, stimulated with 1 μM BmPTSP1 and DmSP for 15 min and then detected by confocal microscopy. (C) BmPTSPR/SPR and BmKurtz transfected sf21 cells were treated with 1 μM BmPTSP1 for the indicated time periods. (D) sf21 cells expressing BmPTSPR/SPR and BmKurtz were treated with various concentrations of BmPTSP1 for 45 min. All data shown are representative examples from three independent experiments.

Figure 6
Internalization and recruitment of BmKurtz in BmPTSPR/SPR expressing sf21 cells

(A) sf21 cells transiently transfected with BmPTSPR/SPR–EGFP and BmKurtz were stimulated with 1 μM BmPTSP1 and DmSP for 45 min and the internalization of BmPTSPR/SPR–EGFP was observed. (B) The BmPTSP1- and DmSP-mediated recruitment of BmKurtz in BmPTSPR/SPR expressing cells was determined by confocal microscopy. Sf21 cells were transiently expressed with BmKurtz–EGFP and BmPTSPR/SPR, stimulated with 1 μM BmPTSP1 and DmSP for 15 min and then detected by confocal microscopy. (C) BmPTSPR/SPR and BmKurtz transfected sf21 cells were treated with 1 μM BmPTSP1 for the indicated time periods. (D) sf21 cells expressing BmPTSPR/SPR and BmKurtz were treated with various concentrations of BmPTSP1 for 45 min. All data shown are representative examples from three independent experiments.

DISCUSSION

BmPTSPR/SPR was first identified as a receptor functionally activated by the DmSP and D. melanogaster ductus ejaculatorius peptide 99B (DmDUP99B) with EC50 values of 63 nM and 261 nM in CHO cells respectively [14]. Previously, using a heterologous expression system, the BmPTSP has been found to be the endogenous ligand for this receptor and was therefore named BmPTSPR/SPR [5]. In the present study, we functionally assessed this receptor with synthetic BmPTSP and DmSP using the mammalian cell line HEK293 and insect cell line sf21 which had been stably or transiently transfected with BmPTSPR/SPR. Our data clearly showed that both BmPTSP1 and DmSP exhibited a comparable potential to activate BmPTSPR/SPR, leading to a significant inhibition of FSK-induced cAMP accumulation with IC50 values of 1.5 nM and 3.5 nM respectively. However, BmPTSP1 displayed a much stronger activity in evoking intracellular Ca2+ mobilization, ERK1/2 phosphorylation, internalization and Kurtz recruitment than did DmSP. Further investigation using the synthetic BmPTSP3 and DmSP peptides tagged with FAM fluorophore at the N-terminal end demonstrated that the binding of FAM–BmPTSP3 and FAM–DmSP to BmPTSPR/SPR was displaced by unlabelled BmPTSP1 and DmSP respectively, suggesting the existence of different binding sites for BmPTSP and DmSP. In addition, in DmSP, a 36-amino-acid peptide responsible for induction of the female post-mating response has been found to be well conserved [1618]. However, this peptide had never previously been identified in the Bombyx genome [5,19]. Previously, DmSP has been reported to activate methuselah, a GPCR associated with longevity in D. melanogaster [20]. This suggests that DmSP appears as a promiscuous agonist for different insect GPCRs. Taken together, these results suggest that the neuropeptide BmPTSP is a specific endogenous ligand for BmPTSPR/SPR. In this case this B. mori neuropeptide receptor is better designated as the BmPTSPR.

BmPTSPR has been long identified to be activated by DmSP and BmPTSP. However, the details of its signalling have thus far only been obtained from heterologous expression systems using mammalian cell lines. In CHO cells co-transfected with chimaeric G-protein Gαqi or Gαqo and the Ca2+ reporter aequorin, BmPTSPR was specifically activated by DmSP and DmDUP99B, leading to robust Ca2+ responses via cAMP pathway into the Ca2+ pathway [14]. Yamanaka et al. [5] demonstrated that BmPTSP stimulation triggered a rise in intracellular Ca2+ mobilization in HEK293 cells co-expressing the promiscuous G-protein Gα15 and BmPTSPR [5]. Using in vitro cAMP assays and ecdysone assays, PTSP has been demonstrated to inhibit both basal and PTTH-induced cAMP accumulation and ecdysone synthesis in the PGs in a dose-dependent manner [6]. In the present study, we established a CRE-driven-Luc assay to analyse intracellular cAMP accumulation in HEK293 cells and sf21 cells expressing BmPTSPR in response to BmPTSP1 and DmSP. Our data demonstrate that BmPTSPR, as expressed in both mammalian HEK293 cells and insect sf21 cells, is specifically activated by BmPTSP and DmSP. Such an activation results in significant inhibition of FSK- or AKH-stimulated CRE-driven Luc activity, an effect that can be reversed via treatment with PTX. This result was also confirmed by direct quantitative analysis of intracellular cAMP using ELISA. Previous studies in Manduca, also showed that the intracellular cAMP level becomes elevated when ecdysteroidogenesis is increased in the PG [21,22]. Therefore, it is more likely that BmPTSPR exerts a regulatory action on ecdysteroidogenesis via Gαi/o-protein-mediated signalling cascades.

We further assessed BmPTSPR-mediated intracellular Ca2+ mobilization and ERK1/2 phosphorylation in both mammalian and insect cells. In BmPTSPR expressing HEK293 and sf21 cells, the addition of the BmPTSP and DmSP peptide evoked a rapid increase in intracellular Ca2+ in a dose-dependent manner. However, BmPTSP exhibited higher activity in inducing intracellular Ca2+ mobilization than did DmSP. This was consistent with the previous observations that even with 10 μM DmSP stimulation, only a very weak response in Ca2+ mobilization was detected in the DmSPR expressing cells [14] whereas a significant increase in intracellular Ca2+ was detected in the BmPTSPR expressing cells when treated with BmPTSPs [5]. In addition, phosphorylation of ERK is known to be physiologically important in GPCR-mediated control of different cell functions. In the present study, despite BmPTSP and DmSP exhibiting comparable time frames to induce a robust ERK1/2 phosphorylation in a time-dependent fashion (with a maximal activation at 5 min and with a subsequent reduction to base line by 15 min), BmPTSPR and BmPTSP1 showed 10-fold higher potency (EC50 ≈ 10 nM) to trigger ERK1/2 activation than did DmSP (EC50 ≈ 100 nM). This was consistent with the observation of BmPTSPR-mediated Ca2+ mobilization. These results suggest that DmSP is equally potent in BmPTSPR-mediated inhibition of FSK- or AKH-stimulated cAMP formation as BmPTSP1, but 10-fold less potent in BmPTSPR-induced intracellular Ca2+ mobilization and ERK1/2 activation than is BmPTSP1.

Activated GPCRs are well known to be phosphorylated at the C-terminal tail by GPCR kinase (GRK), followed by arrestin binding. This results in rapid receptor internalization [23,24]. Using a fusion expression of BmPTSPR with EGFP at the carboxyl terminus for easy detection under confocal microscopy, we demonstrated that BmPTSP, but not DmSP, promoted a rapid internalization of BmPTSPR from the cell membrane into the cytoplasm in a concentration- and time-dependent manner. This observation was confirmed by quantitative ELISA analysis. The human arrestin–GFP recruitment assay was first successfully used to identify endogenous ligands for Drosophila orphan GPCRs in 2003 [25]. Later it was noted that there were no observable differences between the receptor–Kurtz interactions and the receptor–β-arrestin2 associations [26]. Our own previous study showed that Bombyx Kurtz, a novel non-visual arrestin, cloned from B. mori, behaves more similarly to the mammalian β-arrestin2 in the regulation of Bombyx mori corazonin receptor (BmCrzR) internalization than to the mammalian β-arrestin1 [27]. However, in sf21 and HEK293 cells co-transfected with BmPTSPR/SPR and BmKurtz–EGFP or with β-arrestin1/2–EGFP, it was BmKurtz–EGFP, but not β-arrestin1–EGFP and β-arrestin2–EGFP, which was significantly recruited to the plasma membrane in the response to BmPTSP. In contrast, DmSP treatment elicited neither recruitment of BmKurtz nor translocation of β-arrestin1 or β-arrestin2 into the cell membrane. Our data indicate that BmPTSP appears to be an endogenous ligand to potently induce the internalization of BmPTSPR and to act as a biased agonist, whereas DmSP exhibits no activity in induction of Kurtz recruitment and receptor internalization. On the other hand, Bombyx Kurtz can serve as a useful tool for the characterization of insect GPCR internalization and signalling.

In conclusion, in the current study, we functionally characterized BmPTSPR/SPR using the synthetic neuropeptides BmPTSP and DmSP in both mammalian and insect cells. Based on our results, it is more likely that BmPTSP is an endogenous ligand specific to BmPTSPR and that DmSP binds to BmPTSPR at an allosteric site, leading to a Gαi/o-protein-biased signalling pathway but without interfering with Ca2+ mobilization and β-arrestin recruitment. As an allosteric and biased agonist, DmSP might be a useful tool to use for further elucidation of signalling mechanism(s) of BmPTSPR involved in the regulation of insect ecdysteroidogenesis and metamorphosis.

Abbreviations

     
  • AKH

    adipokinetic hormone

  •  
  • AKHR

    AKH receptor

  •  
  • BmPTSP

    Bombyx mori prothoracicostatic peptide

  •  
  • BmPTSPR

    BmPTSP receptor

  •  
  • CHO

    Chinese hamster ovary

  •  
  • CRE

    cAMP-response element

  •  
  • DmDUP99B

    Drosophila melanogaster ductus ejaculatorius peptide 99B

  •  
  • DmSP

    Drosophila melanogaster sex peptide

  •  
  • ERK

    extracellular signal-regulated kinase

  •  
  • FSK

    forskolin

  •  
  • fura-2-am

    fura-2-acetoxymethyl ester

  •  
  • GPCR

    G-protein coupled-receptor

  •  
  • HEK

    human embryonic kidney

  •  
  • HRP

    horseradish peroxidase

  •  
  • Luc

    luciferase

  •  
  • PG

    prothoracic gland

  •  
  • PTSP

    prothoracicostatic peptide

  •  
  • PTTH

    prothoracicotropic hormone

  •  
  • PTX

    pertussis toxin

  •  
  • sf21

    Spodoptera frugiperda 21

  •  
  • SP

    sex peptide

  •  
  • SPR

    SP receptor

AUTHOR CONTRIBUTION

Xiaobai He, Jiashu Zang and Huipeng Yang performed the research. Xiaobai He and Haishan Huang analysed the data. Naiming Zhou and Xiaobai He designed the research and wrote the paper. Ying Shi directed the project.

The authors of this paper would like to thank Aiping Shao, Ming Ding and Hanmin Chen for their technical assistance and the use of their equipment.

FUNDING

This work was supported by the National Natural Science Foundation of China [grant numbers 31172150 and 31301881]; and the Ministry of Science and Technology of China [grant number 2012CB910402].

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