Abstract
G-protein-coupled receptors (GPCRs) are involved in animal steroid hormone signaling, but their mechanism is unclear. In this research, we report that a GPCR called ErGPCR-2 controls steroid hormone 20-hydroxyecdysone (20E) signaling in the cell membrane of the lepidopteran insect Helicoverpa armigera. ErGPCR-2 was highly expressed during molting and metamorphosis. 20E, via ErGPCR-2, regulated rapid intracellular calcium increase, protein phosphorylation, gene transcription and insect metamorphosis. ErGPCR-2 was located in the cell surface and was internalized by 20E induction. GPCR kinase 2 participated in 20E-induced ErGPCR-2 phosphorylation and internalization. The internalized ErGPCR-2 was degraded by proteases to desensitize 20E signaling. ErGPCR-2 knockdown suppressed the entrance of 20E analog [3H] ponasterone A ([3H]Pon A) into the cells. ErGPCR-2 overexpression or blocking of ErGPCR-2 internalization increased the entrance of [3H]Pon A into the cells. However, ErGPCR-2 did not bind to [3H]Pon A. Results suggest that ErGPCR-2 transmits steroid hormone 20E signaling and controls 20E entrance into cells in the cell membrane.
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Introduction
Animal steroid hormones, such as mammal estrogen1 and insect 20-hydroxyecdysone (20E)2, exert their actions via the genomic pathway, wherein hormones fuse into cells and bind to intracellular nuclear receptors, which then bind to DNA to initiate gene transcription3. Recent studies suggest that animal steroid hormones can activate receptors in the cell membrane to initiate rapid nongenomic interactions, such as rapid cellular calcium increase4. G-protein-coupled receptors (GPCRs) are proposed as membrane receptors of animal steroid hormones. For example, GPCR 30 (GPR30/GPER) in the cell membrane binds estrogen and mediates rapid intracellular calcium mobilization in humans5. In Drosophila, the dopamine receptor DmDopEcR binds the 20E analog and is proposed as a 20E membrane receptor6. In Bombyx mori, 20E via unknown GPCRs increases the intracellular Ca2+ level in the anterior silk gland7. In Helicoverpa armigera, 20E induces rapid protein phosphorylation and nuclear translocation of calponin8, cell membrane trafficking of Rab4b9 and nuclear translocation of heat shock cognate protein Hsc7010. Further studies reveal that 20E via GPCRs and Ca2+ signaling regulates rapid phosphorylation of cyclin-dependent kinase 10 (CDK10)11. 20E via phospholipase C-gamma-1 (PLCG1) regulates heterodimeric partner (USP1) phosphorylation and connects the GPCR-mediated nongenomic pathway and the nuclear receptor EcRB1-mediated genomic pathway12. A GPCR called ErGPCR (renamed as ErGPCR-1 to distinguish it from ErGPCR-2) participates in 20E signaling in the cell membrane to regulate calcium increase, protein phosphorylation and subcellular translocation, gene transcription and metamorphosis in H. armigera13. These data suggest the functions of GPCRs in steroid hormone signaling.
GPCRs, which belong to the seven-transmembrane protein family, are involved in signal transduction across cell membranes14. Signal transduction via GPCRs is fundamental for mediating various cellular responses to changes in the extracellular environment15. Different GPCRs exhibit diverse amino acid sequences; however, most GPCRs show similar mechanisms of desensitization by internalization and resensitization by recycling to the cell membrane16. GPCR desensitization17 is regulated by GPCR kinase (GRK) mediating phosphorylation and internalization of GPCRs18. After internalization, GPCRs can be trafficked to lysosomes for degradation or recycled back to the cell surface for resensitization in another round of signaling19. However, animal steroid hormone-induced GPCR internalization remains poorly understood. In this study, we discovered an ecdysone-responsive GPCR (ErGPCR-2), which transmits steroid hormone 20E signaling and controls steroid hormone 20E entrance into the cells. Under 20E stimulation, GRK2 phosphorylates the C-terminus of ErGPCR-2 to regulate ErGPCR-2 internalization. The internalized ErGPCR-2 is then degraded by proteases, which desensitize 20E signaling. ErGPCR-2 participates in 20E signal transmission in the cell membrane for further gene expression and metamorphosis.
Results
ErGPCR-2 is upregulated during 20E-regulated molting and metamorphosis
To study the function of ErGPCR-2, the tissue specificity and developmental expression profiles of ErGPCR-2 were examined. The results showed that ErGPCR-2 was expressed in the midgut, fat body and epidermis. In these three tissues, ErGPCR-2 protein and mRNA exhibited higher expression levels at the fifth instar molting (5M) and metamorphic stages (sixth instar 72 h to 120 h) than at the fifth instar feeding (5 h to 24 h) and sixth instar feeding stages from 6–0 h to 6–48 h (Figures 1A, 1B and 1C). The expression level of ErGPCR-2 was upregulated by 20E injection into the sixth instar 6 h larvae but was unaffected by JH III or Dimethyl sulfoxide DMSO injection (Figures 1D and 1E). These results indicate that ErGPCR-2 expression is upregulated by 20E during molting and metamorphosis.
Expression profile and hormonal regulation of ErGPCR-2.
(A). Western blot with antibody against ErGPCR-2 (specificity of antibody is shown in Supplement Files: Figure S1). (B). Calculation of A according to three independent replicates by ImageJ software (National Institutes of Health, http://imagej.nih.gov/ij/download.html). (C). qRT-PCR. 5F: fifth instar 12 h feeding larvae; 5M: fifth instar 36 h molting larvae; 6-0 h, 6-24 h, 6-48 h, 6-72 h, 6-96 h and 6-120 h: sixth instar larvae from 0 h to 120 h; P-0: zero-day-old pupae. F: feeding; M: molting; MM: metamorphic molting; P: pupae. (D). Western blot analysis of hormonal regulation on ErGPCR-2 in larvae. 20E or JH III (500 ng/larva) was injected into the 6th instar 6 h larvae for 1 h to 24 h. Equal volume of DMSO was injected as control. (E). Statistical analysis of (D) according to three independent replicates by ImageJ software. *Significant differences (P values) were statistically analyzed by Student's t-test. SDS-PAGE gel in Western blot is 12.5%. β-actin was used as control.
ErGPCR-2 participates in 20E-induced metamorphosis
To examine the function of ErGPCR-2 in 20E-regulated metamorphosis, we injected the dsRNA of ErGPCR-2 into the sixth instar 6 h larval hemocoel to knock down ErGPCR-2, followed by 20E induction. The larvae pupated earlier than the DMSO control after injection with 20E alone or dsGFP plus 20E. By contrast, the larvae died before pupation or delayed pupation 37 h after injection with dsErGPCR-2 plus 20E (Figures 2A and 2B). Up to 19% of the larvae died and 81% delayed pupation following ErGPCR-2 knockdown (Figures 2C and 2D). Furthermore, transcript levels of 20E-response genes, including ecdysone nuclear receptor EcRB13, heterodimeric partner USP1 and transcription factors BR-Z7 and HHR320, decreased (Figure 2E). In HaEpi cells21, ErGPCR-2 knockdown also blocked 20E-induced gene expression (Figure 2F). These results suggest that ErGPCR-2 participates in 20E-regulated gene expression and metamorphosis.
ErGPCR-2 silencing represses metamorphosis by repressing 20E response gene expression.
(A). Phenotypes after ErGPCR-2 knockdown (500 ng/larva, thrice at an 18 h interval) and 20E induction (500 ng/larva). Images were obtained at six instar larvae 120 h according to DMSO control group. Scale bar = 1 cm. (B). Statistical analysis of pupation time from 6th instar 0 h larvae developing to pupae 6th 0 h to pupation in (A). (C). Percentages of the phenotype in (A). (D) and (d). Western blot showing the efficacy of ErGPCR-2 knockdown, analyzed by ImageJ software. (E) and (F). qRT-PCR showing mRNA levels of 20E response genes after ErGPCR-2 knockdown in larvae at 6th 72 h in the above treatment and in HaEpi cells (dsRNA 2 μg/mL, 12 h twice, 1 μM 20E for 12 h). β-actin was used as control. Asterisks indicate significant differences (*P value) using Student's t-test based on three replicates, n = 30 × 3 in larvae and n = 3 in the cells. Off-target effect was excluded by examination of another GPCR named ErGPCR-1 (Supplement Files: Figures S2A and B). The HaEpi cell shape was unchanged after incubation with 20E or ErGPCR-2 knockdown (Supplement Files: Figure S2C).
ErGPCR-2 participates in 20E-induced rapid reactions and gene transcription
20E, via GPCRs, induces rapid increase in cellular calcium and phosphorylation of transcription complex proteins USP1 and CDK10 to activate gene transcription12. Thus, the function of ErGPCR-2 in these cascades was detected in HaEpi cells. 20E induced intracellular calcium release and extracellular calcium influx in normal cells (Figure 3A). However, ErGPCR-2 knockdown repressed the 20E-induced intracellular calcium release and the extracellular calcium influx (Figure 3B), suggesting that ErGPCR-2 is involved in 20E-induced calcium increase. The T-type voltage-gated calcium channel inhibitor flunarizine dihydrochloride (FL)22 and the transient receptor potential calcium 3 (TRPC3) channel inhibitor pyrazole compound (Pyr3)23 blocked the calcium influx but not the calcium release (Figure 3C). The intracellular Ca2+-ATPases inhibitor thapsigargin (TG), which depletes the stored intracellular calcium24, repressed the intracellular calcium release and extracellular calcium influx, but did not block extracellular calcium influx in 20E induction (Figure 3C). The GPCR inhibitor suramin blocked both intracellular calcium release and extracellular Ca2+ influx. However, the receptor tyrosine kinase (RTK) inhibitor SU666825 affected neither intracellular calcium release nor extracellular calcium influx (Figure 3D). These results suggest that 20E via ErGPCR-2 induces cellular Ca2+ increase and various calcium channels are involved in this process.
ErGPCR-2 is involved in 20E-induced rapid mobilization of Ca2+ in HaEpi cells.
(A). 20E-induced cytosolic Ca2+ levels increase. AM ester calcium crimson™ dye 3 μM, 20E 1 μM, CaCl2 1 mM, equal volume of DMSO as solvent control. Fluorescence was recorded using a Confocal Microscope at 555 nm and then analyzed using Image Pro-Plus software. F: fluorescence of cells after treatment; F0: average fluorescence of cells before treatment. (B). Effect of the ErGPCR-2 knockdown by dsRNA (2 μg/mL) on the Ca2+ levels. (C). Inhibition of 20E-induced increase in cytosolic Ca2+ levels. FL: T-type calcium channel blocker FL (50 μM); Pyr3: the TRPC3 channel inhibitor (10 μM); and TG: Thapsigargin (2 μM) were added to the medium 30 min before 20E induction. (D). RTK inhibitor SU6668 (5 μM) and suramin (50 μM) were added to the medium 30 min before 20E induction.
Moreover, 20E induced USP1 and CDK10 phosphorylation. By contrast, lambda protein phosphatase (λPPase) treatment degraded USP1 and CDK10 phosphorylation. ErGPCR-2 knockdown repressed 20E-induced USP1 and CDK10 phosphorylation (Figures 4A and 4B), which are essential for the formation of EcRB1/USP1 transcription complex to initiate gene transcription in 20E signaling11,12. These results suggest that ErGPCR-2 is involved in 20E-induced rapid cellular reactions. 20E induced USP1 interaction with EcRB1 to form EcRB1/USP1 transcription complex, thereby initiating gene transcription in 20E signaling3. USP1 and CDK10 are related to 20E-induced transcription11. 20E-induced transcription activity was also decreased by ErGPCR-2 knockdown and reflected by the expression levels of red fluorescence protein using pIEx-HR3pro-RFP report plasmid12, which contains 20E-response element (EcRE), the DNA element that EcR binds to initiate gene transcription26, from Helicoverpa hormone receptor 3 (HR3) and red fluorescence protein (RFP) as reporter (Figures 4C). Overexpression of 7TM-GFP also significantly increased 20E-induced gene expression (Figure 4D).
ErGPCR-2 is involved in 20E-induced rapid reactions and gene transcription in HaEpi cells.
(A) and (B). Western blot analysis 20E-induced phosphorylation of USP1-His and CDK10 (1 μM 20E for 1 h). USP1-His-P: overexpressed phosphorylated USP1-His detected using an anti-His-tag antibody; CDK10-P: phosphorylated CDK10 detected using the anti-CDK10. λPP: protein was incubated with 0.5 μM λPPase at 30 °C for 30 min. SDS-PAGE gel in Western blot was 7.5%. (C). Effect of ErGPCR-2 knockdown on 20E-induced transcription activity. Cells were transfected with pIEx-HR3pro-RFP-His plasmid (2.5 μg/mL, 24 h), inducted with 1 μM 20E for 18 h. The images were statistically analyzed by ImageJ software. (D). The effects of ErGPCR-2-7TM overexpression on 20E-induced gene expression, analyzed by qRT-PCR. β-actin was used as quantitative control. (E). ErGPCR-2 regulates EcRB1 binding to EcRE during 20E induction. (a). ChIP analysis by qRT-PCR detecting the EcRE fragment from the precipitates by anti-RFP under 20E treatment. Cells were transfected with plasmid of pIEx-4-EcRB1-RFP (3 μg/mL) and then treated with DMSO or 20E (1 μM) for 6 h. No antibody was used as the negative control. (b). ErGPCR-2 depletion decreased the 20E-induced EcRB1 binding to EcRE. Cells were transfected with pIEx-4-EcRB1-RFP (b) or pIEx-4-RFP (c) for 24 h. The cells were treated with dsErGPCR-2 (2 μg/mL) or dsGFP (2 μg/mL) for 12 h and then induced by 1 μM 20E or DMSO for 6 h. Western blots indicate protein expression levels. (d). qRT-PCR detected the efficacy of ErGPCR-2 knockdown. Input: The positive control of non-immunoprecipitated chromatin. In all experiments, *P value via Student's t-test based on three replicates.
ChIP experiments were performed to further examine the mechanism of 20E regulates gene transcription through ErGPCR-2. 20E regulates EcRB1/USP1 heterodimer binding to ecdysone response element (EcRE) to regulate gene transcription26. The 5′ regulatory region of Helicoverpa HR3 (HHR3), which contains EcRE (GGGGTCAATGAACTG), was cloned11. The EcRE level was significantly higher by qRT-PCR detection in the immunoprecipitates produced by anti-RFP antibody, which precipitated EcRB1-RFP and the bond EcRE, in 20E induction in the EcRB1-RFP expressing cells (Figure 4E, a). However, in the EcRB1-RFP expressing cells, the qRT-PCR product significantly decreased by 20E induction after knockdown of ErGPCR-2, compared with the dsGFP treatment control (Figure 4E, b). The RFP expression cells were used as negative control (Figure 4E, c) and the efficacy of ErGPCR-2 knockdown is showed in Figure 4E, d. These results from ChIP experiments suggest that 20E via ErGPCR-2 regulates EcRB1 binding to EcRE to regulate the 20E-induced gene transcription.
ErGPCR-2 is localized in the cell membrane and partially internalized by 20E induction
The subcellular location of ErGPCR-2 was analyzed to observe its rapid response to 20E induction. ErGPCR-2 protein was mainly localized in the cell membrane. However, ErGPCR-2 was internalized into the cytoplasm within 15 min of 20E treatment (Figure 5A). Western blot analysis confirmed the internalization of ErGPCR-2 by 20E induction. The molecular mass of ErGPCR-2 in the cytoplasm was also higher than that in the cell membrane (Figure 5B). The increased molecular mass was decreased by lambda protein phosphatase (λPP) (Figure 5C), which probably resulted from protein phosphorylation.
20E regulates ErGPCR-2 phosphorylation and internalization in HaEpi cells.
(A). 20E treatment (1 μM, 15 min). Green: ErGPCR-2 protein stained with an anti-ErGPCR-2 and secondary antibody labeled with Alexa 488. Red: plasma membrane detected with Alexa Fluor 594-conjugated wheat germ agglutinin (WGA). Blue: nucleus stained with 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI). Observed by confocal microscope. (B). and (C). Western blot showing the subcellular distribution and phosphorylation of ErGPCR-2 as the treatment in (A). Gel concentration is 7.5%. M: Membrane protein; Cy: Cytoplasm protein. λPP: λ protein phosphates (5 μM, 30 min at 30 °C). (D). and (E). Mutation and overexpression of ErGPCR-2. Green and blue are the same as those in (A). (F). Degradation of ErGPCR-2. (a), (b), (c) and (d) indicates DMSO: solvent control; 20E 15 min: 20E 2 μM for 15 min; withdrawal 20E: culture 2 h after withdrawal of 20E; inhibitors: anisomycin (10 μM), proteases inhibitor PMSF (1 mM). Green and blue are the same as those in (A). (G). Western blot showing the relative levels of ErGPCR-2 in membrane and cytoplasm after the treatments in (F). Coomassie Brilliant Blue staining was used as loading control for membrane protein quantity and quality. M: membrane; Cy: cytoplasm. Scale bar = 25 μm.
To confirm the 20E-induced internalization of ErGPCR-2, the 7TM (amino acid 359–757, 7TM-GFP), the C-terminus deletion (aa 701–757) of 7TM (7TMΔC-terminal-GFP) and the second extracellular region deletion (aa 565–595) of 7TM (7TMΔe2loop-GFP) (Figure 5D) were overexpressed and fused with green fluorescence protein (GFP). The overexpressed GFP alone was distributed in all the cells without location variation by 20E induction. By contrast, 7TM-GFP was localized in the cell membrane and can be internalized in 15 min by 20E induction. However, deletion of the second extracellular loop caused 7TMΔe2loop-GFP to lose its cell membrane-locating capability. When the C-terminus of 7TM was deleted, the 7TMΔC-terminal-GFP was not internalized from the cell membrane by 20E induction (Figure 5E). These results suggest that the C-terminus of ErGPCR-2 determines the internalization of ErGPCR-2.
To elucidate the fate of ErGPCR-2 after internalization into the cytosol, the subcellular location of ErGPCR-2 was examined following anisomycin (protein translation inhibitor) and PMSF (protein degradation inhibitor). ErGPCR-2 was localized in the cell membrane in DMSO control and was internalized in 15 min by 20E induction. With the exception of those located in the cell membrane, the amount of ErGPCR-2 in the cytosol decreased 2 h after withdrawal of 20E, following the incubation of cells in the inhibitor-free medium. By contrast, the amount of ErGPCR-2 in the cytosol was maintained 2 h after withdrawal of 20E when the protein translation inhibitor and proteases were added into the medium (Figure 5F). Western blot confirmed the degradation of ErGPCR-2 in the inhibitor-free medium, as well as its presence, with the addition of the said inhibitors in the medium (Figure 5G). These results suggest that ErGPCR-2 is not recycled in the cell membrane but is degraded after internalization into the cytosol by 20E induction.
GRK2 regulates phosphorylation and internalization of ErGPCR-2
Given that GRK serves an important function in GPCR phosphorylation and GPCR endocytosis27, we detected the function of GRK2 in 20E-induced ErGPCR-2 phosphorylation and internalization. Immunocytochemical analysis showed that ErGPCR-2 was internalized after 15 min of 20E induction in dsGFP control cells. However, ErGPCR-2 was not internalized after GRK2 knockdown (Figure 6A). Western blot analysis confirmed that ErGPCR-2 was partially phosphorylated and internalized into the cytoplasm by 20E induction in dsGFP-treated cells; however, ErGPCR-2 was kept nonphosphorylated in the cell membrane and could not be internalized by 20E induction in the dsGRK2-treated cells (Figure 6B). GRK2 was also co-precipitated by antibodies against ErGPCR-2 from the 20E-injected larval midgut, but not from DMSO- and JH III-injected larval midgut (Figures 6C and 6D). These results suggest that 20E induces an interaction between ErGPCR-2 and GRK2, resulting in phosphorylation and internalization of ErGPCR-2.
Knockdown of GRK2-blocked 20E-induced ErGPCR-2 internalization.
(A). Cells were treated with dsGFP and dsGRK2 (2 μg/mL) for 24 h and then 1 μM 20E for 15 min, respectively. Green: ErGPCR-2 protein stained with an anti-ErGPCR-2 and secondary antibody labeled with Alexa488. Blue: nucleus stained with DAPI. Scale bar = 25 μm. (B). Western blot of samples in (A). (C). DMSO, 20E, or JH III (500 ng/larva) was injected to 6th 6 h larvae; midgut protein was examined. Input: immunoprecipitates by anti-ErGPCR-2, β-actin was used as control; Output: co-immunoprecipitates. 12.5% gel in SDS-PAGE. (D). Statistical analysis of (C) according to three independent replicate experiments by ImageJ software. Significant differences (*P value) in two samples were statistically analyzed by Student's t-test. (E). Number of moles of phosphorus per mole of 7TM-GFP analyzed by a phosphoprotein phosphate estimation assay kit. 20E concentration was 1 μM. DMSO was used as the control. (F). Phosphorylation of 7TM-His after knockdown of GRK2. Cells treated with dsGFP and dsGRK2 (2 μg/mL) for 24 h and then 1 μM 20E for 15 min, respectively. Statistical significance (*P Value) was based on three biologically independent repeats and analyzed by the Student t test. Gel concentration was 7.5%.
The level of 20E-induced ErGPCR-2 phosphorylation was two phosphates per molecule of 7TM-GFP protein, determined using phosphoprotein phosphate estimation assay kit. When the C-terminus of 7TM was deleted, 7TMΔC-terminal-GFP was not phosphorylated by 20E induction (Figure 6E), suggesting that 20E-induced phosphorylation occurs at the C-terminal of 7TM. When GRK2 was knocked down in the HaEpi cells using dsGRK2, 7TM-His could not be phosphorylated by 20E induction (Figure 6F), suggesting that GRK2 participates in ErGPCR-2 phosphorylation.
ErGPCR-2 determines the entrance of [3H]Pon A into cells
To demonstrate the role of ErGPCR-2 in the entrance of 20E into cells, the levels of [3H] ponasterone A ([3H]Pon A) in the whole cells were assayed. EcRB1 proteins were equally overexpressed in the cells to grasp [3H]Pon A upon its entrance. In ErGPCR-2 knockdown cells, the [3H]Pon A levels decreased significantly compared with those in dsGFP-treated cells (Figure 7A). These results suggest that ErGPCR-2 determines the entrance of [3H]Pon A into the cells.
Binding assay of ErGPCR-2 to [3H]Pon A in HaEpi cells.
(A). [3H]Pon A levels in the whole cells, dsGFP or dsErGPCR-2 (2 μg/mL), for 12 h. The cells (1–100 × 104) were then incubated with 0.1 nM [3H]Pon A (5740 cpm) in 200 μL binding buffer at 27°C for 1 h. (B). Co-immunoprecipitation of ErGPCR-2 and [3H]Pon A with the antibody against ErGPCR-2 after knockdown of ErGPCR-2. The cells were incubated with 1 μM 20E for 12 h, followed by the treatment used in (A). (C). [3H]Pon A levels in the supernatant after co-immunoprecipitation in (B). (D). Co-immunoprecipitation with the antibody against ErGPCR-2 after GRK2 knockdown, similar to the method in (B). (E). [3H]Pon A levels in the supernatant after co-immunoprecipitation in (D). (F). [3H]Pon A levels in the whole HaEpi cells overexpressing GFP, 7TM-GFP and 7TMΔC-terminal-GFP and then treated with the same method used in (A). cpm: counts per minute of [3H]Pon A. Asterisk indicates significant differences (*P value) between two compared samples by Student's t test based on three independent experiments. Pictures on the overexpression of EcRB1, ErGPCR-2 and its mutants and ErGPCR-2 and GRK2 knockdown are shown in Supplement Files: Figure S3.
To determine whether ErGPCR-2 binds to [3H]Pon A, the cells were treated with 20E for 12 h to increase the expression levels of proteins, including ErGPCR-2 and EcRB1, in the 20E pathway, after ErGPCR-2 knockdown. Both ErGPCR-2 and [3H]Pon A were then co-immunoprecipitated in the cell membrane and in the cytosol with antibodies against ErGPCR-2. However, no difference existed on the [3H]Pon A levels in co-immunoprecipitates from normal cells and dsGFP- or dsErGPCR-2-treated cells (Figure 7B), suggesting that ErGPCR-2 does not bind to [3H]Pon A in the cell membrane or in the cytosol. After co-immunoprecipitation (Co-IP), the [3H]Pon A levels in the supernatants significantly decreased in the dsErGPCR-2-treated cells compared with those in the normal cells or dsGFP-treated cells (Figure 7C). These data confirm that although ErGPCR-2 did not bind to [3H]Pon A, this GPCR determined the entrance of 20E analog into the cells.
To address whether ErGPCR-2 internalization brings [3H]Pon A to the cells, we detected the [3H]Pon A levels in the co-immunoprecipitates produced by anti-ErGPCR-2 and the supernatants after Co-IP, which blocked ErGPCR-2 internalization by GRK2 knockdown. The [3H]Pon A levels in the co-immunoprecipitates did not increase after GRK2 knockdown compared with those in the normal cells and dsGFP-treated cells (Figure 7D). This finding suggested that cell membrane-arrested ErGPCR-2 does not bind to [3H]Pon A. However, the [3H]Pon A levels in the supernatants did not decrease but increased in the Co-IP after GRK2 knockdown compared with those in the normal cells or dsGFP-treated cells (Figure 7E). The 7TM-GFP and 7TMΔC-terminal-GFP were overexpressed in HaEpi cells to examine the entrance of [3H]Pon A into the cells. The [3H]Pon A levels in the whole cells were increased by overexpression of 7TM-GFP compared with the GFP control. The [3H]Pon A levels in the whole cells also increased more when 7TMΔC-terminal-GFP was overexpressed compared with those upon overexpression of 7TM-GFP (Figure 7F). These results suggest that the function of ErGPCR-2 in controlling the entrance of [3H]Pon A and ErGPCR-2 internalization is unnecessary for the entrance of [3H]Pon A into the cells.
Discussion
Animal steroid hormones clearly transmit signals via GPCR28. However, the mechanism underlying steroid signal transmission by GPCRs remains unclear. We reveal that ErGPCR-2 is located in the cellular surface and internalized by 20E induction. GRK2 participates in 20E-induced phosphorylation and internalization of ErGPCR-2. The internalized ErGPCR-2 is then degraded, thereby desensitizing 20E signaling. ErGPCR-2 determines [3H]Pon A entrance into the cells. However, ErGPCR-2 does not bind to [3H]Pon A. These results provide evidence that steroid hormone via GPCR transmits signals to direct gene transcription.
Some GPCRs can be internalized to desensitize signaling19 or transmit signals inside the cells continuously29. The mechanism comprises GRK phosphorylation30,31, interaction with β-arrestin and internalization into the cytoplasm32. The internalized GPCR is either degraded by proteases in the lysosome or recycled in the cell membranes16. H. armigera ErRGPCR-2 is identified as a methuselah-like-2 protein by basic local alignment search tool (BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi), with several phosphorylation sites predicted at its C-terminus (Supplement Files: Figure S4). ErGPCR-2 was internalized via GRK by 20E induction and the C-terminus of ErGPCR-2 is critical. The internalized ErGPCR-2 is degraded within 2 h. Whether the internalized ErGPCR-2 still transmits signals inside the cells need further study. Blocking the internalization of ErGPCR-2 by deleting the C-terminus of ErGPCR-2 or by GRK2 knockdown did not block the entrance of [3H]Pon A. These data suggest that the internalization of ErGPCR-2 does not bring [3H]Pon A into the cells but desensitizes 20E signaling after 20E entered the cells. This condition may explain the 20E-upregulating ErGPCR-2 expression, which can compensate for the internalized ErGPCR-2 on the cell membrane. This finding may be ascribed to the different GPCRs that initiate varied signaling on the cell membrane. The activation of GRK2 by 20E induction needs further study.
GPCRs can bind various ligands, such as peptides, lipids, ions, light and odorants14. Some GPCRs bind to animal steroid hormones; for example, GPR30 binds estrogen in humans33 and DmDopEcR binds [3H]Pon A in Drosophila6. 20E (http://en.wikipedia.org/wiki/Ecdysterone) and [3H]Pon A (http://www.scbt.com/zh/datasheet-202768-ponasterone-a.html) are both ecdysones. 20E is a 20E-hydroxyecdysone, whereas [3H]Pon A is a 25-deoxy-20-hydroxyecdysone. [3H]Pon A is used to detect the binding of GPCR to steroid hormone 20E34. However, we did not detect binding of ErGPCR-2 to [3H]Pon A by Co-IP of ErGPCR-2 and [3H]Pon A from the cell membrane or cytosol, which suggests that ErGPCR-2 does not bind or does not tightly bind to [3H]Pon A.
The structure of ErGPCR-2 by Swiss model (http://swissmodel.expasy.org/) showed that ErGPCR-2 appeared as a tubaeform toward the outside of the cell membrane with a wide cave and vertical hole at the center of the structure (Supplement Files: Figure S5). Whether [3H]Pon A enters the cave, passes the hole of ErGPCR-2 and enters the cells by conformational change without tightly binding to ErGPCR-2 need further clarification. The possibility that ErGPCR-2 maintains the cell membrane structure for the entrance of [3H]Pon A should also be studied when the appropriate methods become available.
Another GPCR called ErGPCR-1 participates in 20E signal transduction in H. armigera without binding to [3H]Pon A13. Both ErGPCR-1 and ErGPCR-2 belong to methuselah-2 GPCR in the class B secretin family and are located in the cell membrane. However, ErGPCR-1 contains 489 amino acids with a 19-amino acid signal peptide, whereas ErGPCR-2 contains 757 amino acids without signal peptide as predicted by theoretical analysis (http://smart.embl-heidelberg.de/) (Supplement Files: Figure S4). Both ErGPCR-1 and ErGPCR-2 transmit 20E signals in the cell membrane, including regulation of calcium increase, protein phosphorylation, gene transcription and metamorphosis. However, ErGPCR-2 was internalized by 20E induction to control 20E entrance into the cells, which was not observed in ErGPCR-1 in a previous study13. However, the 20E-mediated internalization of ErGPCR-2 was independent from ErGPCR-1 because ErGPCR-1 knockdown did not affect 20E-mediated ErGPCR-2 internalization. Suramin blocked 20E-mediated internalization of ErGPCR-2, but SU6668 did not. This finding indicates that the GPCR pathway (not RTK pathway) is involved in 20E-mediated internalization of ErGPCR-2 (Supplement Files: Figure S6). These data suggest that various GPCRs are involved in 20E signaling, which initiates signaling coordinately. Whether ErGPCR-1 signaling depends on ErGPCR-2 is undetermined because of the shortage of direct readout on ErGPCR-1 after 20E induction.
The genomic pathway of 20E has been well studied. 20E binds its nuclear receptor EcRB120, which then interacts with USP1 to form EcRB1/USP1 transcription complex, thereby initiating gene transcription in 20E signaling3. The transcription factors include BR-Z7, HHR3, E74 and E75, which initiate insect metamorphosis35. 20E mediates CDK10 phosphorylation to enhance formation of the EcRB1/USP1 transcription complex36. 20E via GPCR-, PLC-, Ca2+- and PKC-signaling mediates USP1 phosphorylation for gene transcription12. At the downstream of ligand activated-GPCR, PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG)37. IP3 binds to its receptor in the endoplasmic reticulum membrane to drive the release of intracellular calcium ions, whereas DAG and Ca2+ bind to the protein kinase C (PKC) to activate PKC38. 20E via PKC regulates CDK1011 and USP1 phosphorylation to form the EcR/USP transcription complex12. In the current study, the larvae died before pupation or delayed pupation after knockdown of ErGPCR-2, suggesting that ErGPCR-2 is involved in gene transcription and metamorphosis in the 20E pathway. 20E-induced transcripts of genes, including EcRB1, USP1 and HR3, were not up-regulated within 15 minutes of 20E induction in the previous study11, therefore, the 20E induced rapid internalization of ErGPCR-2 and Ca2+ signaling are independent of gene transcription and protein expression.
ErGPCR-2 knockdown decreased the entrance of 20E into the cells. The 20E-induced calcium increase and phosphorylation of USP1 and CDK10 were blocked, which repressed the formation of the 20E transcription complex for gene transcription. Therefore, ErGPCR-2 transmits 20E signaling in the cell membrane to regulate gene transcription and metamorphosis.
Conclusions
ErGPCR-2 is a key control factor for the entrance of 20E into cells. Such control is not exerted by directly or tightly binding to 20E. 20E via ErGPCR-2 regulates rapid intracellular Ca2+ increase and phosphorylation of USP1 and CDK10, which induce gene transcription in the 20E pathway, thereby regulating metamorphosis. After performing the task for 20E entrance, ErGPCR-2 is phosphorylated and internalized via GRK2 for degradation to desensitize 20E signaling (Figure 8).
Diagram illustrating the 20E signal transmission mechanism of ErGPCR-2.
ErGPCR-2 controls 20E entering the cells. 20E via ErGPCR-2 regulates rapid increase of cytosolic calcium, CDK10 and USP1 phosphorylation, gene transcription and metamorphosis. ErGPCR-2 is internalized by GRK2-regulated phosphorylation and degraded to desensitize the 20E signal. Pathway Builder Tool 2.0 was used to draw the figure.
Methods
Insect
Cotton bollworms (H. armigera) were obtained from the Henan Agricultural University in China and were raised on an artificial diet composed of wheat germ and soybean powder with various vitamins and inorganic salts. The insects were kept in an insectarium at 26 ± 1°C with 60% to 70% relative humidity and under the light/dark cycles of 14 h/10 h.
RNA interference in larvae and cells
DNA fragment of ErGPCR-2 was amplified as template for dsRNA synthesis by the primers ErGPCR-2RNAiF and ErGPCR-2RNAiR (Supplement Files: Table S1). The dsRNA was sythezsized using MEGA-script RNAi Kit (Ambion Inc, Austin, TX, USA). The dsRNA purity and integrity were determined by agarose gel electrophoresis. The dsRNA were quantified using a spectrophotometer (GeneQuant; Amersham Biosciences). The dsRNA (dsErGPCR-2, dsGFP) was injected using a micro-syringe into the larval hemocoel of the sixth instar thrice at 6, 24 and 42 h at 500 ng/larva. After injection with dsRNA thrice for 12 h, 500 ng of 20E (Sigma, St. Louis, MO, USA) was injected into each larva. Dimethyl sulfoxide (DMSO) was used as control. The phenotypes and developmental rates of the larvae were recorded. The mRNA was isolated from the larvae when the control group grew at the sixth instar for 72 h. The Helicoverpa epidermal cell line (HaEpi) was cultured in Grace's medium with 10% fetal bovine serum (FBS, MDgenics, St. Louis, MO, USA) 2 d before dsRNA transfection. The cells were transfected with dsRNA and RNAfectin transfection reagents (Tiangen, Beijing, China) in Grace's medium without FBS at 2 and 4 μg/mL, respectively. After about 12 h, the cells were re-fed in a fresh medium with FBS for 12 h. Then repeated transfection once using the same dsRNA and RNAfectin concentrations and duration. Finally, the cells were re-fed in a fresh medium with FBS containing 20E at a final concentration of 1 μM at a different time. The controls were treated with equivalent volume of DMSO. Total RNA was isolated and reverse-transcribed for further experiments.
Examination of cellular calcium ions
Upon reaching a density 2 × 106 based on the above protocol, the cells were incubated with 3 μM acetoxymethyl (AM) ester calcium crimson™ dye (Invitrogen, Carlsbad, CA, USA) in Dulbecco's phosphate-buffered saline (DPBS) (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4 and 8 mM Na2HPO4) for 30 min at 27 °C. Cells were washed thrice with DPBS without calcium ions and were exposed at 1 μM 20E to detect the intracellular calcium flux. Calcium chloride was then added to the medium at 1 mM. Fluorescence was detected at 555 nm every 6 s for 360 s using Carl Zeiss LSM 700 laser scan confocal microscope (Thornwood, NY, USA). The data were analyzed using Image Pro-Plus software (Media Cybernetics, United States). After opening the file for editing, the picture was converted into grayscale 8 with automatic counting measurements and the measurement parameters were selected. Automatic statistics were then repeated. Finally, the file was imported into Excel. The Excel file was then submitted for statistical analysis. For the inhibition experiments, the cells were pretreated with different inhibitors for 30 min at 27 °C before washing thrice with DPBS without calcium ions and stimulation with 20E. Suramin (Sigma, St. Louis, MO, USA), RTK inhibitor SU6668 (Selleckchem, Houston, TX, USA), pyrazole compound (Sigma, St. Louis, MO, USA), flunarizine dihydrochloride (Sigma, St Louis, MO, USA) and thapsigargin (Sigma, St Louis, MO, USA) were used as inhibitors.
Protein phosphorylation
Protein phosphorylation was examined by molecular mass variation on 7.5% low gel of SDS–PAGE and λPP degradation as described by Song and Gilbert (1998). Briefly, 40 μL of protein (2 μg/μL) was incubated with 0.5 μL of λPP, 5 μL of buffer and 5 μL of MnCl2 (50 μL total) at 30°C for 30 min according to the manufacturer's specifications (Millipore, Temecula, CA, USA). The sample was boiled for 10 min after adding the SDS sample buffer and then subjected to SDS–PAGE and Western blot analysis. To detect the levels of ErGPCR-2 phosphorylation, 7TM domain was overexpressed in HaEpi cells by transfection with the pIEx-7TM-GEP-His and pIEX-4-7TM-His plasmids and then purified by His-Bind resin (50 μL of resin) after different treatments. The levels of purified 7TM-GFP phosphorylation were analyzed in a 96-well microplate using a phosphoprotein phosphate estimation assay kit (Sangon Biotechnology, Shanghai, China) based on the alkaline hydrolysis of phosphate from seryl and threonyl residues in phosphoproteins. The released phosphates were then quantified using malachite green and ammonium molybdate in accordance with the manufacturer's instructions.
Overexpression of ErGPCR-2 and its mutants
Proofreading DNA polymerase (Tiangen, Beijing) was used to amplify ErGPCR-2 or its mutants via PCR with various primers (Supplementary Table S1). All the fragments were inserted into the pIEx-4 plasmid (Merck, Darmstadt, Germany) and fused with GFP at the C-terminus. The plasmid (5 μg/mL) was transfected into HaEpi cells with Cellfectin following the protocol of the supplier (Invitrogen, Carlsbad, CA, USA) in Grace's medium without FBS at 27°C for 24 h. After 2 d of culture, 1 μM 20E was added to the cells. An equal volume of DMSO was used as solvent control for 20E.
Isolation of cell membrane and cytosol proteins
Membrane and cytoplasmic protein extraction kit (Beyotime, Haimen, China) was used to separate the membranes and cytosolplasmic proteins with phosphatase inhibitors (Roche Diagnostics, Mannheim, Germany) in accordance to the manufacturers' instructions.
Chromatin immunoprecipitation assay
Based on a previous procedure39, chromatin immunoprecipitation (ChIP) assay was implemented. The cells were cultured in a six-well plate. First, the cells were transfected with pIEx-4-EcRB1-RFP and the controls with pIEx-4-RFP for 24 h. Then, the cells were transfected with dsErGPCR-2 and the controls with dsGFP for 24 h. Finally, the cells were treated with 20E and DMSO for 6 h. Through the above process, the cells were cross-linked with 1% formaldehyde at 37°C for 10 min. Up to 0.125 M glycine was then added at 25°C for 10 min to terminate the cross-linking. The cells were washed twice with 1 × PBS and then suspended with SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl, pH 8.1), followed by sonication to obtain the average DNA fragments of 200 bp to 1000 bp. After centrifugation, 100 μL of supernatant was used to detect the effect of sonication. Other supernatants were added to the Protein A resin and incubated at 4°C for 1 h to pre-treat nonspecific binding. After centrifugation, one supernatant was used as a negative control sample for qRT-PCR. Other supernatants were incubated with anti-RFP antibody or without antibody (negative control) at 4°C overnight. Protein A resin was added into the immunoprecipitated protein–DNA complexes and incubated at 4°C for 2 h. The complexes were then washed with elution buffer (1% SDS, 0.1 M NaHCO3). The DNA proteins were reversely cross-linked at 65°C overnight, followed by RNase and proteinase K treatments. The DNA was purified by phenol/chloroform extraction and subjected to qRT-PCR analysis using HHR3F/R primers (Table S1).
[3H]Pon A-binding assays
Ecdysone receptor B1 (EcRB1) fused with histidines (EcRB1-His) was overexpressed in HaEpi cells with pIEX-4-His vector to bind [3H]Pon A. The cells were incubated twice with dsGFP or dsErGPCR-2 (2 μg/mL) for 12 h as described above. The cells (1 × 104 to 100 × 104) were collected and then incubated with 0.1 nM [3H]Pon A (5740 cpm) in 200 μL of binding buffer (20 mM HEPES, 100 mM NaCl, 6 mM MgCl2, 1 mM EDTA and 1 Mm EGTA) at 27°C for 1 h. Subsequently, the cells were subjected to [3H]Pon A detection. For Co-IP with antibodies against ErGPCR-2, the cells were incubated with 1 μM 20E for 12 h to increase the expression of ErGPCR-2 and EcRB1, followed by incubation with 2 μg/mL of dsGFP, dsErGPCR-2 and dsGRK2 twice for 12 h each. The cells were collected and incubated with 0.1 nM [3H]Pon A in 200 μL binding buffer at 27°C for 1 h. The cells were lysated in RIPA (Radio immunoprecipition Assay) buffer, after which 40 μL of anti-ErGPCR-2 and 50 μL of the Protein A resin was added at 4°C for 4 h to co-immunoprecipitate (CoIP) ErGPCR-2 in the membrane and cytosol which possible binding to [3H]Pon A. The precipitates were washed twice with RIPA buffer. After Co-IP, [3H]Pon A was detected in the supernatant. For the overexpression of the seven-transmembrane (7TM) domain of ErGPCR-2 and its mutants, the plasmids of pIEX-4-GFP, pIEX-4-7-TM-GFP, OVErGPCR-2-7TMΔC-terminal-GFP and OVErGPCR-2-7TMΔe2loop-GFP (5 μg/mL) were transfected into the HaEpi cells with Cellfectin following the protocol of the supplier (Invitrogen, Carlsbad, CA, USA) in Grace's medium without FBS at 27°C for 24 h. Then, the cells were collected and incubated with 0.1 nM [3H]Pon A (5740 cpm) in 200 μL binding buffer at 27°C for 1 h. After all the above mentioned treatments, particular cell proteins were collected on glass fiber filters. After air drying in the dark, the different filters were then added into 5 mL of scintillation fluid. Radioactivity was measured using SN-6930 liquid scintillation counter (Shanghai Hesuo Rihuan Photoelectric Instrument Co., Ltd., China). The efficacy of RNA interference of ErGPCR-2 and GRK2 was examined via Western blot.
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Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (No. 31230067), the National Basic Research Program of China (973 Program, No. 2012CB114101) and the Ph.D. Programs Foundation of the Ministry of Education of China (No. 20120131110025).
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Di Wang performed the major work and drafted the manuscript. Mei-juan Cai detected the Ca2+ levels. Wen-Li Zhao supplied the antibodies and dsRNA of GRK2. Jin-Xing Wang directed the research. Xiao-Fan Zhao designed the studies and edited the manuscript. All authors reviewed the manuscript.
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Wang, D., Zhao, WL., Cai, MJ. et al. G-protein-coupled receptor controls steroid hormone signaling in cell membrane. Sci Rep 5, 8675 (2015). https://doi.org/10.1038/srep08675
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DOI: https://doi.org/10.1038/srep08675
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