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Cited by 44Receptor Mechanisms and SignalingNeurobiology and Insect Physiology ResearchEstrogen and related hormone effects

G-protein-coupled receptor participates in 20-hydroxyecdysone signaling on the plasma membrane

Open access
Cai, Mei-Juan, Dong, Du-Juan, Wang, Yu, Liu, Peng-Cheng, Liu, Wen, Wang, Jin-Xing, Zhao, Xiao-Fan·2014·Cell Communication and Signaling
DOI 10.1186/1478-811x-12-9PMC PMC3937218
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Abstract

BACKGROUND: Animal steroid hormones are conventionally known to initiate signaling via a genomic pathway by binding to the nuclear receptors. The mechanism by which 20E initiates signaling via a nongenomic pathway is unclear. RESULTS: We illustrate that 20E triggered the nongenomic pathway through a plasma membrane G-protein-coupled receptor (named ErGPCR) in the lepidopteran insect Helicoverpa armigera. The transcript of ErGPCR was increased at the larval molting stage and metamorphic molting stage by 20E regulation. Knockdown of ErGPCR via RNA interference in vivo blocked larval-pupal transition and suppressed 20E-induced gene expression. ErGPCR overexpression in the H. armigera epidermal cell line increased the 20E-induced gene expression. Through ErGPCR, 20E modulated Calponin nuclear translocation and phosphorylation, and induced a rapid increase in cytosolic Ca2+ levels. The inhibitors of T-type voltage-gated calcium channels and canonical transient receptor potential calcium channels repressed the 20E-induced Ca2+ increase. Truncation of the N-terminal extracellular region of ErGPCR inhibited its localization on the plasma membrane and 20E-induced gene expression. ErGPCR was not detected to bind with the steroid hormone analog [3H]Pon A. CONCLUSION: These results suggest that ErGPCR participates in 20E signaling on the plasma membrane.

AI snapshot

Written from the paper’s full text.

TL;DR

ErGPCR, a plasma membrane G-protein-coupled receptor in *Helicoverpa armigera*, participates in 20-hydroxyecdysone (20E) nongenomic signaling and is required for 20E-induced gene expression, rapid Calponin nuclear translocation/phosphorylation, cytosolic Ca²⁺ increase, and larval–pupal transition. The authors propose that 20E acts through ErGPCR to modulate downstream genomic responses, although ErGPCR was not detected to bind the 20E analog [³H]Pon A.

Key findings

  • 20E induced *EcRB1*, *BrZ2*, *HHR3*, and *USP1* expression in HaEpi cells, and the GPCR inhibitor suramin (50 μM) repressed this induction.
  • In vivo ErGPCR knockdown blocked larval–pupal transition: only 29% of dsErGPCR larvae pupated, 50% died, and 21% became larval–pupal chimeras, versus 90% pupation and 10% death in dsGFP controls; surviving pupaters were delayed by 23 h (fifth to sixth instar) and 52 h (sixth instar to pupation).
  • ErGPCR localized to the plasma membrane; its knockdown decreased 20E-induced *EcRB1*, *USP1*, *HHR3*, *BrZ2*, and *E75B*, while overexpression increased *EcRB1*, *BrZ2*, *HHR3*, and *USP1*.
  • 20E via ErGPCR caused rapid cytosolic Ca²⁺ increase (peaking at ~50 s and declining by 120 s, followed by extracellular Ca²⁺ influx after adding 1 mM CaCl₂), Calponin nuclear translocation and phosphorylation, and EcRB1 binding to EcRE; T-type channel blocker FL (50 μM) and TRPC3 blocker Pyr3 (10 μM) inhibited Ca²⁺ influx and downstream gene expression/phosphorylation, whereas L-type blocker verapamil (100 μM) and 2-APB (10–100 μM) did not.
  • Truncation of the ErGPCR N-terminal extracellular region (aa 20–197) caused cytoplasmic localization and reduced 20E-induced *EcRB1*, *BrZ2*, *HHR3*, and *USP1*; truncation of the second inner loop (aa 275–308) did not affect these transcripts. ErGPCR overexpression did not increase [³H]Pon A binding in whole cells or membrane fractions.

Methods

  • *Helicoverpa armigera* larvae and the HaEpi epidermal cell line were used; analyses included qRT-PCR, RNAi in cells and larvae, ErGPCR overexpression and truncation, immunocytochemistry, western blot, Ca²⁺ imaging, ChIP, and [³H]Pon A binding assays.
  • Treatments included 1 μM 20E, suramin (50 μM), flunarizine dihydrochloride (50 μM), verapamil hydrochloride (100 μM), 2-APB (10–100 μM), and Pyr3 (10 μM); dsRNA was used at 5 μg/mL in cells and 0.4 μg/μL (5 μL injected) in larvae.
  • Statistical comparisons used Student’s t test, with one-way ANOVA and Duncan’s test for binding data; experiments used three independent replicates, and larval RNAi experiments used 30 larvae per group.

Limitations

  • Stated by the authors: ErGPCR was not detected to bind [³H]Pon A; they note the analytical method may explain this and requires further study, and whether other GPCRs bind 20E needs further exploration.
  • Stated by the authors: the mechanism underlying GPCRs in the 20E membrane pathway requires further study.
  • Evident from the text: the study relies heavily on pharmacological inhibitors, overexpression, and truncation in a cell line, and does not show direct physical interaction between ErGPCR and G proteins or calcium channels; the ligand-binding status of ErGPCR remains unresolved.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1186/1478-811x-12-9

Cites

56 of 56 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 35 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Phylogenetic analysis indicated that ErGPCR does not cluster with DmDopEcR, GPR30, and AR.

    “Phylogenetic analysis indicated that ErGPCR does not cluster with DmDopEcR, GPR30, and AR.”
    Quote in the paperNot a substantial claim
  • Findingown data

    20E significantly promoted the expression of EcRB1, BrZ2, HHR3, and USP1 compared with the DMSO solvent control.

    “20E significantly promoted the expression of EcRB1, BrZ2, HHR3, and USP1 compared with the DMSO solvent control.”
    Quote in the paperSubstantial claim
  • Findingown data

    Through ErGPCR, 20E induces a rapid increase in cytosolic Ca2+ levels.

    “Through ErGPCR, 20E modulated Calponin nuclear translocation and phosphorylation, and induced a rapid increase in cytosolic Ca2+ levels.”
    • Also stated in: § 20E regulates cellular Ca2+ release and influx via ErGPCR to regulate gene expression · plain
      “When ErGPCR was knocked down by RNAi, the 20E-induced Ca2+ increase, including intracellular Ca2+ release, and extracellular Ca2+ influx, was also inhibited compared with the control (Figure 7B).”
    • Also stated in: § 20E regulates cellular Ca2+ release and influx via ErGPCR to regulate gene expression · hedged
      “These findings suggest that 20E induces rapid intracellular Ca2+ release and extracellular Ca2+ influx via ErGPCR.”
    Quote in the paperSubstantial claim
  • Findingown data

    Through ErGPCR, 20E modulates Calponin phosphorylation.

    “Through ErGPCR, 20E modulated Calponin nuclear translocation and phosphorylation, and induced a rapid increase in cytosolic Ca2+ levels.”
    • Also stated in: § 20E regulates protein nuclear translocation and phosphorylation via ErGPCR · plain
      “Moreover, the 20E-mediated Calponin phosphorylation was suppressed when ErGPCR was silenced (Figure 6B).”
    • Also stated in: § 20E regulates protein nuclear translocation and phosphorylation via ErGPCR · hedged
      “These results suggest that 20E regulates Calponin nuclear translocation and phosphorylation via ErGPCR.”
    Quote in the paperSubstantial claim
  • Findingown data

    ErGPCR is located on the plasma membrane of HaEpi cells.

    “Immunocytochemical analysis using rabbit anti-ErGPCR polyclonal antibodies showed that ErGPCR was located on the plasma membrane of HaEpi cells.”
    • Also stated in: § ErGPCR is located on the membrane and is necessary for 20E-induced gene expression · plain
      “Moreover, the overexpression of ErGPCR-GFP (C-terminal with GFP) was also located on the cell plasma membrane (Figure 5B).”
    Quote in the paperSubstantial claim
  • May combine two claims. 31
  • The quote only partly states this claim. 2
  • Classified as probably not a substantial claim. 29

  • May combine two claims.

    Findingown data

    D. melanogaster DmDopEcR, H. sapiens GPR30, and H. sapiens beta-2 adrenergic receptor are not found by BLASTX analysis.

    “However, D. melanogaster DmDopEcR, Homo sapiens GPR30, and H. sapiens beta-2 adrenergic receptor (AR) are not found by BLASTX analysis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After ErGPCR knockdown, the duration of development is significantly delayed compared with the dsGFP control: a 23 h delay from fifth instar to sixth instar and a 52 h delay from sixth instar to pupal stage.

    “Of the 29% that pupated after ErGPCR knockdown, the duration of development was significantly delayed compared with the dsGFP control: a 23 h delay from fifth instar to the sixth instar, and a 52 h delay from the sixth instar to the pupal stage (Figure 4C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    JH III injection into sixth-instar larvae did not affect ErGPCR transcript levels, but repressed the 20E-induced upregulation of ErGPCR.

    “JH III injection into the sixth-instar larvae did not affect the ErGPCR transcript levels, but repressed the 20E-induced upregulation of ErGPCR (Figure 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The ErGPCR transcript is increased at the larval molting stage and metamorphic molting stage.

    “The transcript of ErGPCR was increased at the larval molting stage and metamorphic molting stage by 20E regulation.”
    • Also stated in: § ErGPCR is involved in 20E-regulated gene expression · plain
      “The transcript level of ErGPCR was increased at the larval molting stage (5 M) and metamorphic molting stage (sixth-instar 72 h larvae to pupae) in the tissues (Figure 2).”
    • Also stated in: § ErGPCR is involved in 20E-regulated gene expression · plain
      “ErGPCR is highly expressed during molting and metamorphosis in epidermis, midgut and fat body detected by qRT-PCR.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The mRNA levels of six GPCR candidates were upregulated by 20E induction, and two non-GPCR ESTs were unaffected.

    “The mRNA levels of six GPCR candidates were upregulated by 20E induction, and two non-GPCR ESTs were unaffected.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the dsGFP-injected control, 90% of the larvae pupated and 10% died.

    “In the dsRNA of green fluorescent protein (dsGFP)-injected control, 90% of the larvae pupated, whereas 10% died.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    ErGPCR has 57% identity with Spodoptera frugiperda GPCR, 32% with Tribolium castaneum GPCR, and 30% with Drosophila melanogaster GPCR.

    “ErGPCR has 57% identity with Spodoptera frugiperda GPCR, 32% with Tribolium castaneum GPCR, and 30% with Drosophila melanogaster GPCR (Additional file 1: Figure S4).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In dsErGPCR treatment, 29% of larvae pupated, 50% died, and 21% displayed larval-pupal chimeras.

    “However, in dsErGPCR treatment, only 29% of the larvae pupated, 50% died, and 21% displayed larval–pupal chimeras (Figure 4B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The 20E-induced Ca2+ influx was restrained by 50 μM FL without affecting intracellular Ca2+ release.

    “The 20E-induced Ca2+ influx was restrained by 50 μM FL without affecting intracellular Ca2+ release.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    10 μM Pyr3 suppressed the 20E-induced Ca2+ influx, but had no effect on intracellular Ca2+ release.

    “However, 10 μM Pyr3 suppressed the 20E-induced Ca2+ influx, but had no effect on intracellular Ca2+ release (Figure 7D).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    The 20E-induced phosphorylation of Calponin was inhibited by FL and Pyr3.

    “Meanwhile, the 20E-induced phosphorylation of Calponin was inhibited (Figure 7F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    When cells were incubated in calcium-free buffer, cytosolic Ca2+ level increased rapidly by 20E treatment, peaked at approximately 50 s, then declined to a lower level at 120 s.

    “When the cells were incubated in calcium-free buffer (DPBS), cytosolic Ca2+ level increased rapidly by 20E treatment, and peaked at approximately 50 s, then declined to a lower level at 120 s.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Ve and 2-APB inhibitors had no effect on the 20E-induced gene expression and 20E-induced Calponin phosphorylation.

    “By contrast, Ve and 2-APB inhibitors had no effect on the 20E-induced gene expression and 20E-induced Calponin phosphorylation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Calponin is mainly localized in the cytoplasm in DMSO-negative control cells, but is translocated into the nucleus after 20E induction.

    “Calponin was mainly localized in the cytoplasm in the DMSO-negative control cells, but was translocated into the nucleus after 20E induction.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Following the addition of 1 mM calcium into DPBS at 120 s, the cytosolic Ca2+ levels gradually increased and then remained constant.

    “Following the addition of 1 mM calcium into DPBS at 120 s, the cytosolic Ca2+ levels gradually increased and then remained constant.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    20E regulates rapid intracellular Ca2+ release and extracellular Ca2+ influx through ErGPCR.

    “Our data show that 20E regulates rapid intracellular Ca2+ release and extracellular Ca2+ influx through ErGPCR, and T-type voltage-gated Ca2+ channels are involved in Ca2+ influx.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The inhibitors used (suramin, flunarizine, verapamil, 2-APB, Pyr3) specifically inhibit their intended targets without off-target effects.

  • The quote only partly states this claim.

    Findingreasoning

    T-type voltage-gated Ca2+ channels are involved in 20E-induced Ca2+ influx.

    “T-type voltage-gated Ca2+ channels are involved in Ca2+ influx.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The inhibitors used (suramin, flunarizine, verapamil, 2-APB, Pyr3) specifically inhibit their intended targets without off-target effects.

  • May combine two claims.

    Findingown data

    The deduced amino acid sequence of ErGPCR contains a signal peptide at the N-terminus and seven transmembrane domains.

    “The deduced amino acid sequence of ErGPCR contains a signal peptide at the N-terminus and seven transmembrane domains (Additional file 1: Figure S3).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    When cellular Ca2+ was blocked by inhibitors, 20E-induced gene expression and phosphorylation of Calponin were blocked.

    “In our study, when the cellular Ca2+ was blocked by inhibitors, 20E-induced gene expression and the phosphorylation of Calponin were blocked.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The inhibitors used (suramin, flunarizine, verapamil, 2-APB, Pyr3) specifically inhibit their intended targets without off-target effects.

  • May combine two claims.

    Findingown data

    ErGPCR differs from GPR30, beta-2 AR, or Drosophila DmDopEcR.

    “Identification and phylogenetic analysis using amino acid sequences show that ErGPCR differs from GPR30, beta-2 AR, or Drosophila DmDopEcR, which may explain the differences in ligand binding activity.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    GPR30 can act as the membrane receptor for estrogen.

    “A G-protein-coupled seven transmembrane receptor (GPR30) can act as the membrane receptor for estrogen [12]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] Prossnitz ER, Maggiolini M: Mechanisms of estrogen signaling and gene expression via GPR30. Mol Cell Endocrinol. 2009, 308: 32-38. 10.1016/j.mce.2009.03.026. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The complete structure of ErGPCR is essential to its plasma membrane location, and the N-terminal extracellular region is required for ErGPCR function in the 20E signaling pathway.

    “These results indicate that the complete structure of ErGPCR is essential to its plasma membrane location, and the N-terminal extracellular region is required for ErGPCR function in the 20E signaling pathway.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The 20E-induced rapid intracellular Ca2+ increase is required for 20E-regulated gene expression and protein phosphorylation.

    “These results show that the 20E-induced rapid intracellular Ca2+ increase is required for 20E-regulated gene expression and protein phosphorylation.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationreasoning

    The reason that knockdown of EcRB1 repressed ErGPCR but did not repress mod(mdg4)1a might be the time difference of mRNA transcription and protein translation of ErGPCR.

    “The reason that knockdown of EcRB1 repressed ErGPCR but did not repress mod(mdg4)1a might because the time difference of mRNA transcription and protein translation of ErGPCR.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Previous studies indicated that animal steroid hormones can trigger nongenomic actions through the cytoplasmic membrane.

    “Previous studies indicated that animal steroid hormones can trigger nongenomic actions through the cytoplasmic membrane [10].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [10] Losel R, Wehling M: Nongenomic actions of steroid hormones. Nat Rev Mol Cell Biol. 2003, 4: 46-56. 10.1038/nrm1009. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Plant steroid hormones, such as brassinosteroids, employ a nongenomic pathway to initiate signaling by combining with plasma membrane receptor kinases for gene transactivation.

    “However, plant steroid hormones, such as brassinosteroids, employ a nongenomic pathway to initiate signaling by combining with plasma membrane receptor kinases for gene transactivation [8].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [8] Bishop GJ, Koncz C: Brassinosteroids and plant steroid hormone signaling. Plant Cell. 2002, 14 (Suppl): S97-110. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Nuclear receptors act by forming homodimers or heterodimers.

    “Nuclear receptors act by forming homodimers or heterodimers. For example, the glucocorticoid receptor [1] and estrogen receptor [2] form a homodimer, and insect ecdysone receptor (EcR) forms a heterodimer with an ultraspiracle protein (USP), the ortholog of retinoid X receptor in vertebrates [3].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [1] Ratman D, Vanden Berghe W, Dejager L, Libert C, Tavernier J, Beck IM, De Bosscher K: How glucocorticoid receptors modulate the activity of other transcription factors: a scope beyond tethering. Mol Cell Endocrinol. 2013, 380: 41-54. 10.1016/j.mce.2012.12.014. source

      Not checked in the free run.

    • cites [2] Parker MG, Arbuckle N, Dauvois S, Danielian P, White R: Structure and function of the estrogen receptor. Ann N Y Acad Sci. 1993, 684: 119-126. 10.1111/j.1749-6632.1993.tb32276.x. source

      Not checked in the free run.

    • cites [3] Oro AE, McKeown M, Evans RM: Relationship between the product of the Drosophila ultraspiracle locus and the vertebrate retinoid X receptor. Nature. 1990, 347: 298-301. 10.1038/347298a0. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Knockdown of ErGPCR represses the binding of EcRB1 to EcRE, thus blocking 20E-induced expression of EcRB1, USP1, HHR3, BrZ2, and E75B in the cell line and larvae, which results in blocking the 20E genomic pathway, thereby inhibiting metamorphosis.

    “Knockdown of ErGPCR repressed the binding of EcRB1 to EcRE thus blocked 20E-induced expression of EcRB1, USP1, HHR3, BrZ2, and E75B in the cell line and larvae, which resulted in blocking the 20E genomic pathway, thereby inhibiting metamorphosis.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    20E induces intracellular Ca2+ release into the cytoplasm via an unknown GPCR pathway in the anterior silk gland of silkworms.

    “20E induces intracellular Ca2+ release into the cytoplasm via an unknown G-protein-coupled receptor (GPCR) pathway in the anterior silk gland of silkworms [23].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [23] Manaboon M, Iga M, Iwami M, Sakurai S: Intracellular mobilization of Ca2+ by the insect steroid hormone 20-hydroxyecdysone during programmed cell death in silkworm anterior silk glands. J Insect Physiol. 2009, 55: 122-128. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    20E via cellular Ca2+ signaling modulates the binding of EcRB1 to EcRE, and regulates the 20E-induced gene transcription.

    “These results suggest that 20E via cellular Ca2+ signaling modulates the binding of EcRB1 to EcRE, and regulates the 20E-induced gene transcription.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationown data

    Cells or cell membrane fractions could bind with [3H]Pon A in a cell- or cell membrane fraction-dependent manner.

    “These results suggest that cells or cell membrane fractions could bind with [3H]Pon A in a cell- or cell membrane fraction-dependent manner.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In our previous study, we demonstrated that 20E regulates the rapid nuclear translocation and phosphorylation of Calponin for gene expression in Helicoverpa armigera.

    “In our previous study, we demonstrated that 20E regulates the rapid nuclear translocation and phosphorylation of Calponin for gene expression in Helicoverpa armigera[26].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [26] Liu PC, Wang JX, Song QS XFZ: The Participation of Calponin in the cross talk between 20-hydroxyecdysone and juvenile hormone signaling pathways by phosphorylation variation. PLoS One. 2011, 6: 1-12.

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    TRP channels are non-voltage-gated Ca2+ channels involved in Ca2+ entry.

    “TRP channels are non-voltage-gated Ca2+ channels involved in Ca2+ entry [42].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [42] Cioffi DL, Barry C, Stevens T: Store-operated calcium entry channels in pulmonary endothelium: the emerging story of TRPCS and Orai1. Adv Exp Med Biol. 2010, 661: 137-154. 10.1007/978-1-60761-500-2_9. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    20E functions in the membrane via a nongenomic pathway to regulate protein translocation and phosphorylation, which may contribute to the activation of transcription factors and formation of transcription complexes.

    “This finding suggests that 20E functions in the membrane via a nongenomic pathway to regulate protein translocation and phosphorylation, which may contribute to the activation of transcription factors and formation of transcription complexes.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The increase in cellular Ca2+ is another characteristic of the nongenomic pathway of steroid hormones.

    “The increase in cellular Ca2+ is another characteristic of the nongenomic pathway of steroid hormones [32].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] Vasudevan N, Pfaff DW: Membrane-initiated actions of estrogens in neuroendocrinology: emerging principles. Endocr Rev. 2007, 28: 1-19. 10.1210/er.2005-0021. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Voltage-gated calcium channels are essential in regulating extracellular Ca2+ influx in a wide variety of tissues.

    “Voltage-gated calcium channels are essential in regulating extracellular Ca2+ influx in a wide variety of tissues [40].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [40] Christel C, Lee A: Ca2 + −dependent modulation of voltage-gated Ca2+ channels. Biochim Biophys Acta. 2012, 1820: 1243-1252. 10.1016/j.bbagen.2011.12.012. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    GPCRs serve as chaperones and interact with voltage-gated calcium channels to form complexes.

    “GPCRs serve as chaperones and interact with voltage-gated calcium channels to form complexes [41].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [41] Altier C: GPCR and Voltage-Gated Calcium Channels (VGCC) Signaling Complexes. Subcell Biochem. 2012, 63: 241-262. 10.1007/978-94-007-4765-4_13. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Ecdysteroids trigger rapid Ca2+ increase, including intracellular Ca2+ release, and extracellular Ca2+ influx through GPCR in mouse skeletal muscle cells.

    “Ecdysteroids trigger rapid Ca2+ increase, including intracellular Ca2+ release, and extracellular Ca2+ influx through GPCR in mouse skeletal muscle cells [25].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [25] Gorelick-Feldman J, Cohick W, Raskin I: Ecdysteroids elicit a rapid Ca2+ flux leading to Akt activation and increased protein synthesis in skeletal muscle cells. Steroids. 2010, 75: 632-637. 10.1016/j.steroids.2010.03.008. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    GPCRs directly or indirectly modulate several TRP channels.

    “GPCRs directly or indirectly modulate several TRP channels [44,45].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [44] Clapham DE: TRP channels as cellular sensors. Nature. 2003, 426: 517-524. 10.1038/nature02196. source

      Not checked in the free run.

    • cites [45] Yekkirala AS: Two to tango: GPCR oligomers and GPCR-TRP channel interactions in nociception. Life Sci. 2013, 92: 438-445. 10.1016/j.lfs.2012.06.021. source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    20E initiates the genomic pathway by binding with its nuclear hormone receptor EcR to regulate gene expression for metamorphosis.

    “20E initiates the genomic pathway by binding with its nuclear hormone receptor EcR to regulate gene expression for metamorphosis [37].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [37] Bender M, Imam FB, Talbot WS, Ganetzky B, Hogness DS: Drosophila ecdysone receptor mutations reveal functional differences among receptor isoforms. Cell. 1997, 91: 777-788. 10.1016/S0092-8674(00)80466-3. source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Suramin disrupts GPCR binding with the G protein by blocking the association of G protein α and βγ subunits.

    “Suramin disrupts GPCR binding with the G protein by blocking the association of G protein α and βγ subunits [28].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [28] Chung WC, Kermode JC: Suramin disrupts receptor-G protein coupling by blocking association of G protein alpha and betagamma subunits. J Pharmacol Exp Ther. 2005, 313: 191-198. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The nongenomic pathway is characterized by rapid protein translocation and phosphorylation.

    “The nongenomic pathway is characterized by rapid protein translocation and phosphorylation [31].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [31] Raz L, Khan MM, Mahesh VB, Vadlamudi RK, Brann DW: Rapid estrogen signaling in the brain. Neurosignals. 2008, 16: 140-153. 10.1159/000111559. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    GPCRs form homodimers, heterodimers, oligomers, or complexes in the signalsomes of the membrane.

    “GPCRs form homodimers, heterodimers, oligomers, or complexes in the signalsomes of the membrane [54].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [54] Moreno JL, Holloway T, Gonzalez-Maeso J: G protein-coupled receptor heterocomplexes in neuropsychiatric disorders. Prog Mol Biol Transl Sci. 2013, 117: 187-205. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Several GPCRs bind with 20E, such as DmDopEcR binding with [3H]Pon A, or an unknown GPCR in the anterior silk gland of silkworms binding with [3H]Pon A.

    “Numerous studies have reported the binding of several GPCRs with 20E, such as the binding of DmDopEcR with [3H]Pon A [24], or an unknown GPCR in the anterior silk gland of silkworms binding with [3H]Pon A [22].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [24] Srivastava DP, Yu EJ, Kennedy K, Chatwin H, Reale V, Hamon M, Smith T, Evans PD: Rapid, nongenomic responses to ecdysteroids and catecholamines mediated by a novel Drosophila G-protein-coupled receptor. J Neurosci. 2005, 25: 6145-6155. 10.1523/JNEUROSCI.1005-05.2005. source

      Not checked in the free run.

    • cites [22] Elmogy M, Iwami M, Sakurai S: Presence of membrane ecdysone receptor in the anterior silk gland of the silkworm Bombyx mori. Eur J Biochem. 2004, 271: 3171-3179. 10.1111/j.1432-1033.2004.04249.x. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    The negative control cells were transfected with the same volume of pIEx-4-RFP, and the cells received the same treatment as above.

    “The negative control cells were transfected with the same volume of pIEx-4-RFP, and the cells received the same treatment as above.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Three independent experiments were performed using 30 larvae each for RNAi in larvae.

    “Three independent experiments were performed using 30 larvae each.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    TRP channels are associated with steroid hormones in mammals.

    “TRP channels are associated with steroid hormones in mammals [46].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [46] Wagner TF, Loch S, Lambert S, Straub I, Mannebach S, Mathar I, Dufer M, Lis A, Flockerzi V, Philipp SE, Oberwinkler J: Transient receptor potential M3 channels are ionotropic steroid receptors in pancreatic beta cells. Nat Cell Biol. 2008, 10: 1421-1430. 10.1038/ncb1801. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    DmDopEcR shows homology with vertebrate ARs.

    “DmDopEcR shows homology with vertebrate ARs [24].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [24] Srivastava DP, Yu EJ, Kennedy K, Chatwin H, Reale V, Hamon M, Smith T, Evans PD: Rapid, nongenomic responses to ecdysteroids and catecholamines mediated by a novel Drosophila G-protein-coupled receptor. J Neurosci. 2005, 25: 6145-6155. 10.1523/JNEUROSCI.1005-05.2005. source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductown data

    The specificity of the antibody was determined via western blot analysis.

    “The specificity of the antibody was determined via western blot analysis using horseradish peroxidase–labeled goat anti-rabbit polyclonal secondary antibodies (Zhongshan, Beijing).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    In classical GPCR signaling pathways, ligands bind to cell surface transmembrane receptors, such as β2 ARs, and cause conformational changes in their transmembrane and intracellular domains.

    “In classical GPCR signaling pathways, ligands bind to cell surface transmembrane receptors, such as the β2 ARs, and cause conformational changes in their transmembrane and intracellular domains [48].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [48] Rasmussen SG, DeVree BT, Zou Y, Kruse AC, Chung KY, Kobilka TS, Thian FS, Chae PS, Pardon E, Calinski D, Mathiesen JM, Shah ST, Lyons JA, Caffrey M, Gellman SH, Steyaert J, Skiniotis G, Weis WI, Sunahara RK, Kobilka BK: Crystal structure of the beta2 adrenergic receptor-Gs protein complex. Nature. 2011, 477: 549-555. 10.1038/nature10361. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The qRT-PCR experiment was repeated three times using three independent RNA samples for statistical analysis.

    “The experiment was repeated three times using three independent RNA samples for statistical analysis.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A major challenge in the study of steroid hormone nongenomic pathways is the binding assay of GPCR with the steroid hormone.

    “A major challenge in the study of steroid hormone nongenomic pathways is the binding assay of GPCR with the steroid hormone [50].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [50] Norman AW, Mizwicki MT, Norman DP: Steroid-hormone rapid actions, membrane receptors and a conformational ensemble model. Nat Rev Drug Discov. 2004, 3: 27-41. 10.1038/nrd1283. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    GPR30 has shown negligible binding to estrogen (17β-estradiol) in several studies.

    “GPR30 has shown negligible binding to estrogen (17β-estradiol) in several studies [49], which may be due to the different analytical methods [17].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [49] Otto C, Fuchs I, Kauselmann G, Kern H, Zevnik B, Andreasen P, Schwarz G, Altmann H, Klewer M, Schoor M, Vonk R, Fritzemeier KH: GPR30 does not mediate estrogenic responses in reproductive organs in mice. Biol Reprod. 2009, 80: 34-41. 10.1095/biolreprod.108.071175. source

      Not checked in the free run.

    • cites [17] Filardo EJ, Thomas P: Minireview: G protein-coupled estrogen receptor-1, GPER-1: its mechanism of action and role in female reproductive cancer, renal and vascular physiology. Endocrinology. 2012, 153: 2953-2962. 10.1210/en.2012-1061. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Calcium functions as a secondary messenger in gene expression and protein phosphorylation.

    “These findings confirm the function of calcium on gene expression and protein phosphorylation as the secondary messenger, and reveal that 20E regulates the cellular calcium via ErGPCR to regulate the genomic pathway.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Calcium is an important secondary messenger that regulates numerous essential physiologic processes, including protein kinase C activation, for further protein phosphorylation and gene transcription.

    “Calcium is an important secondary messenger that regulates numerous essential physiologic processes, including protein kinase C activation, for further protein phosphorylation [47] and gene transcription.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [47] Clapham DE: Calcium signaling. Cell. 2007, 131: 1047-1058. 10.1016/j.cell.2007.11.028. source

      Not checked in the free run.

55 of 55

  • The paper relies on this without support.

    Rapid calcium release or influx in cells is the outcome of nongenomic signaling.

    “Rapid calcium release or influx in the cells is the outcome of nongenomic signaling.”

  • The paper relies on this without support.

    The inhibitors used (suramin, flunarizine, verapamil, 2-APB, Pyr3) specifically inhibit their intended targets without off-target effects.

    “For the inhibition experiments, the cells were pretreated with different inhibitors for 1 h prior to 20E treatment. The GPCR inhibitor suramin, T-type voltage-gated calcium channel inhibitor flunarizine dihydrochloride, L-type calcium channel inhibitor verapamil hydrochloride, and TRP channel inhibitors 2-APB and Pyr3 were purchased from Sigma Chemical (St. Louis, MO, USA).”

  • The paper relies on this without support.

    dsRNA-mediated knockdown of ErGPCR is specific and does not affect other genes.

    “After ErGPCR knockdown, the cells were treated with 1 μM 20E for 0.5 h to 3 h.”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • [3H]Pon A concentration differs between binding-assay results and methods

    Major

    The results and Figure 10 caption specify 0.1 nM [3H]Pon A, whereas the binding-assay methods specify 1 nM for membrane fractions and say the whole-cell experiments used the same method.

  • ErGPCR knockdown duration differs for the ChIP experiment

    Minor

    The Figure 8A caption says cells received dsErGPCR or dsGFP for 12 hours before 20E treatment, but the ChIP methods say the dsRNA treatment lasted 24 hours.

  • Second inner-loop deletion starts at different amino acids

    Minor

    The results identify the second inner-loop deletion as amino acids 275-308, while the Figure 9B caption identifies the corresponding construct as deleting amino acids 276-308.

3 of 3

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Authors
Cai, M.-J., Dong, D.-J., Wang, Y., Liu, P.-C., Liu, W., Wang, J.-X., & Zhao, X.-F.
Affiliations
Shandong University (CN)
Corresponding author
Zhao, Xiao-Fan
ORCID
Venue
Cell Communication and Signaling
Publisher
Springer Science and Business Media LLC
Published
2014-02-10
Volume
12
Issue
1
Pages
9-9
ISSN
1478-811X (ISSN-L)
DOI
10.1186/1478-811x-12-9
PMC
PMC3937218
OpenAlex
W2097907702
Semantic Scholar
4fba552af634b8e8936bb0c52c267f561b9ae312
Cited by
44
References
56
Citations by year
2026: 1 · 2025: 5 · 2024: 3 · 2022: 4 · 2021: 3 · 2020: 6 · 2019: 3 · 2018: 2 · 2017: 5 · 2016: 4 · 2015: 5 · 2014: 3
FWCI
2.32
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Cell Communication and Signaling (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • National Natural Science Foundation of China (973 Program, 31230067)
  • Ministry of Education of the People's Republic of China (20120131110025)
  • National Key Research and Development Program of China (2012CB114101)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neurobiology and Insect Physiology Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Estrogen and related hormone effects
Keywords
20-hydroxyecdysone, non-genomic steroid action, G protein-coupled receptors, Helicoverpa armigera, plasma membrane receptors, larval-pupal transition, molting, calcium signaling, calponin, T-type calcium channels, canonical transient receptor potential channels, steroid hormone signaling
MeSH terms
Calponins, Amino Acid Sequence, Animals, Base Sequence, Calcium, Calcium-Binding Proteins, Cell Membrane, Cells, Cultured, Ecdysterone, Lepidoptera, Microfilament Proteins, Molecular Sequence Data, Insect Proteins, Calcium Signaling, Receptors, G-Protein-Coupled

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Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-10.

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