G protein-coupled receptors function as cell membrane receptors for the steroid hormone 20-hydroxyecdysone

May combine two claims.

GPCRs can bind 20E in the cell membrane and after being isolated, suggesting GPCRs as cell membrane receptors of 20E.

“GPCRs can bind 20E in the cell membrane and after being isolated, suggesting GPCRs as cell membrane receptors of 20E.”

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

GPCRs are cell membrane receptors for various ligands.

“G protein-coupled receptors (GPCRs) are cell membrane receptors for various ligands.”

• Also stated in: § Background · plain

  “GPCRs have been identified as the cell membrane receptors of various ligands, including biological amines, amino acids, ions, lipids, peptides/proteins, light, odorant, pheromones, nucleotides, and opiates [5].”

• cites [5] Marinissen MJ, Gutkind JS. G-protein-coupled receptors and signaling networks: emerging paradigms. Trends Pharmacol Sci (1184). 2001;22:368–76. sourceNot checked yet

  Not checked in the free run.

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

Extensive evidence indicates that animal steroids activate receptors on the cell membrane.

“Extensive evidence indicates that animal steroids activate receptors on the cell membrane.”

May combine two claims.

20E induces a signal via GPCRs to regulate rapid cellular responses, including rapid Ca2+ release from the endoplasmic reticulum and influx from the extracellular medium, rapid protein phosphorylation, and subcellular translocation.

“Studies indicate that 20E induces a signal via GPCRs to regulate rapid cellular responses, including rapid Ca2+ release from the endoplasmic reticulum and influx from the extracellular medium, as well as rapid protein phosphorylation and subcellular translocation.”

Classified as probably not a substantial claim. May combine two claims.

Certain GPCRs bind steroid hormones; GPER1 binds estrogen in humans and DopEcR binds 20E in insects.

“Certain GPCRs have been shown to bind steroid hormones, for example, G protein-coupled estrogen receptor 1 (GPER1) binds estrogen in humans, and Drosophila dopamine/ecdysteroid receptor (DopEcR) binds the molting hormone 20-hydroxyecdysone (20E) in insects.”

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

Estrogen activates PI3K to recruit AKT/PKB to the cell membrane in mammals via a mechanism independent of the genomic actions of hormones.

“For example, estrogen activates phosphoinositide 3 kinase (PI3K) to recruit protein kinase B (AKT/PKB) to the cell membrane in mammals via a mechanism independent of the genomic actions of hormones [14].”

• cites [14] Aronica SM, Kraus WL, Katzenellenbogen BS. Estrogen action via the cAMP signaling pathway: stimulation of adenylate cyclase and cAMP-regulated gene transcription. Proc Natl Acad Sci U S A. 1994;91:8517–21. sourceNot checked yet

  Not checked in the free run.

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

Plant steroid hormones, brassinosteroids, initiate signaling by combining with plasma membrane receptors.

“However, the plant steroid hormones, brassinosteroids, initiate signaling by combining with plasma membrane receptors [11].”

• cites [11] Bishop GJ, Koncz C. Brassinosteroids and plant steroid hormone signaling. Plant Cell. 2002;14:S97–110. sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Animal steroid hormones exert their actions via binding to their intracellular nuclear receptors.

“Animal steroid hormones are known exert their actions via binding to their intracellular nuclear receptors [7], for example, estrogen binds to its nuclear estrogen receptors ERα and ERβ [8], androgens bind to androgen receptors (AR) [9], and glucocorticoids bind to glucocorticoid receptors [10].”

• cites [7] Weigel NL, Moore NL. Kinases and protein phosphorylation as regulators of steroid hormone action. Nucl Recept Signal. 2007;5:e005. sourceNot checked yet

  Not checked in the free run.

• cites [8] Dahlman-Wright K, Cavailles V, Fuqua SA, Jordan VC, Katzenellenbogen JA, Korach KS, et al. International union of pharmacology. LXIV. Estrogen receptors. Pharmacol Rev (18393). 2006;58:773–81. sourceNot checked yet

  Not checked in the free run.

• cites [9] Aarnisalo P, Palvimo JJ, Janne OA. CREB-binding protein in androgen receptor-mediated signaling. Proc Natl Acad Sci U S A. 1998;95:2122–7. sourceNot checked yet

  Not checked in the free run.

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

Pregnenolone regulates gene expression via a nuclear receptor-mediated genomic pathway and via a TRP cation channel-mediated nongenomic pathway.

“Pregnenolone, the precursor of androgens, estrogens, progesterone, mineralocorticoids, and glucocorticoids [15], regulates gene expression via a nuclear receptor-mediated genomic pathway and via a transient receptor potential (TRP) cation channels-mediated nongenomic pathway [16].”

• cites [16] Aquila S, Sisci D, Gentile M, Middea E, Catalano S, Carpino A, et al. Estrogen receptor (ER) alpha and ER beta are both expressed in human ejaculated spermatozoa: evidence of their direct interaction with phosphatidylinositol-3-OH kinase/Akt pathway. J Clin Endocrinol Metab. 2004;89:1443–51. sourceNot checked yet

  Not checked in the free run.

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

Several GPCRs are reported as progesterone receptors (mPRα, mPRβ, mPRγ, mPRδ, and mPRε).

“Several GPCRs are reported as progesterone receptors (mPRα, mPRβ, mPRγ, mPRδ, and mPRε) [17].”

• cites [17] Kasubuchi M, Watanabe K, Hirano K, Inoue D, Li X, Terasawa K, et al. Membrane progesterone receptor beta (mPRbeta/Paqr8) promotes progesterone-dependent neurite outgrowth in PC12 neuronal cells via non-G protein-coupled receptor (GPCR) signaling. Sci Rep. 2017;7:5168. sourceNot checked yet

  Not checked in the free run.

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

Estrogen transmits signals via GPERs to transactivate epidermal growth factor receptors for cell proliferation in female reproductive cancers.

“Estrogen transmits signals via GPERs to transactivate epidermal growth factor receptors for cell proliferation in female reproductive cancers [26].”

• cites [26] 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 (4159). 2012;153:2953–62. sourceNot checked yet

  Not checked in the free run.

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

Testosterone mediates nongenomic effects via a calcium and amino acid sensing GPCR (GPRC6A).

“Testosterone mediates nongenomic effects via a calcium and amino acid sensing GPCR (GPRC6A) [22].”

• cites [22] Pi M, Parrill AL, Quarles LD. GPRC6A mediates the non-genomic effects of steroids. J Biol Chem. 2010;285:39953–64. sourceNot checked yet

  Not checked in the free run.

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

GPR30, renamed GPER1, transmits estrogen signals from the membrane.

“The estrogen receptor GPCR (GPR30) [23], which transmits estrogen signals from the membrane [24], has been renamed as G protein-coupled estrogen receptor 1 (GPER1) [25].”

• cites [23] Revankar CM, Cimino DF, Sklar LA, Arterburn JB, Prossnitz ER. A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science. 2005;307:1625–30. sourceNot checked yet

  Not checked in the free run.

• cites [24] Prossnitz ER, Maggiolini M. Mechanisms of estrogen signaling and gene expression via GPR30. Mol Cell Endocrinol. 2009;308:32–8. sourceNot checked yet

  Not checked in the free run.

• cites [25] Maggiolini M, Picard D. The unfolding stories of GPR30, a new membrane-bound estrogen receptor. J Endocrinol. 2010;204:105–14. sourceNot checked yet

  Not checked in the free run.

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

20E binds to its nuclear receptor EcR to exert its effect on gene transcription in the classical genomic pathway.

“20E binds to its nuclear receptor, ecdysone receptor (EcR), to exert its effect on gene transcription in the classical genomic pathway.”

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

Androgen transmits signals via cell membrane receptors that are distinct from the androgen nuclear receptors.

“Androgen transmits signals via cell membrane receptors [19], which are distinct from the androgen nuclear receptors [20].”

• cites [19] Michels G, Hoppe UC. Rapid actions of androgens. Front Neuroendocrinol. 2008;29:182–98. sourceNot checked yet

  Not checked in the free run.

• cites [20] Bennett NC, Gardiner RA, Hooper JD, Johnson DW, Gobe GC. Molecular cell biology of androgen receptor signalling. Int J Biochem Cell Biol. 2010;42:813–27. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The evidence suggests that 20E transmits signals via cell membrane receptors and that a nongenomic pathway exists.

“The evidence suggests that 20E transmits signals via cell membrane receptors and that a nongenomic pathway exists.”

• AssumptionRests on an assumption: The cell membrane receptor-mediated nongenomic pathway of 20E is not fully understood.

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

The EcR/USP complex binds to ecdysone response elements (EcRE) to regulate 20E-responsive gene transcription, such as HR3.

“This complex binds to ecdysone response elements (EcRE) to regulate 20E-responsive gene transcription [36], such as hormone receptor 3 (HR3), an early-late gene in the 20E pathway [38].”

• cites [36] Riddiford LM, Hiruma K, Zhou X, Nelson CA. Insights into the molecular basis of the hormonal control of molting and metamorphosis from Manduca sexta and Drosophila melanogaster. Insect Biochem Mol Biol. 2003;33:1327–38. sourceNot checked yet

  Not checked in the free run.

• cites [38] Liu W, Cai MJ, Wang JX, Zhao XF. In a nongenomic action, steroid hormone 20-hydroxyecdysone induces phosphorylation of cyclin-dependent kinase 10 to promote gene transcription. Endocrinology (4159). 2014;155:1738–50. sourceNot checked yet

  Not checked in the free run.

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

Earlier evidence that 20E triggers rapid nongenomic actions before gene transcription was obtained in studies of the anterior silk gland of Bombyx mori.

“The earlier evidence that 20E triggers rapid nongenomic actions before gene transcription was obtained in studies of the anterior silk gland of Bombyx mori.”

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

AipsDopEcR is involved in the expression of sexual behavior in the male moth.

“AipsDopEcR is involved in the expression of sexual behavior in the male moth [44].”

• cites [44] Abrieux A, Debernard S, Maria A, Gaertner C, Anton S, Gadenne C, et al. Involvement of the G-protein-coupled dopamine/ecdysteroid receptor DopEcR in the behavioral response to sex pheromone in an insect. PLoS One. 2013;8:e72785. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

20E triggers intracellular rapid increases in Ca2+ and cAMP, and increases ERK phosphorylation in DmDopEcR overexpressing Sf9 cells.

“In addition, 20E triggers intracellular rapid increases in Ca2+ and cyclic adenosine monophosphate (cAMP), and increases ERK phosphorylation in the DmDopEcR overexpressing Sf9 cells [42].”

• cites [42] Srivastava DP, Yu EJ, Kennedy K, Chatwin H, Reale V, Hamon M, et al. Rapid, nongenomic responses to ecdysteroids and catecholamines mediated by a novel Drosophila G-protein-coupled receptor. J Neurosci. 2005;25:6145–55. sourceNot checked yet

  Not checked in the free run.

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

The plasma membrane can bind [3H] ponasterone A, suggesting the existence of an unknown membrane receptor.

“The plasma membrane can bind [3H] ponasterone A ([3H] Pon A), suggesting the existence of an unknown membrane receptor [39].”

• cites [39] 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;271:3171–9. sourceNot checked yet

  Not checked in the free run.

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

EcR must interact with USP/RXR to form a heterodimeric transcription complex, EcR/USP.

“EcR must interact with the ultraspiracle protein (USP), retinoid X receptor (RXR) in Hemimetabola, the ortholog of the retinoid X receptor in vertebrates, to form a heterodimeric transcription complex, EcR/USP [37].”

• cites [37] Oro AE, McKeown M, Evans RM. Relationship between the product of the Drosophila ultraspiracle locus and the vertebrate retinoid X receptor. Nature (38138). 1990;347:298–301. sourceNot checked yet

  Not checked in the free run.

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

20E initiates insect larval molting from one instar to the next and metamorphic molting from larva to adult.

“20-hydroxyecdysone (20E), which is also known as the insect molting hormone, initiates insect larval molting from one instar to the next (molting), or the metamorphic molting from larva to adult (metamorphosis) [35, 36].”

• cites [35] Gilbert LI, Granger NA, Roe RM. The juvenile hormones: historical facts and speculations on future research directions. Insect Biochem Mol Biol. 2000;30:617–44. sourceNot checked yet

  Not checked in the free run.

• cites [36] Riddiford LM, Hiruma K, Zhou X, Nelson CA. Insights into the molecular basis of the hormonal control of molting and metamorphosis from Manduca sexta and Drosophila melanogaster. Insect Biochem Mol Biol. 2003;33:1327–38. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

ErGPCR-1 and ErGPCR-2 are 20E-responsive GPCRs revealed in Helicoverpa armigera.

“The 20E-responsive GPCR (initially designated ErGPCR, and later, ErGPCR-1) [47] and ErGPCR-2 [48] are further revealed in Helicoverpa armigera.”

• cites [47] Cai MJ, Dong DJ, Wang Y, Liu PC, Liu W, Wang JX, et al. G-protein-coupled receptor participates in 20-hydroxyecdysone signaling on the plasma membrane. Cell Commun Signal (3661). 2014;12:9. sourceNot checked yet

  Not checked in the free run.

• cites [48] Wang D, Zhao WL, Cai MJ, Wang JX, Zhao XF. G-protein-coupled receptor controls steroid hormone signaling in cell membrane. Sci Rep. 2015;5:8675. sourceNot checked yet

  Not checked in the free run.

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

GPER1-mediated nongenomic activity is independent of the estrogen nuclear receptor.

“GPER1-mediated nongenomic activity is independent of the estrogen nuclear receptor [26].”

• cites [26] 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 (4159). 2012;153:2953–62. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The function of PLCG1 in the 20E signaling pathway is mediated via ErGPCR-1, Gαq, and Src-family kinases, and 20E mediates tyrosine phosphorylation at the SH2 domain of PLCG1.

“Studies in H. armigera demonstrate that the function of PLCG1 in the 20E signaling pathway is mediated via ErGPCR-1, Gαq, and Src-family kinases, and that 20E mediates tyrosine phosphorylation at the SH2 domain of PLCG1.”

May combine two claims.

Activated-PLCG1 migrates toward the cell membrane to initiate intracellular Ca2+ signaling and calcium channel-controlled Ca2+ influx, which triggers PKC-mediated USP phosphorylation to modulate USP binding to EcRE for subsequent gene transcription.

“Activated-PLCG1 migrates toward the cell membrane to initiate intracellular Ca2+ signaling and calcium channel-controlled Ca2+ influx, which triggers PKC-mediated USP phosphorylation to modulate USP binding to EcRE for subsequent gene transcription.”

May combine two claims.

ErGPCR-2 has a similar function to ErGPCR-1, such as regulation of rapid increases in intracellular Ca2+ levels and phosphorylation of USP.

“ErGPCR-2 has a similar function to ErGPCR-1, such as regulation of rapid increases in intracellular Ca2+ levels, and phosphorylation of USP [48].”

• cites [48] Wang D, Zhao WL, Cai MJ, Wang JX, Zhao XF. G-protein-coupled receptor controls steroid hormone signaling in cell membrane. Sci Rep. 2015;5:8675. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

USP lysine acetylation at amino acid 303 is necessary for its interaction with EcR to form the transcription complex and for the binding of the EcR-USP complex to EcRE.

“This modification is necessary for its interaction with EcR to form the transcription complex and for the binding of the EcR-USP complex to EcRE [56].”

• cites [56] Jing YP, Liu W, Wang JX, Zhao XF. The steroid hormone 20-hydroxyecdysone via nongenomic pathway activates Ca2+/calmodulin-dependent protein kinase II to regulate gene expression. J Biol Chem. 2015;290:8469–81. sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

G proteins directly transmit GPCR signals.

“G proteins directly transmit GPCR signals [1].”

• cites [1] Preininger AM, Meiler J, Hamm HE. Conformational flexibility and structural dynamics in GPCR-mediated G protein activation: a perspective. J Mol Biol (4517). 2013;425:2288–98. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

ErGPCR-1 is essential for 20E pathway gene expression and larval-pupal transition.

“ErGPCR-1 is essential for 20E pathway gene expression and larval-pupal transition.”

May combine two claims.

20E induces a rapid increase in cytosolic Ca2+ levels and promotes calponin nuclear translocation and phosphorylation via ErGPCR-1.

“20E induces a rapid increase in cytosolic Ca2+ levels and promotes calponin nuclear translocation and phosphorylation via ErGPCR-1 [47].”

• cites [47] Cai MJ, Dong DJ, Wang Y, Liu PC, Liu W, Wang JX, et al. G-protein-coupled receptor participates in 20-hydroxyecdysone signaling on the plasma membrane. Cell Commun Signal (3661). 2014;12:9. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

ErGPCR-2 and DopEcR in H. armigera could bind 20E in the cell membrane or as isolated proteins, using a 20E enzyme immunoassay.

“A recent study shows that ErGPCR-2 and DopEcR in H. armigera could bind 20E in the cell membrane or as isolated proteins, using a 20E enzyme immunoassay (20E-EIA).”

May combine two claims.

20E competes with dopamine to bind to DopEcR to block dopamine-mediated motor function and reward-motivated behavior, and initiates the 20E pathway.

“That study also demonstrates one of the mechanisms by which 20E represses larval feeding and promotes metamorphosis: 20E competes with dopamine to bind to DopEcR to block the dopamine-mediated motor function and reward-motivated behavior, and initiates the 20E pathway [49].”

• cites [49] Kang XL, Zhang JY, Wang D, Zhao YM, Han XL, Wang JX, et al. The steroid hormone 20-hydroxyecdysone binds to dopamine receptor to repress lepidopteran insect feeding and promote pupation. PLoS Genet. 2019;15:e1008331. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

High levels of 20E switch autophagy to apoptosis in the H. armigera midgut by increasing the Ca2+ levels in cells, thereby inducing apoptosis.

“The high levels of 20E switches autophagy to apoptosis in the H. armigera midgut by increasing the Ca2+ levels in cells [59], thereby inducing apoptosis [60].”

• cites [59] Li YB, Li XR, Yang T, Wang JX, Zhao XF. The steroid hormone 20-hydroxyecdysone promotes switching from autophagy to apoptosis by increasing intracellular calcium levels. Insect Biochem Mol Biol. 2016;79:73–86. sourceNot checked yet

  Not checked in the free run.

• cites [60] Wang D, Pei XY, Zhao WL, Zhao XF. Steroid hormone 20-hydroxyecdysone promotes higher calcium mobilization to induce apoptosis. Cell Calcium. 2016;60:1–12. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

RNAi-mediated silencing of PLCG1 blocks 20E-induced pupation, larval death, and pupation.

“In that study, RNAi-mediated silencing of PLCG1 blocks 20E-induced pupation, larval death, and pupation.”

May combine two claims.

20E, through GPCRs, induces intracellular Ca2+ release, which causes STIM1 phosphorylation and aggregation.

“20E, through GPCRs, induces intracellular Ca2+ release, which causes stromal interaction molecule 1 (STIM1) phosphorylation and aggregation.”

May combine two claims.

CaMKII expression and phosphorylation increase during metamorphosis in H. armigera.

“CaMKII expression and phosphorylation increase during metamorphosis in H. armigera [56].”

• cites [56] Jing YP, Liu W, Wang JX, Zhao XF. The steroid hormone 20-hydroxyecdysone via nongenomic pathway activates Ca2+/calmodulin-dependent protein kinase II to regulate gene expression. J Biol Chem. 2015;290:8469–81. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

20E regulates phosphorylation of CaMKII at threonine 290, which induces CaMKII translocation into the nucleus.

“20E regulates phosphorylation of CaMKII at threonine 290, which induces CaMKII translocation into the nucleus.”

May combine two claims.

RNAi-mediated CaMKII knockdown prevents larval-pupal transition and 20E-responsive gene expression.

“RNAi-mediated CaMKII knockdown prevents larval-pupal transition and 20E-responsive gene expression.”

May combine two claims.

Gαq participates in the 20E-induced increase in intracellular Ca2+ levels and is necessary for larval development, metamorphosis, and 20E pathway gene expression, and plays roles downstream of ErGPCR-1 in 20E signaling.

“Gαq participates in the 20E-induced increase in intracellular Ca2+ levels and is necessary for larval development, metamorphosis, and 20E pathway gene expression, and plays roles downstream of ErGPCR-1 in 20E signaling [50].”

• cites [50] Ren J, Li XR, Liu PC, Cai MJ, Liu W, Wang JX, et al. G-protein alphaq participates in the steroid hormone 20-hydroxyecdysone nongenomic signal transduction. J Steroid Biochem Mol Biol. 2014;144:313–23. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Interference with Arrb1 by dsRNA injection into larvae causes advanced pupation and a chimeric larva-pupa phenotype.

“Further study showed that in contrast to the 20E-promoted pupation, interference with Arrb1 (encoding β-arrestin-1) by dsRNA injection into larvae causes advanced pupation and a chimeric larva-pupa phenotype.”

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

Ecdysone is the precursor of 20E.

“Ecdysone (E) is the precursor of 20E [70].”

• cites [70] Riddiford LM, Cherbas P, Truman JW. Ecdysone receptors and their biological actions. Vitam Horm. 2001;60:1–73.Not checked yet

  Not checked in the free run.

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

Recent work suggests that 20E entry into cells is controlled by a 12 transmembrane protein, Ecdysone Importer (EcI), in Drosophila.

“Recent work suggests that 20E entry into cells is controlled by a 12 transmembrane protein, Ecdysone Importer (EcI), in Drosophila [67].”

• cites [67] Okamoto N, Viswanatha R, Bittar R, Li Z, Haga-Yamanaka S, Perrimon N, et al. A membrane transporter is required for steroid hormone uptake in Drosophila. Dev Cell. 2018;47:294–305 e297. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Through ErGPCR2, 20E regulates PKAC1 phosphorylation and its nuclear translocation.

“Through ErGPCR2, 20E regulates PKAC1 phosphorylation and its nuclear translocation.”

May combine two claims.

PKAC1 expression increases during metamorphosis, and PKAC1 knockdown blocks pupation and represses 20E-responsive gene expression.

“The expression of the catalytic subunit 1 of PKA (PKAC1) increases during metamorphosis, and that PKAC1 knockdown blocks pupation and represses 20E-responsive gene expression.”

May combine two claims.

GRK2 interacts with ErGPCR-2 and phosphorylates ErGPCR-2 to induce its internalization.

“GRK2 then interacts with ErGPCR-2 and phosphorylates ErGPCR-2 to induce its internalization.”

May combine two claims.

β-arrestin-1 depletion increases the mRNA levels of 20E-responsive genes, while their levels are decreased by Arrb1 mRNA overexpression.

“β-arrestin-1 depletion increases the mRNA levels of 20E-responsive genes, while their levels are decreased by Arrb1 mRNA overexpression.”

May combine two claims.

Via a negative feedback mechanism, 20E induces β-arrestin-1 phosphorylation and cell membrane migration, which blocks 20E signaling by the interaction between β-arrestin-1 and ErGPCR-1.

“Therefore, via negative feedback mechanism, 20E induces β-arrestin-1 phosphorylation and cell membrane migration, which blocks 20E signaling by the interaction between β-arrestin-1 and ErGPCR-1 [62].”

• cites [62] Zhang XQ, Li XR, Ren J, Li YB, Cai MJ, Wang JX. Zhao XF: beta-Arrestin1 interacts with G protein-coupled receptor to desensitize signaling of the steroid hormone 20-hydroxyecdysone in the lepidopteran insect Helicoverpa armigera. Cell Signal. 2015;27:878–86. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

PKAC1 induces the phosphorylation of CREB at serine 143, which allows it to bind to the cAMP response element (CRE) to enhance 20E-responsive gene transcription.

“PKAC1 induces the phosphorylation of cAMP response element-binding protein (CREB) at serine 143, which allows it to bind to the cAMP response element (CRE) to enhance 20E-responsive gene transcription.”

May combine two claims.

Via ErGPCR1, 20E regulates β-arrestin-1 phosphorylation at serines 170 and 234, and mutation of these residues inhibits 20E-induced β-arrestin-1 migration to the cell membrane.

“Via ErGPCR1, 20E regulates β-arrestin-1 phosphorylation at serines 170 and 234, and mutation of these residues inhibits 20E-induced β-arrestin-1 migration to the cell membrane.”

May combine two claims.

Through ErGPCR2, 20E increases cellular cAMP levels, which induces PKA-mediated CREB phosphorylation and, in turn, promotes 20E-responsive gene expression.

“Through ErGPCR2, 20E increases cellular cAMP levels, which induces PKA-mediated CREB -phosphorylation and, in turn, promotes 20E-responsive gene expression.”

May combine two claims.

20E stimulates a rapid increase in cAMP levels and activates the protein kinase A (PKA) pathway in H. armigera.

“In addition to triggering rapid increases in intracellular Ca2+ levels to activate the PKC pathway, 20E also stimulates a rapid increase in cAMP levels and activates the protein kinase A (PKA) pathway in H. armigera [61].”

• cites [61] Jing YP, Wang D, Han XL, Dong DJ, Wang JX, Zhao XF. The steroid hormone 20-hydroxyecdysone enhances gene transcription through the cAMP response element-binding protein (CREB) signaling pathway. J Biol Chem. 2016;291:12771–85. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

GRK2 is phosphorylated at serine 680 by PKC after induction by 20E, which leads to the translocation of GRK2 to the cell membrane.

“GRK2 is phosphorylated at serine 680 by PKC after induction by 20E, which leads to the translocation of GRK2 to the cell membrane.”

May combine two claims.

The genomic action/pathway of 20E is regulated by the nongenomic action/pathway because the 20E-induced rapid calcium increase in cells activates protein kinases, thereby promoting EcR-USP transcription complex formation, which initiates gene transcription in the 20E pathway.

“From the studies in H. armigera, the genomic action/pathway of 20E is regulated by the nongenomic action/pathway, because the 20E-induced rapid calcium increase in cells activates protein kinases [52, 56], therefore promotes EcR-USP transcription complex formation, which initiates gene transcription in 20E pathway finally [64].”

• cites [52] Liu W, Cai MJ, Zheng CC, Wang JX, Zhao XF. Phospholipase C gamma 1 connects the cell membrane pathway to the nuclear receptor pathway in insect steroid hormone signaling. J Biol Chem. 2014;289:13026–41. sourceNot checked yet

  Not checked in the free run.

• cites [56] Jing YP, Liu W, Wang JX, Zhao XF. The steroid hormone 20-hydroxyecdysone via nongenomic pathway activates Ca2+/calmodulin-dependent protein kinase II to regulate gene expression. J Biol Chem. 2015;290:8469–81. sourceNot checked yet

  Not checked in the free run.

• cites [64] Chen CH, Pan J, Di YQ, Liu W, Hou L, Wang JX, et al. Protein kinase C delta phosphorylates ecdysone receptor B1 to promote gene expression and apoptosis under 20-hydroxyecdysone regulation. Proc Natl Acad Sci U S A. 2017;114:E7121–30. sourceNot checked yet

  Not checked in the free run.

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

Different concentrations of 20E in cells induce different gene transcription via EcR.

“suggesting that the different concentrations of 20E in cells induce different gene transcription via EcR, and 20E entry into cells is controlled by some unknown mechanism, which needs further study.”

• cites [69] Di YQ, Han XL, Kang XL, Wang D, Chen CH, Wang JX, et al. Autophagy triggers CTSD (cathepsin D) maturation and localization inside cells to promote apoptosis. Autophagy. 2020;1:1–23.Not checked yet

  Not checked in the free run.

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

20E is an ecdysteroid hydroxylated at the 20th carbon in addition to other carbons.

“20E is an ecdysteroid hydroxylated at the 20th carbon in addition to other carbons [71].”

• cites [71] Gilbert LI. Halloween genes encode P450 enzymes that mediate steroid hormone biosynthesis in Drosophila melanogaster. Mol Cell Endocrinol. 2004;215:1–10. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

GRK2 knockdown in larvae causes accelerated pupation, an increase in 20E-responsive gene expression, and advanced apoptosis and metamorphosis.

“GRK2 knockdown in larvae causes accelerated pupation, an increase in 20E-responsive gene expression, and advanced apoptosis and metamorphosis.”

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

20E, which is produced in both insects and plants, such as Cyanotis vaga, can be used to disrupt the development of insect pests.

“20E, which is produced in both insects and plants, such as Cyanotis vaga, can be used to disrupt the development of insect pests [86].”

• cites [86] Thummel CS, Chory J. Steroid signaling in plants and insects--common themes, different pathways. Genes Dev. 2002;16:3113–29. sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

The challenge to achieving full elucidation of the steroid hormone-GPCR pathway is critically dependent on establishing a method to detect the highly dynamic conformations of GPCRs and their interaction with steroids.

“The challenge to achieving full elucidation of the steroid hormone-GPCR pathway is critically dependent on establishing a method to detect the highly dynamic conformations of GPCRs and their interaction with steroids.”

Classified as probably not a substantial claim. May combine two claims.

Recent researches in insects have revealed the axis of the 20E signaling pathway via GPCRs, which will encourage the study of steroid hormone signaling pathways that act via GPCRs.

“Recent researches in insects have revealed the axis of the 20E signaling pathway via GPCRs, which will encourage the study of steroid hormone signaling pathways that act via GPCRs.”

• AssumptionRests on an assumption: The GPCR-mediated mechanism of 20E action in insects is representative of steroid hormone signaling via GPCRs in other organisms.

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

GPCRs undergo highly dynamic structural changes during signal transduction upon ligand binding and interactions with intracellular effectors.

“GPCRs undergo highly dynamic structural changes during signal transduction upon the binding of their ligands and interactions with intracellular effectors [77].”

• cites [77] Miao Y, McCammon JA. G-protein coupled receptors: advances in simulation and drug discovery. Curr Opin Struct Biol. 2016;41:83–9. sourceNot checked yet

  Not checked in the free run.

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

GPR30 is suggested as the cell membrane receptor for the steroid hormone estrogen by detection of the binding of fluorescence-labeled estrogen in Cos7 cells.

“GPR30 is suggested as the cell membrane receptor for the steroid hormone estrogen by detection of the binding of fluorescence-labeled estrogen in Cos7 cells [25].”

• cites [25] Maggiolini M, Picard D. The unfolding stories of GPR30, a new membrane-bound estrogen receptor. J Endocrinol. 2010;204:105–14. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

20E-EIA detects the binding of GPCRs to 20E directly, presenting an alternative method to that using the isotope-labeled analog Pon A.

“20E-EIA detects the binding of GPCRs to 20E directly, presenting an alternative method to that using the isotope-labeled analog Pon A [49].”

• cites [49] Kang XL, Zhang JY, Wang D, Zhao YM, Han XL, Wang JX, et al. The steroid hormone 20-hydroxyecdysone binds to dopamine receptor to repress lepidopteran insect feeding and promote pupation. PLoS Genet. 2019;15:e1008331. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

20E binds to GPCRs to trigger the PKA and PKC pathways via Gαs and Gαq, respectively.

“20E binds to GPCRs to trigger the PKA and PKC pathways via Gαs and Gαq, respectively.”

May combine two claims.

Ecdysone accelerates metamorphic timing and results in elevation of lethality during metamorphosis in D. melanogaster, suggesting both 20E and E are essential for the regulation of metamorphic timing in D. melanogaster.

“Although 20E is the active molting hormone and ecdysone is relatively inactive, E accelerates the metamorphic timing and results in elevation of lethality during metamorphosis in D. melanogaster, suggesting both 20E and E are essential for the regulation of metamorphic timing in D. melanogaster [72].”

• cites [72] Ono H. Ecdysone differentially regulates metamorphic timing relative to 20-hydroxyecdysone by antagonizing juvenile hormone in Drosophila melanogaster. Dev Biol. 2014;391:32–42. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

DopEcR in H. armigera can bind 20E in the cell membrane or as an isolated protein.

“DopEcR in H. armigera can bind 20E in the cell membrane or as an isolated protein [49].”

• cites [49] Kang XL, Zhang JY, Wang D, Zhao YM, Han XL, Wang JX, et al. The steroid hormone 20-hydroxyecdysone binds to dopamine receptor to repress lepidopteran insect feeding and promote pupation. PLoS Genet. 2019;15:e1008331. sourceNot checked yet

  Not checked in the free run.

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

In H. armigera, high concentrations of 20E induce Ca2+ influx to switch autophagy to apoptosis in the midgut.

“In H. armigera, high concentrations of 20E induce Ca2+ influx to switch autophagy to apoptosis in the midgut [59, 60].”

• cites [59] Li YB, Li XR, Yang T, Wang JX, Zhao XF. The steroid hormone 20-hydroxyecdysone promotes switching from autophagy to apoptosis by increasing intracellular calcium levels. Insect Biochem Mol Biol. 2016;79:73–86. sourceNot checked yet

  Not checked in the free run.

• cites [60] Wang D, Pei XY, Zhao WL, Zhao XF. Steroid hormone 20-hydroxyecdysone promotes higher calcium mobilization to induce apoptosis. Cell Calcium. 2016;60:1–12. sourceNot checked yet

  Not checked in the free run.

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

Both ecdysone and 20E exist in plants to play roles in the plants or participate in the defense of plants against insects.

“Both E and 20E exist in plants to play roles in the plants or participate in the defense of plants against insects [73].”

• cites [73] Dinan L. Phytoecdysteroids: biological aspects. Phytochemistry. 2001;57:325–39. sourceNot checked yet

  Not checked in the free run.

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

Oligomerization of GPCRs results in the diversification of receptor signaling.

“Oligomerization of GPCRs results in the diversification of receptor signaling [84].”

• cites [84] Van Craenenbroeck K. GPCR oligomerization: contribution to receptor biogenesis. Subcell Biochem. 2012;63:43–65. sourceNot checked yet

  Not checked in the free run.

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

20E regulates gene transcription via its nuclear receptor EcR in H. armigera.

“20E regulates gene transcription via its nuclear receptor EcR in H. armigera [64].”

• cites [64] Chen CH, Pan J, Di YQ, Liu W, Hou L, Wang JX, et al. Protein kinase C delta phosphorylates ecdysone receptor B1 to promote gene expression and apoptosis under 20-hydroxyecdysone regulation. Proc Natl Acad Sci U S A. 2017;114:E7121–30. sourceNot checked yet

  Not checked in the free run.

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

DmDopEcR in Drosophila binds the 20E analog [3H]-labeled Pon A.

“DmDopEcR in Drosophila is reported to bind the 20E analog [3H]-labeled Pon A [30].”

• cites [30] Srivastava DP, Evans PD. G-protein oestrogen receptor 1: trials and tribulations of a membrane oestrogen receptor. J Neuroendocrinol (3172). 2013;25:1219–30. sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S4 (based).

20E signaling is terminated by desensitization of GPCRs via a negative feedback mechanism.

“20E signaling is terminated by desensitization of GPCRs via a negative feedback mechanism.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

GPCRs represent targets to develop new types of insecticides.

“In addition, GPCRs represent targets to develop new types of insecticides.”

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

GPCRs function not as a single molecule, but in the form of homo- or hetero-oligomers.

“GPCRs are considered to function not as a single molecule, but in the form of homo- or hetero-oligomers [82].”

• cites [82] Farran B. An update on the physiological and therapeutic relevance of GPCR oligomers. Pharmacol Res (4816). 2017;117:303–27. sourceNot checked yet

  Not checked in the free run.

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

Ligand binding to beta-adrenergic receptors results in a conformational change that activates Gs protein complexes.

“For example, ligand binding to beta-adrenergic receptors (ARs) results in a conformational change that activates Gs protein complexes [78].”

• cites [78] Rasmussen SG, DeVree BT, Zou Y, Kruse AC, Chung KY, Kobilka TS, et al. Crystal structure of the beta2 adrenergic receptor-Gs protein complex. Nature. 2011;477:549–55. sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S2 (checkable).

The identification of GPCRs as 20E cell membrane receptors will improve our understanding of the roles of 20E and its mechanism of action in mammals and in insects.

“The identification of GPCRs as 20E cell membrane receptors will improve our understanding of the roles of 20E and its mechanism of action in mammals and in insects.”

• AssumptionRests on an assumption: The GPCR-mediated mechanism of 20E action in insects is representative of steroid hormone signaling via GPCRs in other organisms.

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

GPCRs exist as an ensemble of temporally interchanging conformations.

“GPCRs exist as an ensemble of temporally interchanging conformations [85].”

• cites [85] Tian H, Furstenberg A, Huber T. Labeling and single-molecule methods to monitor G protein-coupled receptor dynamics. Chem Rev (37369). 2017;117:186–245. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Via the GPCR-mediated nongenomic pathway, 20E regulates the phosphorylation and lysine acetylation of nuclear transcription factors to form the transcription complex EcR/USP and binding to EcRE for further gene transcription.

“Via the GPCR-mediated nongenomic pathway, 20E regulates the phosphorylation and lysine acetylation of nuclear transcription factors to form the transcription complex, EcR/USP, and the binding to EcRE for further gene transcription”