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Cited by 102Ubiquitin and proteasome pathwaysIon Channels and ReceptorsCellular transport and secretion

RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation

Open access
Lu, Justine P., Wang, Yuan, Sliter, Danielle A., Pearce, Margaret M.P., Wojcikiewicz, Richard J.H.·2011·Journal of Biological Chemistry
DOI 10.1074/jbc.m111.251983PMC PMC3129221
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Abstract

Inositol 1,4,5-trisphosphate (IP(3)) receptors are endoplasmic reticulum membrane calcium channels that, upon activation, are degraded via the ubiquitin-proteasome pathway. While searching for novel mediators of IP(3) receptor processing, we discovered that RNF170, an uncharacterized RING domain-containing protein, associates rapidly with activated IP(3) receptors. RNF170 is predicted to have three membrane-spanning helices, is localized to the ER membrane, and possesses ubiquitin ligase activity. Depletion of endogenous RNF170 by RNA interference inhibited stimulus-induced IP(3) receptor ubiquitination, and degradation and overexpression of a catalytically inactive RNF170 mutant suppressed stimulus-induced IP(3) receptor processing. A substantial proportion of RNF170 is constitutively associated with the erlin1/2 (SPFH1/2) complex, which has been shown previously to bind to IP(3) receptors immediately after their activation. Depletion of RNF170 did not affect the binding of the erlin1/2 complex to stimulated IP(3) receptors, whereas erlin1/2 complex depletion inhibited RNF170 binding. These results suggest a model in which the erlin1/2 complex recruits RNF170 to activated IP(3) receptors where it mediates IP(3) receptor ubiquitination. Thus, RNF170 plays an essential role in IP(3) receptor processing via the ubiquitin-proteasome pathway.

AI snapshot

Written from the paper’s full text.

TL;DR

RNF170 is identified as an ER membrane RING-domain ubiquitin ligase that associates with activated IP3 receptors and mediates their ubiquitination and degradation. The erlin1/2 complex appears to recruit RNF170 to activated IP3 receptors, linking recognition to ERAD processing.

Key findings

  • RNF170 was found as a 21.5-kDa protein that binds rapidly to activated IP3R1 in GnRH-stimulated αT3-1 cells and carbachol-stimulated SH-SY5Y cells, with kinetics similar to erlin2 binding and before IP3R1 polyubiquitination/p97 binding.
  • RNF170 is an integral ER membrane protein with a canonical RING-HC domain and three predicted transmembrane domains; immunopurified RNF170 exhibited ubiquitin ligase activity, while the Cys-101/His-103 mutant (*RNF170) did not.
  • RNF170 depletion inhibited GnRH-induced IP3R1 polyubiquitination to 57 ± 7% of control and inhibited IP3R1 down-regulation by ~50% in αT3-1 cells; it also increased basal IP3R1 levels by 27 ± 11%. In mHeLa cells, RNF170 depletion inhibited IP3 receptor ubiquitination and completely blocked down-regulation.
  • A substantial proportion of RNF170 is constitutively associated with the erlin1/2 complex; erlin2 depletion inhibited RNF170 binding to activated IP3 receptors, whereas RNF170 depletion did not affect erlin2 binding, supporting a model in which erlin1/2 recruits RNF170.

Methods

  • Biochemical cell biology in αT3-1, HeLa, Rat1, SH-SY5Y, and mHeLa cells: IP3R1 immunoprecipitation, silver staining, mass spectrometry, immunoblotting, RT-PCR cloning, immunofluorescence, and subcellular fractionation.
  • In vitro ubiquitin ligase assays using immunopurified RNF170-FLAG or *RNF170-FLAG with E1 (UBE1), E2 (UbcH5b), and HA-ubiquitin.
  • RNA interference against RNF170 in αT3-1 and mHeLa cells, erlin2 depletion, and stable Rat1 clones expressing RNF170-FLAG or *RNF170-FLAG; quantitated data reported as mean ± S.E. with Student’s t test.

Limitations

  • The paper states that RNF170 depletion did not completely block IP3 receptor ubiquitination, which could reflect residual RNF170 after RNAi or activity of another ligase; whether RNF170 is the only E3 for IP3 receptors remains unclear.
  • The paper states that how the erlin1/2 complex and RNF170 interact has yet to be defined, and that effects of the sensory ataxia-associated RNF170 mutation on activity and neuronal substrates have not been examined.
  • Evident from the text: the evidence relies on cultured cell lines and RNAi/overexpression rather than in vivo models, and the in vitro ligase assay shows RNF170 activity with generic HA-ubiquitin rather than direct ubiquitination of IP3 receptor substrate.

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

Integrity signals

Registry

Crossref · 10.1074/jbc.m111.251983

Cites

30 of 30 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

26 of 48 cited sources checked · 23 supported · 1 flagged · accuracy not yet measured

  • Premisecitation
    § DISCUSSION · p. 7

    Basal IP3 receptor turnover is mediated by transfer to lysosomes.

    “This suggests that RNF170 also plays a role in basal IP 3 receptor turnover, which thus far has appeared to be independent of the UPP and which available data suggest is mediated by transfer to lysosomes (26).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [26] Kahn, 2003, Biochem. J. source

      The Discussion says the differing effects of two lysosomal inhibitors suggest that the lysosomal pathway was probably not involved. It also reports that a receptor fragment remained associated with the ER fraction and concludes that at least the initial steps of basal degradation occur in the ER, not by transfer to lysosomes. The paper does not establish where every later step occurs. (LLM, conf=0.88)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
  • Premisecitation
    § RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation * · p. 1

    Upon activation, inositol 1,4,5-trisphosphate (IP3) receptors are degraded via the ubiquitin-proteasome pathway.

    “Inositol 1,4,5-trisphosphate (IP3) receptors are endoplasmic reticulum membrane calcium channels that, upon activation, are degraded via the ubiquitin-proteasome pathway.”
    • Also stated in: § RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation * · p. 1 · boosted
      “In recent years it has become clear that G-protein-coupled receptor-induced activation of endogenous IP 3 receptors can lead to their rapid degradation via the ubiquitin-proteasome pathway (UPP) (2).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [2] Wojcikiewicz, 2009, Cell Calcium source
  • Premisecitation
    § RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation * · p. 1

    The erlin1/2 complex binds to IP3 receptors immediately after their activation.

    “whichhasbeenshownpreviouslytobindtoIP3 receptors immediately after their activation.”
    • Also stated in: § RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation * · p. 2 · hedged
      “we have recently found that the ER membrane-located erlin1/2 (SPFH1/2) complex associates rapidly with activated IP 3 receptors and appears to be the element that selects them for ERAD (14, 15).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [14] Pearce, 2007, J. Biol. Chem. source
    • cites [15] Pearce, 2009, J. Biol. Chem. source
  • Premisecitation
    § RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation * · p. 1

    The ERAD pathway accounts for the ubiquitination and proteasomal degradation of certain metabolically regulated native ER membrane proteins, including IP3 receptors.

    “a range of evidence indicates that the ERAD pathway also accounts for the ubiquitination and proteasomal degradation of certain metabolically regulated native ER membrane proteins, including IP 3 receptors (2) and HMG-CoA reductase (12).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [2] Wojcikiewicz, 2009, Cell Calcium source
    • cites [12] Hampton, 2009, Chem. Rev. source
  • Premisecitation
    § DISCUSSION · p. 6

    The erlin1/2 complex is located in the ER membrane.

    “Because IP 3 receptors (1) and the erlin1/2 complex (15) are located in the ER membrane”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [15] Pearce, 2009, J. Biol. Chem. source
  • Classified as probably not a substantial claim. 27
  • May combine two claims. 13
  • The quote only partly states this claim. 1

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

    Premisecitation
    § RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation * · p. 2

    The ER membrane-located erlin1/2 (SPFH1/2) complex associates rapidly with activated IP3 receptors and appears to be the element that selects them for ERAD.

    “Additionally, we have recently found that the ER membrane-located erlin1/2 (SPFH1/2) complex associates rapidly with activated IP 3 receptors and appears to be the element that selects them for ERAD (14, 15).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [14] Pearce, 2007, J. Biol. Chem. source
    • cites [15] Pearce, 2009, J. Biol. Chem. source

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

    Premisecitation
    § RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation * · p. 1

    The generally accepted summary of the UPP is that substrates are first polyubiquitinated and then processed by the proteasome.

    “The generally accepted summary of the UPP is that substrates are first polyubiquitinated and then processed by the proteasome, a multi-subunit protease that can recognize and degrade polyubiquitinated proteins (7, 8).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [7] Finley, 2009, Annu. Rev. Biochem. source
    • cites [8] Komander, 2009, Biochem. Soc. Trans. source

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

    Premisecitation
    § RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation * · p. 1

    Ubiquitination is achieved through the hierarchical action of three enzymes: E1, E2, and E3.

    “Ubiquitination, the key step in targeting a protein for proteasomal degradation, is achieved through the hierarchical action of three enzymes, termed ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (Ubc or E2), and ubiquitin ligase (E3) (7, 8).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [7] Finley, 2009, Annu. Rev. Biochem. source
    • cites [8] Komander, 2009, Biochem. Soc. Trans. source

  • Classified as probably not a substantial claim.

    Premisecitation
    § RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation * · p. 1

    The ERAD pathway is a facet of the UPP responsible for the degradation of aberrant proteins in the ER.

    “The ERAD pathway is a facet of the UPP responsible for the degradation of aberrant proteins in the ER (11)”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [11] Vembar, 2008, Nat. Rev. Mol. Cell Biol. source

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

    Premisecitation
    § RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation * · p. 1

    IP3 receptor down-regulation has been demonstrated in many mammalian cell types, including GnRH-stimulated αT3-1 mouse pituitary gonadotropes, ET1-stimulated Rat1 fibroblasts, muscarinic agonist-stimulated SH-SY5Y human neuroblastoma cells, and mHeLa cells.

    “This IP 3 receptor 'down-regulation' has been demonstrated in many mammalian cell types, including gonadotropin-releasing hormone (GnRH)-stimulated 𝛂 T3-1 mouse pituitary gonadotropes (3), endothelin 1 (ET1)-stimulated Rat1 fibroblasts (4), and muscarinic agonist-stimulated SH-SY5Y human neuroblastoma cells (5) and mHeLa cells (6).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [3] Wojcikiewicz, 2003, J. Biol. Chem. source
    • cites [4] Alzayady, 2005, J. Biol. Chem. source
    • cites [5] Zhu, 2000, Biochem. J. source

      Citation (6) is attached to mHeLa cells. The Experimental and Results passages describe SH-SY5Y cells, and a search of our copy for “mHeLa cells” found no passage addressing IP3 receptor down-regulation in mHeLa cells. (LLM, conf=0.75)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation
    § RESULTS · p. 5

    In mHeLa cells, carbachol induces IP3 receptor ubiquitination and down-regulation.

    “we have previously shown that carbachol, a muscarinic receptor agonist, induces IP 3 receptor ubiquitination and down-regulation (6).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [6] Wang, 2009, Biochim. Biophys. Acta. source

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

    Premisecitation
    § RESULTS · p. 3

    In previously authenticated RING domain-containing E3s, wild-type in vitro ubiquitin ligase activity and inhibition by mutation of cognate Cys/His residues have been seen.

    “The in vitro ubiquitin ligase activity of wild-type protein and the inhibitory effect of mutation of residues cognate with Cys-101 and His-103 has been seen with previously authenticated RING domain-containing E3s (9, 18)”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [9] Deshaies, 2009, Annu. Rev. Biochem. source
    • cites [18] Fang, 2000, J. Biol. Chem. source

  • The quote only partly states this claim.

    Interpretationreasoning
    § RESULTS · p. 6

    By extrapolation, depletion of RNF170 did not inhibit the association of the erlin1/2 complex with activated IP3 receptors.

    “extrapolation, the erlin1/2 complex (15) with activated IP 3 receptors (Fig. 5 C , lane 3 )”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [15] Pearce, 2009, J. Biol. Chem. source

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

    Premisecitation
    § DISCUSSION · p. 6

    IP3 receptors are ubiquitinated and degraded in response to cell stimulation.

    “That IP 3 receptors are ubiquitinated and degraded in response to cell stimulation has been known for over a decade”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [2] Wojcikiewicz, 2009, Cell Calcium source

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

    Premisecitation
    § RESULTS · p. 3

    Carbachol induces IP3 receptor ubiquitination and down-regulation in SH-SY5Y cells.

    “SH-SY5Y cells in which carbachol has been shown previously to induce IP 3 receptor ubiquitination and down-regulation (5).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [5] Zhu, 2000, Biochem. J. source

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

    Premisecitation
    § RESULTS · p. 3

    α-transaldolase is a cytosolic marker.

    “the cytosolic marker 𝛂 -transaldolase (14, 15)”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [14] Pearce, 2007, J. Biol. Chem. source
    • cites [15] Pearce, 2009, J. Biol. Chem. source

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

    Premisecitation
    § RESULTS · p. 3

    p97 associates with IP3 receptors after they have been polyubiquitinated.

    “which associates with IP 3 receptors after they have been polyubiquitinated (4, 14, 15).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [4] Alzayady, 2005, J. Biol. Chem. source
    • cites [14] Pearce, 2007, J. Biol. Chem. source
    • cites [15] Pearce, 2009, J. Biol. Chem. source

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

    Premisecitation
    § DISCUSSION · p. 8

    The intralumenal regions of the erlin1/2 complex interact with intralumenal regions of activated IP3 receptors.

    “It is thought that the intralumenal regions of the erlin1/2 complex interact with intralumenal regions of activated IP 3 receptors and that the erlin1/2 complex acts as a 'recognition factor' that selects activated IP 3 receptors for degradation via the ERAD pathway (2, 15).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [2] Wojcikiewicz, 2009, Cell Calcium source
    • cites [15] Pearce, 2009, J. Biol. Chem. source

  • Classified as probably not a substantial claim.

    Premisecitation
    § DISCUSSION · p. 8

    The erlin1/2 complex acts as a recognition factor that selects activated IP3 receptors for degradation via the ERAD pathway.

    “It is thought that the intralumenal regions of the erlin1/2 complex interact with intralumenal regions of activated IP 3 receptors and that the erlin1/2 complex acts as a 'recognition factor' that selects activated IP 3 receptors for degradation via the ERAD pathway (2, 15).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [2] Wojcikiewicz, 2009, Cell Calcium source
    • cites [15] Pearce, 2009, J. Biol. Chem. source

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

    Findingown data
    § RNF170 Is an E3 Ubiquitin Ligase for IP 3 Receptors · p. 2

    In Rat1 cells stably expressing the tagged proteins, RNF170 FLAG expression levels were consistently higher than those of *RNF170 FLAG.

    “Expression levels of RNF170 FLAG were consistently higher than those of *RNF170 FLAG”
    Quote in the paperNot a substantial claim

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

    Findingown data
    § RESULTS · p. 3

    RNF170 has three putative transmembrane domains.

    “has three putative transmembrane domains”
    Quote in the paperNot a substantial claim

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

    Findingown data
    § RNF170 Is an E3 Ubiquitin Ligase for IP 3 Receptors · p. 2

    Rat1 cells expressing *RNF170 FLAG grew relatively slowly.

    “*RNF170 FLAG -expressing cells grew relatively slowly”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation
    § DISCUSSION · p. 6

    Depletion of Hrd1, gp78, TEB4, or RNF5 did not significantly affect IP3 receptor ubiquitination or degradation.

    “we previously investigated several of these mammalian E3 ligases (Hrd1, gp78, TEB4, and RNF5) for a role in IP 3 receptor processing using RNAinterference,butdepletionofnonehadasignificanteffecton IP 3 receptor ubiquitination or degradation 4”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS · p. 3

    SDS, but not Na2CO3, releases RNF170 FLAG from the pellet.

    “SDS, but not Na2CO3, released RNF170 FLAG from the pellet (Fig. 3 B , lanes 3-8 ).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS · p. 3

    RNF170 ubiquitin ligase activity requires E1, E2, and HA-ubiquitin; omission of any of these blocks formation of the high molecular mass species.

    “omission of E1, E2, or HA-ubiquitin blocked the formation of the high molecular mass species ( lanes 2-4 )”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS · p. 3

    In SH-SY5Y cells, RNF170 rapidly associates with IP3 receptors after cell stimulation, and this association parallels erlin2.

    “As in 𝛂 T3-1 cells, RNF170 associated rapidly with IP 3 receptors after cell stimulation, and the association paralleled that of erlin2 (Fig. 1 G ).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS · p. 3

    After centrifugation of hypotonically lysed cells, RNF170 FLAG localizes to the pellet with integral ER membrane E3 Hrd1 and not to the supernatant with cytosolic marker α-transaldolase.

    “Centrifugation of hypotonically lysed cells showed that RNF170 FLAG localizes to the pellet together with the integral ER membrane E3 Hrd1 (19) and not to the supernatant with the cytosolic marker 𝛂 -transaldolase (14, 15) (Fig. 3 B , lanes 1 and 2 ).”
    Quote in the paperSubstantial claim

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

    Findingown data
    § DISCUSSION · p. 8

    p < 0.05 comparing IP3R1 immunoreactivity without or with ET1 for each clone.

    “*, p &lt; 0.05 comparing IP 3 R1 immunoreactivity without or with ET1 for each clone.”
    Quote in the paperNot a substantial claim

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

    Premisecitation
    § RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation * · p. 1

    The presence of a RING domain in RING E3s is essential for ubiquitin ligase activity.

    “The RING domain is a 40-100aminoacidmotifthatcoordinateszincandwhosepresence in RING E3s is essential for ubiquitin ligase activity.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [9] Deshaies, 2009, Annu. Rev. Biochem. source

  • May combine two claims.

    Interpretationreasoning
    § RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation * · p. 1

    The erlin1/2 complex recruits RNF170 to activated IP3 receptors, where RNF170 mediates IP3 receptor ubiquitination.

    “These results suggest a model in which the erlin1/2 complex recruits RNF170 to activated IP 3 receptors where it mediates IP 3 receptor ubiquitination.”
    Quote in the paperSubstantial claim

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

    Interpretationown data
    § RNF170 Is an E3 Ubiquitin Ligase for IP 3 Receptors · p. 2

    *RNF170 FLAG may be detrimental to cell health.

    “suggesting that *RNF170 FLAG may be detrimental to cell health”
    Quote in the paperNot a substantial claim

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

    Premisecitation
    § RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation * · p. 2

    The p97-Ufd1-Npl4 complex participates in the delivery of polyubiquitinated IP3 receptors to the proteasome.

    “This p97-Ufd1-Npl4 complex participates in the delivery of polyubiquitinated IP 3 receptors to the proteasome (4).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [4] Alzayady, 2005, J. Biol. Chem. source

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

    Interpretationnone given
    § RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation * · p. 1

    RNF170 is predicted to have three membrane-spanning helices.

    “RNF170 is predicted to have three membrane-spanning helices”
    Quote in the paperNot a substantial claim

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

    Premisecitation
    § RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation * · p. 1

    The selection of substrates for ubiquitination rests primarily with E3s.

    “The selection of substrates for ubiquitination appears to rest primarily with E3s, and two major families are known: those that contain RING domains (9), and those that contain HECT domains (10).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [9] Deshaies, 2009, Annu. Rev. Biochem. source
    • cites [10] Bernassola, 2008, Cancer Cell source

  • May combine two claims.

    Interpretationown data
    § RESULTS · p. 3

    Topological predictions place RNF170 in the ER membrane with its N terminus in the ER lumen and its RING domain and C terminus in the cytosol.

    “Topological predictions indicate that RNF170 resides in the ER membrane with its NterminusintheERlumenandwiththeRINGdomainandthe C terminus in the cytosol (Fig. 1 C ).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data
    § RESULTS · p. 5

    siRNA targeting RNF170 specifically depletes RNF170 without changing erlin2 levels in αT3-1 cells.

    “RNF170 immunoreactivity was markedly reduced without a change in the levels of erlin2, which served as a loading control (Fig. 5 A and B , lower panels ).”
    • Also stated in: § RESULTS · p. 5 · plain
      “In 𝛂 T3-1 cells, introduction by electroporation of a vector encoding a siRNA targeting RNF170 resulted in specific depletion of RNF170.”
    Quote in the paperSubstantial claim

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

    Premisecitation
    § RESULTS · p. 3

    Hrd1 is an integral ER membrane E3.

    “the integral ER membrane E3 Hrd1 (19)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [19] Kikkert, 2004, J. Biol. Chem. source

  • Classified as probably not a substantial claim.

    Premisecitation
    § DISCUSSION · p. 6

    IP3 receptors are located in the ER membrane.

    “Because IP 3 receptors (1) and the erlin1/2 complex (15) are located in the ER membrane”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [1] Foskett, 2007, Physiol. Rev. source

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

    Study design and conductown data
    § RNF170 Is an E3 Ubiquitin Ligase for IP 3 Receptors · p. 3

    All experiments were repeated at least once.

    “All experiments were repeated at least once, and representative images of gels or micrographs are shown.”
    Quote in the paperNot a substantial claim

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

    Premisecitation
    § DISCUSSION · p. 8

    An Arg-to-Cys point mutation in human RNF170 causes autosomal dominant sensory ataxia.

    “A very recent report presented evidence that an Arg-to-Cys point mutation in human RNF170 is the cause of autosomal dominant sensory ataxia, a rare, progressive ataxia caused by degeneration of the posterior columns of the spinal cord (27).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] Valdmanis, 2011, Brain. source

  • Classified as probably not a substantial claim.

    Study design and conductown data
    § DISCUSSION · p. 8

    Data shown are representative of at least six vector-, RNF170 FLAG-, or *RNF170 FLAG-expressing clones.

    “Data shown are representative of at least six vector-, RNF170 FLAG -, or *RNF170 FLAG -expressing clones.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation
    § DISCUSSION · p. 8

    Deranged IP3 receptor function and Ca2+ signaling contributes to various neurodegenerative disorders.

    “as deranged IP 3 receptor function and Ca 2 + signaling contributes to various neurodegenerative disorders (28).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [28] Bezprozvanny, 2011, Neurochem. Res.

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

37 of 37

  • The paper relies on this without support.

    Overexpressed triple-FLAG-tagged RNF170 behaves like endogenous RNF170 in ubiquitin ligase activity and subcellular localization.

    “To facilitate the identification and purification of RNF170, a triple FLAG tag was appended to the C terminus, generating RNF170 FLAG that migrated at 26 kDa”

  • The paper relies on this without support.

    The anti-RNF170 antibody used specifically recognizes RNF170.

    “generated a 21.5-kDa anti-RNF170 immunoreactive band”

  • The paper relies on this without support.

    Down-regulation of IP3R1 immunoreactivity reflects degradation of the receptor.

    “This RNF170 depletion inhibited GnRH-induced IP 3 R1 polyubiquitination (Fig. 5 A , lane 3 , uppermost panel ) and down-regulation (Fig. 5 B , lanes 5-8 , uppermost panel ).”

  • The paper relies on this without support.

    Depletion of erlin2 is an adequate proxy for loss of erlin1/2 complex function in recruiting RNF170.

    “depletion of erlin2 strongly inhibited association of RNF170 with activated IP 3 receptors (Fig. 5 C , lane 4 )”
    “Thus, the erlin1/2 complex appears to recruit RNF170 to activated IP 3 receptors.”

  • The paper relies on this without support.

    Basal IP3 receptor turnover is independent of the UPP.

    “which thus far has appeared to be independent of the UPP”
5 of 5

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.

  • Figure 4 assigns lanes 3 and 6 to two different conditions

    Minor

    The Figure 4D caption identifies lanes 3 and 6 as samples incubated without antibody and also as samples incubated with anti-HA antibody. Both assignments cannot be correct.

1 of 1

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. Bezprozvanny, 2011, Neurochem. Res.

Cited by

Sorted by citations

Related

Authors
Lu, J. P., Wang, Y., Sliter, D. A., Pearce, M. M. P., & Wojcikiewicz, R. J. H.
Affiliations
SUNY Upstate Medical University (US)
Corresponding author
Wojcikiewicz, Richard J.H.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2011-07
Volume
286
Issue
27
Pages
24426-24433
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m111.251983
PMC
PMC3129221
OpenAlex
W2001909617
Semantic Scholar
73d4617ca86d6b4773070e4b1750858641bfd3b7
Cited by
102
References
29
Citations by year
2026: 4 · 2025: 5 · 2024: 13 · 2023: 4 · 2022: 10 · 2021: 8 · 2020: 9 · 2019: 4 · 2018: 8 · 2017: 4 · 2016: 10 · 2015: 7 · 2014: 6 · 2013: 1 · 2012: 6
FWCI
1.88
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion)
  • PubMed Central (submittedVersion)
Funders
National Institute of Diabetes and Digestive and Kidney Diseases (DK049194, R29 DK049194, R01 DK049194)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Life Sciences › Neuroscience › Sensory Systems › Ion Channels and Receptors
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
Keywords
IP3 receptor, ubiquitination, ubiquitin ligase, endoplasmic reticulum membrane, RING domain, ubiquitin-proteasome system, calcium channels, protein degradation
MeSH terms
Animals, Endoplasmic Reticulum, HeLa Cells, Humans, Membrane Proteins, Mutation, Nerve Tissue Proteins, Protein Binding, Protein Structure, Secondary, Ubiquitin-Protein Ligases, Multiprotein Complexes, Proteasome Endopeptidase Complex, Rats, Inositol 1,4,5-Trisphosphate Receptors, Ubiquitination

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-18.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-18.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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