CoChat
Sign Up
Cited by 83Heat shock proteins researchEndoplasmic Reticulum Stress and DiseaseCholesterol and Lipid Metabolism

The DOI matches its Crossref record; 57 of the 57 references it cites were found in a registry or an index; none of the 75 sources its claims cite is checked for support yet.

Proteomic Plasma Membrane Profiling Reveals an Essential Role for gp96 in the Cell Surface Expression of LDLR Family Members, Including the LDL Receptor and LRP6

Open access
Weekes, Michael P., Antrobus, Robin, Talbot, Suzanne, Hör, Simon, Simecek, Nikol, Smith, Duncan L., Bloor, Stuart, Randow, Felix, Lehner, Paul J.·2012·Journal of Proteome Research
DOI 10.1021/pr201135ePMC PMC3292266
Open this paper in CoChat — free accountOpen PDF

Abstract

The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

Using plasma membrane profiling, the authors compared gp96-deficient and gp96-reconstituted murine pre-B cells and found that gp96 is required for cell surface expression of 29 proteins, including four extended LDL receptor family members. They propose beta-propeller and leucine-rich repeat motifs as common structural features among gp96-dependent clients.

Key findings

  • Plasma membrane profiling identified 706 PM/CS/XC/ShG proteins after peptide fractionation; 29 proteins identified by ≥2 peptides were downregulated >3-fold in gp96-deficient cells, of which 24 were annotated PM/CS/XC and 26 PM/CS/XC/ShG (93%).
  • Four extended LDLR family members showed decreased cell surface expression in the absence of gp96: LDLR (25.2–35.0 fold), LRP6 (21.9–65.6 fold), Sorl1 (29.5–32.3 fold), and LRP8/ApoER2 (5.4 fold).
  • The study confirmed gp96 dependence of integrins α4, αV, αL, β2 and TLR6, and gp96 independence of integrins β1, α6 and α5; CD180/Ly86 was identified as a novel gp96 client.
  • Structural enrichment analysis found significant over-representation (p < 0.001) of beta-propeller, leucine-rich repeat, and α-integrin domains among proteins downregulated in gp96-deficient cells.

Methods

  • Plasma membrane profiling (PMP) combined SILAC labeling with selective cell surface aminooxy-biotinylation of sialylated glycoproteins, streptavidin capture, tryptic digestion, PNGase F release, and LC–MS/MS.
  • M36 gp96-deficient murine pre-B cells (light SILAC) were compared with M36/gp96+ reconstituted cells (heavy SILAC); an independently derived gp96-deficient E4.126 line was also used for flow cytometry validation.
  • Data were searched/processed with MaxQuant, MASCOT, and Andromeda; peptide prefractionation used high-pH reversed-phase HPLC; structural annotation used InterPro and BiNGO, and interactions used Cytoscape/MiMI.

Limitations

  • The paper states the proteomic experiment was performed as a single replicate, while flow cytometry confirmation used at least three biological replicates.
  • The other six LDLR family members (VLDLR, MEGF7, LRP1, LRP1B, LRP2, LRP5) were not detected, likely due to low expression in pre-B cells or poor enrichment of large proteins; no suitable flow cytometry antibodies were available for other mouse family members.
  • The authors state that biochemical confirmation is needed to show the surface-expression changes are due to loss of specific gp96 chaperone activity rather than changes in transcription or translation; the text also notes that fractionation increased non-PM contaminants and that GO annotation is likely incomplete.

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

Integrity signals

Registry

Crossref · 10.1021/pr201135e

Cites

57 of 57 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Verification

Claims the paper makes

0 of 75 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Flow cytometry confirmed absolute dependence of cell surface LDLR expression on gp96 in both gp96-deficient cell lines M36 and E4.126.

    “The absolute dependence of cell surface LDLR expression on gp96 was readily confirmed by flow cytometry in both gp96 deficient cells (M36 and E4.126) (Figure 3).”
    Quote in the paperSubstantial claim
  • Findingcitation

    Comparison of gp96-knockout and gp96-reconstituted murine pre-B cells found that the cell surface expression of 29 proteins was decreased >3.0 fold in the absence of gp96.

    “A comparison of gp96-knockout and gp96-reconstituted murine pre-B cells3,13 found that the cell surface expression of 29 proteins was decreased >3.0 fold in the absence of gp96.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [3] Randow F., 2001, Nat. Cell Biol. source
    • cites [13] Bloor S., 2010, Proc. Natl. Acad. Sci. U.S.A. source
    • Rests on an assumption: The M36 and M36/gp96+ cell lines differ only in gp96 expression, so observed differences between them are attributable to gp96.
    • Rests on an assumption: The SILAC ratio measured by PMP reflects the relative cell surface expression level of a protein.
  • Findingown data

    Flow cytometric analysis of integrin β2 (CD18) demonstrated decreased cell surface expression to the level of the unstained control in two independently derived gp96-deficient cell lines.

    “Furthermore, flow cytometric analysis of integrin β2 (CD18) demonstrated decreased cell surface expression to the level of the unstained control in two independently derived gp96-deficient cell lines (Figure 3).”
    Quote in the paperSubstantial claim
  • Findingown data

    Following fractionation of the tryptic peptide sample, the percentage of proteins annotated PM/CS/XC fell from 70 to 35%.

    “Following fractionation of the tryptic peptide sample, the percentage of proteins annotated PM/CS/XC fell from 70 to 35%.”
    Quote in the paperNot a substantial claim
  • Findingown data

    CD180/Ly86 is a novel gp96 client protein.

    “Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide.”
    Quote in the paperNot a substantial claim
  • It may state two separate claims as one. 41
  • Probably not a substantial claim. 40

  • It may state two separate claims as one.

    Findingown data

    gp96 (Endoplasmin) was detected at the plasma membrane in M36/gp96+ cells and showed decreased plasma membrane expression (3.8-5.3 fold) in M36 cells.

    “Finally, gp96 (Endoplasmin) was detected at the PM in M36/gp96+ cells and showed decreased plasma membrane expression (3.8–5.3 fold) in M36 cells (Table 2).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    CD180 (RP105, Ly78) and Ly86 (MD1) both show decreased cell surface expression in the absence of gp96.

    “In addition to LDLR family members, CD180 (RP105, Ly78) and Ly86 (MD1) both show decreased cell surface expression in the absence of gp96 (Table 2).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Among the other proteins dependent on gp96 for cell surface expression, attractin contains a beta-propeller (Kelch-repeat type); Laminin alpha 5, attractin, N-glycan processing alpha-mannosidase IIx, and EGF-containing fibulin-like extracellular matrix protein 1 contain EGF-like domains.

    “Among the other proteins dependent on gp96 for cell surface expression, attractin contains a beta-propeller (of the Kelch-repeat type rather than WD-repeat type); Laminin alpha 5, attractin, N-glycan processing alpha-mannosidase IIx and EGF-containing fibulin-like extracellular matrix protein 1 contain EGF-like domains (Table 2).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Findingown data

    Forty-six of the 1271 proteins were downregulated >3 fold, 29 of which were identified by ≥2 peptides.

    “Forty-six of the 1271 proteins were downregulated >3 fold (Table S2, Supporting Information), 29 of which were identified by ≥2 peptides (Figure 2 and Table 2).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Several proteins were not dependent on gp96 for their surface expression, including CD44, ICAM1, CXCR4, IL7R and MHC class I allotypes, and this was confirmed by flow cytometry for CD44 and H2-Kb.

    “Several proteins were not dependent on gp96 for their surface expression, including CD44, ICAM1, CXCR4, IL7R as well as MHC class I allotypes3,11 and this could be confirmed by flow cytometry for CD44 and H2-Kb (Figure 3).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [3] Randow F., 2001, Nat. Cell Biol. source
    • cites [11] Liu B., 2008, Blood source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    Including the 19 proteins with a short GO annotation gave an upper estimate of 113 PM/CS/XC/ShG proteins (84%).

    “Including the 19 proteins with a “short GO” (ShG) annotation (described above) gave a useful upper estimate of the number of identified PM proteins, 113 PM/CS/XC/ShG proteins (84%).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: GOCC annotations accurately identify plasma membrane proteins.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Findingown data

    When data were reprocessed with requantify turned off, there was much better correspondence between Mascot and Andromeda ratios, albeit with a reduced number of quantified proteins.

    “When we reprocessed data with requantify turned off, there was much better correspondence between Mascot and Andromeda ratios (Figure S3, Supporting Information) albeit with a reduced number of quantified proteins.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Findingown data

    Protein ratios generally corresponded well when data were processed using either Mascot or Andromeda, except when proteins were downregulated >3 fold, in which case correspondence was less good.

    “Protein ratios generally corresponded well when data were processed using either Mascot or Andromeda (Table S1, Supporting Information) except when proteins were downregulated >3 fold, in which case the ratios generated using either software corresponded less well (Table 2 and Table S2, Supporting Information).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    The data validated the dependence on gp96 for cell surface expression of integrins α4, αV, αL, and β2, and gp96 independence of integrins β1, α6 and α5.

    “Our data was validated by confirming the dependence on gp96 for cell surface expression of integrins α4, αV, αL, and β2,2,3,9,11 and gp96 independence of integrins β1, α6 and α52 (Table 2 and Table S1, Supporting Information).”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [2] Staron M., 2009, Blood source
    • cites [3] Randow F., 2001, Nat. Cell Biol. source
    • cites [9] Morales C., 2009, J. Immunol. source
    • cites [11] Liu B., 2008, Blood source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    Analysis of unfractionated tryptic peptides initially identified 113 PM proteins.

    “Analysis of unfractionated tryptic peptides initially identified 113 PM proteins”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Findingcitation

    The reported gp96 dependence of integrins α4, αV, αL, β2 and TLR6 and TLR4, and the lack of dependence of α6 and β1 integrins, were confirmed.

    “We confirmed the reported gp96 dependence of integrins α4, αV, αL, β2 and toll-like receptors TLR6 and TLR4, and the lack of dependence of the α6 and β1 integrins.2−4,9,11”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [2] Staron M., 2009, Blood source
    • cites [3] Randow F., 2001, Nat. Cell Biol. source
    • cites [4] Yang Y., 2007, Immunity source
    • cites [9] Morales C., 2009, J. Immunol. source
    • cites [11] Liu B., 2008, Blood source

  • It may state two separate claims as one.

    Findingown data

    Fractionation substantially increased total and PM protein identifications; combining Mascot and Andromeda data, 563 proteins annotated PM/CS/XC and 706 proteins annotated PM/CS/XC/ShG were identified.

    “Fractionation substantially increased the total and PM protein identifications—combining the Mascot and Andromeda data, a total of 563 proteins annotated PM/CS/XC and 706 proteins annotated PM/CS/XC/ShG were identified (Table 1, Table S1, Supporting Information).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: GOCC annotations accurately identify plasma membrane proteins.

  • It may state two separate claims as one.

    Findingown data

    We identified 706 PM proteins and found a requirement for gp96 in the cell surface expression of 29 of these proteins, including integrins, TLRs, and four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8).

    “We identified 706 PM proteins and found a requirement for gp96 in the cell surface expression of 29 of these proteins, including integrins, TLRs, and four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Findingown data

    Of the 29 proteins identified by ≥2 peptides and downregulated >3 fold, 24 were annotated PM/CS/XC and 26 PM/CS/XC/ShG (93%).

    “Of these 29 proteins, 24 were annotated PM/CS/XC and 26 PM/CS/XC/ShG (93%).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: GOCC annotations accurately identify plasma membrane proteins.

  • It may state two separate claims as one.

    Findingown data

    Additional LRR-containing proteins downregulated in gp96-deficient cells include Leucine-rich repeat-containing protein 4C, Extracellular leucine-rich repeat and fibronectin type-III domain-containing protein 1, Adhesion molecule with Ig like domain 1, and Leucine-rich repeats and immunoglobulin-like domains protein 2.

    “Additional LRR-containing proteins downregulated in gp96-deficient cells include: Leucine-rich repeat-containing protein 4C, Extracellular leucine-rich repeat and fibronectin type-III domain-containing protein 1, Adhesion molecule with Ig like domain 1, Leucine-rich repeats and immunoglobulin-like domains protein 2 (Table 2).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    Four-hundred fifty proteins annotated PM/CS/XC and 565 proteins annotated PM/CS/XC/ShG were quantified, out of a total of 1271 quantified proteins.

    “Four-hundred fifty proteins annotated PM/CS/XC and 565 proteins annotated PM/CS/XC/ShG were quantified, out of a total of 1271 quantified proteins.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: GOCC annotations accurately identify plasma membrane proteins.

  • It may state two separate claims as one.

    Findingown data

    In gp96-deficient murine pre-B cells, cell surface expression of four extended LDLR family proteins-LDLR, LRP6, Sorl1, and LRP8-is decreased, with fold ranges of 25.2-35.0 for LDLR, 21.9-65.6 for LRP6, 29.5-32.3 for Sorl1, and 5.4 for LRP8.

    “Most notable of the novel proteins whose cell surface expression is decreased in the absence of gp96 were 4 proteins of the extended LDLR family: the LDLR itself (25.2–35.0 fold), LRP6 (21.9–65.6 fold), Sorl1 (29.5–32.3 fold) and LRP8 (apolipoprotein E receptor 2) (5.4 fold).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Findingown data

    The normal cell surface expression of integrin α1 and decreased expression of integrin β3 in gp96-deficient cells contrasts with a previous report.

    “The normal cell surface expression of integrin α1 and decreased expression of integrin β3 in gp96-deficient cells contrasts with a previous report.2”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2] Staron M., 2009, Blood source

  • It may state two separate claims as one.

    Findingown data

    In the unfractionated sample, 135 proteins were identified, of which 89 were annotated plasma membrane, 2 cell surface, and 3 extracellular, giving 94 PM/CS/XC proteins, representing 70% of all identified proteins with a GOCC annotation.

    “We identified 135 proteins, of which 89 were annotated “plasma membrane” (PM). An additional 2 proteins were annotated “cell surface” (CS), and 3 proteins “extracellular” (XC), giving a total of 94 PM/CS/XC proteins, representing 70% of all identified proteins with a GOCC annotation (Table 1).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: GOCC annotations accurately identify plasma membrane proteins.

  • It may state two separate claims as one.

    Findingown data

    In gp96-deficient cells, scavenger receptor class B type I is upregulated 1.9 fold, Hmgcs1 is upregulated 1.8 fold, and acetoacetyl-CoA synthetase and fatty acid synthase are among proteins implicated in cholesterol metabolism.

    “These include scavenger receptor class B type I (upregulated 1.9 fold) which mediates the bidirectional transfer of cholesterol between cells and HDL cholesterol,44 hydroxymethylglutaryl-CoA synthase (Hmgcs1, upregulated 1.8 fold) which condenses acetyl-CoA with acetoacetyl-CoA to form HMG-CoA, the substrate for HMG-CoA reductase,45 acetoacetyl-CoA synthetase and fatty acid synthase.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    In summary, Gp96 is required for cell surface expression of at least four members of the extended LDL receptor family, as well as CD180/Ly86 and most integrins and TLRs.

    “In summary, Gp96 is required for cell surface expression of at least four members of the extended LDL receptor family, as well CD180/Ly86 and most integrins and TLRs.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Sorl1 is implicated in the pathogenesis of Alzheimer's disease.

    “the sortilin related receptor Sorl1 implicated in the pathogenesis of Alzheimer’s disease,16”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16] Rogaeva E., 2007, Nat. Genet. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    Serum immunoglobulins are normal in mice with a B-cell selective gp96-depletion, suggesting that folding of immunoglobulins is gp96-independent.

    “However, serum immunoglobulins are normal in mice with a B-cell selective gp96-depletion, suggesting that folding of immunoglobulins is gp96-independent.11”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [11] Liu B., 2008, Blood source

  • It may state two separate claims as one.

    Interpretationreasoning

    The non-detection of the other six LDLR family members likely reflects either low expression levels in pre-B cells or solubility problems due to their large size (211-522 kDa) preventing efficient enrichment by the PMP approach.

    “This likely reflects either low expression levels in pre-B cells, or solubility problems since these proteins are large, ranging from 211 to 522 kDa, and may therefore not be efficiently enriched by our PMP approach.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Interpretationcitation

    The downregulation of fibronectin might be explained by it being a ligand for integrin αV/β3.

    “Fibronectin is a ligand for integrin αV/β3,36 which might explain its downregulation (19.6–39.8 fold) in gp96 deficient cells.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [36] Danen E. H., 2005, J. Cell. Biol. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    LRP6 participates with Frizzled proteins as coreceptors in Wnt signaling.

    “the Wnt signaling protein lipoprotein receptor-related protein-6 (LRP6) which participates with Frizzled proteins as coreceptors in Wnt signaling,15”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [15] Logan C. Y., 2004, Ann. Rev. Cell Dev. Biol. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    The best recognized role of gp96 is in the folding and assembly of membrane proteins of the secretory pathway.

    “Gp96 has several biological functions, of which the best recognized is its role in the folding and assembly of membrane proteins of the secretory pathway.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Eletto D., 2010, Semin. Cell Dev. Biol. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductnone given

    Protein quantitation utilized razor and unique peptides and required a minimum of 2 ratio counts and normalized protein ratios reported.

    “Protein quantitation utilized razor and unique peptides and required a minimum of 2 ratio counts and normalized protein ratios reported.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Other than the presence of disulfide bonds, no common structural feature of gp96 clients has emerged.

    “Other than the presence of disulfide bonds, no common structural feature of gp96 clients has emerged.12”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] May P., 2003, Traffic source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    The E4.126 clone was independently derived from 70Z/3 cells by ICR191 mutagenesis and also carries two frameshifting mutations in gp96.

    “The E4.126 clone was independently derived from 70Z/3 cells by ICR191 mutagenesis, and also carries two frameshifting mutations in gp96.3,13”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [3] Randow F., 2001, Nat. Cell Biol. source
    • cites [13] Bloor S., 2010, Proc. Natl. Acad. Sci. U.S.A. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    All α-integrins contain a single seven-bladed beta-propeller, and β-integrins contain four cysteine-rich EGF repeats.

    “All α-integrins contain a single seven-bladed beta-propeller, and β-integrins contain four cysteine-rich EGF repeats31”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [31] Barczyk M., 2010, Cell Tissue Res. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    Gp96 is a ubiquitously expressed chaperone and the only ER resident member of the HSP90 family.

    “Gp96 (also known as gpr94 or HSP90b1) is a ubiquitously expressed chaperone and the only ER resident member of the HSP90 family.1”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Eletto D., 2010, Semin. Cell Dev. Biol. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    Many integrins require chaperoning by gp96, while some α- and β-integrins are gp96 independent.

    “While many integrins require chaperoning by gp96, some α- and β-integrins are gp96 independent.2,9”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [2] Staron M., 2009, Blood source
    • cites [9] Morales C., 2009, J. Immunol. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    LRP8 (apolipoprotein E receptor 2) is required for neural development through Reelin signaling.

    “LRP8 (apolipoprotein E receptor 2) required for neural development through Reelin signaling.17”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [17] Niu S., 2004, Neuron source

  • It may state two separate claims as one.

    Interpretationown data

    Our data indicate that gp96 is required for assembly of this functionally important class of proteins and suggest that it may be required to chaperone folding of some beta-propeller domains and leucine-rich repeats.

    “Our data indicates that gp96 is required for assembly of this functionally important class of proteins and suggests that it may be required to chaperone folding of some beta-propeller domains and leucine-rich repeats.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Interpretationreasoning

    Common structural motifs in gp96 client proteins, including the beta-propeller and leucine-rich repeat, may be recognized by gp96.

    “We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Interpretationreasoning

    All α-integrins and all members of the extended LDLR family contain β-propeller motifs, suggesting that the beta-propeller and leucine-rich repeat may require gp96 for proper folding.

    “All α-integrins and all members of the extended LDLR family contain β-propeller motifs, suggesting that the beta-propeller and leucine-rich repeat may require gp96 for proper folding.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    CD180 is related to TLRs, contains leucine-rich repeats, and binds Ly86 (MD1).

    “CD180 is related to TLRs, contains leucine-rich repeats32,33 and binds Ly86 (MD1).34”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [32] Miyake K., 1995, J. Immunol. source
    • cites [33] Ogata H., 2000, J. Exp. Med. source
    • cites [34] Miyake K., 1998, J. Immunol. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Interpretationown data

    The 3 nuclear proteins not annotated PM are likely to represent contaminants.

    “The 3 nuclear proteins not annotated PM are likely to represent contaminants (Table 1).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    CD180/Ly86 affect the immune response to lipopolysaccharide (LPS) by interfering with an LPS-derived signal delivered through the CD14-MD2-TLR4 complex.

    “Together CD180/Ly86 affect the immune response to lipopolysaccharide (LPS) by interfereing with an LPS-derived signal delivered through the CD14-MD2-TLR4 complex.35”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [35] Gorczynski R. M., 2006, J. Immunol. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductown data

    Incorporation of heavy label was >98% for both arginine and lysine-containing peptides.

    “Incorporation was >98% for both arginine and lysine-containing peptides.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductnone given

    Reversed decoy databases were used and the false discovery rate for both peptides and proteins was set at 0.01.

    “Reversed decoy databases were used and the false discovery rate for both peptides and proteins were set at 0.01.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Interpretationreasoning

    Ly86 may show decreased cell surface expression in gp96-deficient cells due to interaction with its LRR-containing partner CD180.

    “Ly86 may show decreased cell surface expression in gp96 deficient cells due to interaction with its LRR containing partner, CD180.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Interpretationreasoning

    It is likely that the six other LDLR family members are also gp96-dependent, given their structural similarities to LDLR and ApoER2, though confirmation is required because gp96 is selective for certain α-integrins.

    “Given the structural similarities of other members of the LDLR family to LDLR and ApoER2, it seems likely that these six proteins are also gp96-dependent, though confirmation is required given the selectivity of gp96 for certain α-integrins.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Interpretationreasoning

    There must be further complexity because some α-integrins containing a β-propeller and some TLR proteins containing the leucine-rich repeat are not dependent on gp96 for cell surface expression.

    “However, there must be further complexity as some α-integrins containing a β-propeller, and some TLR proteins containing the leucine-rich repeat are not dependent on gp96 for cell surface expression.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    LDLR plays a central role in regulating the concentration of plasma LDL and when absent leads to familial hypercholesterolaemia.

    “the founding member LDLR, which plays a central role in regulating the concentration of plasma LDL and when absent leads to familial hypercholesterolaemia,14”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [14] Soutar A. K., 2007, Nat. Clin. Pract. Cardiovasc. Med. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. The paper may give no reason why it holds. It may state two separate claims as one.

    Interpretationreasoning

    Where a majority of proteins identified from a given sample are annotated PM/CS/XC, it is likely that a substantial proportion of proteins with a short GOCC term are also integral PM proteins, so including these proteins provides a useful upper estimate of the number of identified PM proteins.

    “Where a majority of proteins identified from a given sample are annotated PM/CS/XC, it is likely that a substantial proportion of the proteins with a short GOCC term are also integral PM proteins—including these proteins provides a useful upper estimate of the number of identified PM proteins.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: GOCC annotations accurately identify plasma membrane proteins.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Interpretationcitation

    Gp96 may be required for CD180/MD1 complex assembly, by analogy to TLR4/MD2, as in the absence of gp96, TLR4 and MD2 cannot associate.

    “Gp96 may be required for CD180/MD1 complex assembly, by analogy to TLR4/MD2, as in the absence of gp96, TLR4 and MD2 cannot associate.3”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [3] Randow F., 2001, Nat. Cell Biol. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    Biotinylation was confirmed by staining aliquots of cells prior to and after biotinylation with streptavidin-allophycocyanin.

    “Biotinylation was confirmed by staining aliquots of cells prior to and after biotinylation with streptavidin-allophycocyanin (eBioscience, San Diego, CA).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim.

    Study design and conductnone given

    Plasma membrane profiling (PMP) combines SILAC labeling with selective cell surface aminooxy-biotinylation.

    “we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    The chaperone activity of gp96 is selective, with targets including integrins, TLRs, IGFs, thyroglobulin and the platelet glycoprotein Ib-IX-V complex.

    “However, the chaperone activity of gp96 is selective, with targets including integrins, toll-like receptors (TLRs), insulin-like growth factors (IGFs), thyroglobulin and the platelet glycoprotein Ib-IX-V complex.2−8”
    Quote in the paperNot a substantial claim0 of 7 sources checked
    • cites [2] Staron M., 2009, Blood source
    • cites [3] Randow F., 2001, Nat. Cell Biol. source
    • cites [4] Yang Y., 2007, Immunity source
    • cites [5] Harding C. V., 2007, Immunity source
    • cites [6] Ostrovsky O., 2009, Mol. Biol. Cell source
    • cites [7] Ostrovsky O., 2009, Biochim. Biophys. Acta source
    • cites [8] Staron M., 2011, Blood source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    Gp96 is critical for proper folding and functional expression of a number of integrins and TLRs; in gp96-deficient cells, these proteins are minimally expressed at the cell surface as assessed by flow cytometry.

    “Gp96 is critical for proper folding and functional expression of a number of integrins and TLRs; in gp96-deficient cells, these proteins are minimally expressed at the cell surface as assessed by flow cytometry.3,5”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [3] Randow F., 2001, Nat. Cell Biol. source
    • cites [5] Harding C. V., 2007, Immunity source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    All members of the LDLR family and more distantly related receptors contain at least one YWTD-motif-containing beta-propeller domain with flanking EGF-like cysteine-rich repeats.

    “All members of the LDLR family and more distantly related receptors are characterized by the presence of at least one YWTD-motif containing beta-propeller domain, with flanking EGF-like cysteine-rich repeats.12”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] May P., 2003, Traffic source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Boca/Mesd is specifically required for maturation of the LDLR family beta-propeller/EGF modules.

    “Boca/mesd is specifically required for maturation of the LDLR family beta-propeller/EGF modules,53−55”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [53] Culi J., 2004, EMBO J. source
    • cites [54] Koduri V., 2007, Biochemistry source
    • cites [55] Liu C. C., 2009, J. Biol. Chem. source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Interpretationcitation

    One or more of the extended LDLR family, or other gp96-dependent proteins identified in this analysis, may be the receptor for VSV-G.

    “Gp96-deficient (M36 and E4.126) cells are resistant to infection by vesicular stomatitis virus (VSV) and viruses pseudotyped with VSV-G13 suggesting that one or more of the extended LDLR family, or indeed other gp96-dependent proteins identified in this analysis may be the receptor for VSV-G.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [13] Bloor S., 2010, Proc. Natl. Acad. Sci. U.S.A. source

  • It may state two separate claims as one.

    Interpretationreasoning

    Not all propeller domain-containing proteins require gp96 for correct folding, but the unique interface between the propeller and adjacent modules may require gp96-assisted assembly.

    “Clearly, not all propeller domain-containing proteins require gp96 for correct folding, but the unique interface between the propeller and adjacent modules may require gp96-assisted assembly.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationreasoning

    The increased identification of non-PM contaminants following high resolution fractionation suggests that contaminants form a minor part of the total protein content and highlights the efficiency of PM protein enrichment.

    “The increased identification of non-PM contaminants seen following high resolution fractionation suggests that contaminants form a minor part of the total protein content and highlights the efficiency of PM protein enrichment.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Interpretationreasoning

    Cellular cholesterol uptake is likely to be significantly diminished when cell surface expression of LDLR and other family members is reduced, and may explain the observed increase in proteins implicated in cholesterol metabolism in gp96-deficient cells.

    “Cellular cholesterol uptake is likely to be significantly diminished when cell surface expression of LDLR and other family members is reduced, and may explain the observed increase in proteins implicated in cholesterol metabolism in gp96-deficient cells (Table S1, Supporting Information, Table 2).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Interpretationreasoning

    Increased Ech1 expression might provide acetyl-CoA as a substrate for Hmgcs1 and cholesterol synthesis, although other metabolic pathways could also generate this intermediate.

    “Increased Ech1 expression might also provide acetyl-CoA as a substrate for Hmgcs1 and cholesterol synthesis, although other metabolic pathways could also generate this important intermediate.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    LDLR family members have a specific requirement for the molecular ER chaperone boca (Drosophila melanogaster) and its mammalian orthologue Mesd for folding and trafficking to the cell surface.

    “LDLR family members also have a specific requirement for the molecular ER chaperone boca (Drosophila melanogaster), and its mammlian orthologue Mesd, for folding and trafficking to the cell surface.50−52”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [50] Culi J., 2003, Cell source
    • cites [51] Hsieh J. C., 2003, Cell source
    • cites [52] Li Y., 2005, J. Cell Sci. source

  • It may state two separate claims as one.

    Interpretationcitation

    The known gp96 clients, integrins and TLRs, fail to reach the cell surface in the absence of gp96 due to misfolding; it is therefore likely that LDLR family members are similarly affected in the absence of gp96.

    “The known gp96 clients, integrins and TLRs, fail to reach the cell surface in the absence of gp96 due to misfolding1−8—it is therefore likely that LDLR family members are similarly affected in the absence of gp96.”
    Quote in the paperSubstantial claim0 of 8 sources checked
    • cites [1] Eletto D., 2010, Semin. Cell Dev. Biol. source
    • cites [2] Staron M., 2009, Blood source
    • cites [3] Randow F., 2001, Nat. Cell Biol. source
    • cites [4] Yang Y., 2007, Immunity source
    • cites [5] Harding C. V., 2007, Immunity source
    • cites [6] Ostrovsky O., 2009, Mol. Biol. Cell source
    • cites [7] Ostrovsky O., 2009, Biochim. Biophys. Acta source
    • cites [8] Staron M., 2011, Blood source
61 of 61

  • The paper relies on this without support.

    The M36 and M36/gp96+ cell lines differ only in gp96 expression, so observed differences between them are attributable to gp96.

    “M36/gp96+ cells were derived by stable transduction of M36 cells with gp96.13”
    “M36 and M36/gp96+ cells were used for the majority of the analysis in our study.”

  • The paper relies on this without support.

    The SILAC ratio measured by PMP reflects the relative cell surface expression level of a protein.

    “This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells.”
    “A comparison of gp96-knockout and gp96-reconstituted murine pre-B cells3,13 found that the cell surface expression of 29 proteins was decreased >3.0 fold in the absence of gp96.”

  • The paper relies on this without support.

    GOCC annotations accurately identify plasma membrane proteins.

    “as defined by gene ontology (GO) terms”
    “To assess the number of PM proteins identified, we summed the proteins with GOCC terms plasma membrane (PM), cell surface but not PM (CS) and extracellular but not PM/CS (XC).”
3 of 3

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.

  • Percentage of downregulated membrane proteins is incorrect

    Minor

    The stated 26 proteins are 26/29, or about 90%, not 93%.

    Reported 93 · recomputed 89.655172

  • Aminooxy-biotin concentration differs by 1,000-fold

    Minor

    The preparation specifies 100 mM aminooxy-biotin, whereas the discussion says the method uses 100 μM.

  • Discussion counts all 29 downregulated proteins as PM proteins

    Minor

    The discussion says 29 of the identified PM proteins required gp96, but the results classify only 26 of the 29 downregulated proteins as PM/CS/XC/ShG, the broader membrane-protein estimate.

3 of 3

Citations

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. Weekes M. P., 2010, J. Biomol. Tech., 21, 108

Cited by

Sorted by citations

Related

Metadata

Authors
Weekes, M. P., Antrobus, R., Talbot, S., Hör, S., Simecek, N., Smith, D. L., Bloor, S., Randow, F., & Lehner, P. J.
Affiliations
  • University of Cambridge (GB)
  • University of Manchester (GB)
  • MRC Laboratory of Molecular Biology (GB)
Corresponding author
  • Weekes, Michael P.
  • Lehner, Paul J.
ORCID
Venue
Journal of Proteome Research
Publisher
American Chemical Society (ACS)
Published
2012-02-07
Volume
11
Issue
3
Pages
1475-1484
ISSN
  • 1535-3893 (ISSN-L)
  • 1535-3907
DOI
10.1021/pr201135e
PMC
PMC3292266
OpenAlex
W1998249786
Cited by
82
References
57
Citations by year
2026: 4 · 2025: 6 · 2024: 8 · 2023: 1 · 2022: 4 · 2021: 8 · 2020: 5 · 2019: 14 · 2018: 4 · 2017: 4 · 2016: 4 · 2015: 8 · 2014: 5 · 2013: 6 · 2012: 1
FWCI
2.10
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Proteome Research (publishedVersion, other-oa)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • Apollo (University of Cambridge) (submittedVersion, cc-by)
  • Apollo
Funders
Wellcome Trust (084957, 084957/Z/08/Z, 086615, 086615/Z/08/Z)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Heat shock proteins research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Endoplasmic Reticulum Stress and Disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Cholesterol and Lipid Metabolism
Keywords
gp96, endoplasmic reticulum chaperone, surface protein expression, LDL receptor family, LDL receptor, LRP6, SORL1, LRP8, beta-propeller, leucine-rich repeats, pre-B cells, Toll-like receptors, integrins, lipopolysaccharide response
MeSH terms
Animals, Cell Line, Cell Membrane, Chromatography, High Pressure Liquid, Membrane Glycoproteins, Membrane Proteins, Peptide Fragments, Receptors, LDL, Down-Regulation, Antigens, CD, Integrins, Protein Interaction Mapping, Proteomics, Toll-Like Receptors, Mice, Tandem Mass Spectrometry, Low Density Lipoprotein Receptor-Related Protein-6

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.

A language model drew the claims from the paper on 2026-11-10, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).

A second model labelled each claim on 2026-11-10 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).

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 ›

Are you an author, or do you see a problem with this page? Request a review