Clathrin adapters AP-1 and GGA2 support expression of epidermal growth factor receptor for cell growth

Depletion of AP-1 or GGA2 increases CIMPR in both total and cell surface fractions.

“One of well-known cargoes of AP-1/GGA2, cation independent (CI) MPR was rather increased by the depletion of AP-1/GGA2 in both total and cell surface fraction, which is consistent with previous data [25–27].”

• cites [25] Ghosh P, Griffith J, Geuze HJ, Kornfeld S. Mammalian GGAs act together to sort mannose 6-phosphate receptors. J Cell Biol. 2003;163:755–66. sourceNot checked yet
• cites [26] Meyer C, Eskelinen EL, Guruprasad MR, von Figura K, Schu P. Mu 1A deficiency induces a profound increase in MPR300/IGF-II receptor internalization rate. J Cell Sci. 2001;114:4469–76. sourceNot checked yet
• cites [27] Navarro Negredo P, Edgar JR, Wrobel AG, Zaccai NR, Antrobus R, Owen DJ, et al. Contribution of the clathrin adaptor AP-1 subunit micro1 to acidic cluster protein sorting. J Cell Biol. 2017;216:2927–43. sourceNot checked yet

Depletion of AP-1 or GGA2 significantly suppresses EGFR recycling to the plasma membrane regardless of EGF stimulation.

“Biochemical recycling assay revealed that the depletion of AP-1 or GGA2 significantly suppressed EGFR recycling to the plasma membrane regardless of the EGF stimulation.”

• Also stated in: § AP-1 and GGA2 support recycling of EGFR back to the PM · hedged

  “These results together with the PLA data under EGF stimulation as described above (Supplementary Fig. S4C) suggest that AP-1 and GGA2 support the recycling of EGFR regardless of the EGF stimulation.”

EGFR associates preferentially with both AP-1 and GGA2 in vitro.

“Here, we show that EGFR associates preferentially with both AP-1 and GGA2 in vitro.”

• Also stated in: § AP-1-depletion causes a reduction of EGFR protein · boosted

  “These assays revealed a strong EGFR interaction with AP-1 and GGA2 and a weak interaction with AP-4 and AP-5, and little interaction with AP-3, GGA1, or GGA3 in both cell-types (Fig. 1B).”

The interaction of EGFR with AP-1 or GGA2 occurs more frequently in Rab11-positive recycling endosomes than in Rab5-positive early endosomes.

“Triple immunofluorescence microscopy and proximity ligation assays demonstrated that the interaction of EGFR with AP-1 or GGA2 occurred more frequently in Rab11-positive recycling endosomes than in Rab5-positive early endosomes.”

• Also stated in: § AP-1 and GGA2 interact with EGFR at the recycling endosomes · hedged

  “Quantification revealed that about 49–52% of the signal for AP-1/GGA2–EGFR interaction overlapped with Rab11-positive structures, while it was 13–15% with Rab5-positive ones (Fig. 4D), indicating that the interactions between AP-1/GGA2 and EGFR occur preferentially in the recycling endosomes.”

Depletion of AP-1 or GGA2 reduces MET in H1975 cells.

“In H1975 cells that have minimal expression of Erb-B4 (Supplementary Fig. S6B), MET were significantly decreased by AP-1/GGA2 depletion (P < 0.01 or 0.001; Fig. 7B and Supplementary Fig. S6A).”

Depletion of AP-1 or GGA2 suppresses growth of H1975 cancer cells in culture.

“and therefore, suppressed the growth of H1975 cancer cells in culture and xenograft model.”

Depletion of AP-1 or GGA2 reduces cell surface and total EGFR protein in ARPE-19 and H1975 cells.

“As shown in Fig. 3, EGFR in the cell surface was significantly decreased (P < 0.001) by depletion of either AP-1 or GGA2 in both ARPE-19 and H1975 cells, which is comparable with the decrease in the total protein.”

• Also stated in: § Depletion of AP-1 or GGA2 downregulates cell surface expression of EGFR · hedged

  “Together, this assay indicates that the depletion of AP-1 or GGA2 reduces the cell surface amounts as well as the total amounts of EGFR protein.”

Depletion of AP-1 or GGA2 reduces cellular contents of MET and ErbB4 in ARPE-19 cells.

“Depletion of AP-1 or GGA2 also reduced cell contents of other tyrosine kinases, MET and ErbB4”

• Also stated in: § Depletion of AP-1 or GGA2 causes downregulation of other receptor tyrosine kinase (RTK) · plain

  “We found that Erb-B4 and MET were also decreased significantly (P < 0.05 or 0.001) by depletion of either AP-1 or GGA2 in ARPE-19 cells (Fig. 7A, and Supplementary Fig. S6A).”

AP-1 depletion causes a reduction in EGFR protein in ARPE-19 cells.

“AP-1 depletion caused a reduction in the EGFR protein by promoting its lysosomal degradation.”

• Also stated in: § AP-1-depletion causes a reduction of EGFR protein · hedged

  “These results indicate that the depletion of AP-1 causes a reduction of EGFR protein in ARPE-19 cells.”

• Also stated in: § AP-1-depletion causes a reduction of EGFR protein · plain

  “When ARPE-19 cells were treated with siRNAs against γ1 (AP-1)-, δ (AP-3)-, ε (AP-4)-, and ζ (AP-5)-adaptin, γ1- and ζ-adaptin depletions led to marked decrease of EGFR (Fig. 1C).”

Depletion of AP-1 or GGA2 suppresses growth of H1975 cancer cell xenografts.

“and therefore, suppressed the growth of H1975 cancer cells in culture and xenograft model.”

Depletion of AP-1 or GGA2 causes downregulation of other RTK proteins.

“Depletion of AP-1 or GGA2 causes downregulation of other RTK proteins.”

AP-1 and AP-5, as well as GGA2, support the steady-state cell surface expression of EGFR.

“We found that AP-1 and AP-5, as well as GGA2 support the steady-state cell surface expression of EGFR.”

AP-1 was changed in its cellular distribution in many cases of HCC, NSCLC, and CRC.

“We found by immunohistological analysis that AP-1 was changed in its cellular distribution in many cases of HCC, NSCLC, and CRC, and also upregulated in NSCLC.”

Depletion of γ1-adaptin significantly lowers growth rates of ARPE-19 and H1975 cells in culture.

“In cell culture conditions, growth rates of ARPE-19 or H1975 cells depleted of γ1-adaptin were significantly lower than those of control cells (P < 0.05–0.001; Fig. 8A and Supplementary Fig. S7B).”

AP-1 is primarily localized to EEA1-positive endosomes in HCC, NSCLC, and CRC.

“Moreover, AP-1 is primarily localized to EEA1-positive endosomes in HCC, NSCLC, and CRC, which is in line with the recycling endosome-localized AP-1 observed in cultured cells.”

AP-1 was upregulated in NSCLC.

“We found by immunohistological analysis that AP-1 was changed in its cellular distribution in many cases of HCC, NSCLC, and CRC, and also upregulated in NSCLC.”

AP-1 and GGA2 support cell surface expression of other tyrosine kinases, MET and ErbB4.

“In addition, AP-1 and GGA2 also support cell surface expression of other tyrosine kinases, MET and ErbB4.”

• Also stated in: § Discussion · hedged

  “The present study also suggests AP-1/GGA2 can support the expression of other RTKs, MET and ErbB4, which might have a significant impact on cancer growth in HCC, since MET is known to be overexpressed in this type of tumor [42].”

GGA3 hardly associated with EGFR at steady-state in this study.

“since it hardly associated with EGFR at steady-state in this study (Fig. 1A).”

AP-1 and GGA2 support EGFR recycling back to the plasma membrane regardless of EGF stimulation.

“Strikingly, both AP-1 and GGA2 interact with EGFR at Rab11-positive recycling endosomes, and support the receptor recycling back to the PM regardless of the EGF stimulation.”

• Also stated in: § Discussion · boosted

  “We confirmed these associations both in vitro and in vivo, and further found that these associations specifically occur in Rab11a-positive recycling endosomes, and support the EGFR recycling.”

AP-1 depletion significantly suppressed tumor growth rates in H1975 xenografts.

“In xenoplantation experiments using H1975 cells, AP-1-depletion significantly suppressed growth rates of tumors (P < 0.001; Fig. 8B and Supplementary Fig. S7C).”

AP-1 and GGA2 interact with EGFR at Rab11-positive recycling endosomes.

“Strikingly, both AP-1 and GGA2 interact with EGFR at Rab11-positive recycling endosomes, and support the receptor recycling back to the PM regardless of the EGF stimulation.”

• Also stated in: § Discussion · boosted

  “We confirmed these associations both in vitro and in vivo, and further found that these associations specifically occur in Rab11a-positive recycling endosomes, and support the EGFR recycling.”

In tissue arrays, cytoplasmic granular AP-1-positive structures were more evident in tumor regions compared to non-tumor regions in 68-84% of cancer tissues (HCC, NSCLC, CRC).

“Cytoplasmic granular structures positive for AP-1 were more evident in tumor regions compared to non-tumor regions in 68–84% of cancer tissues (Fig. 8C).”

In AP-1-depleted ARPE-19 cells, phosphorylation of MAPK, a downstream signaling of EGFR, was significantly reduced.

“Phosphorylation of MAPK, a downstream signaling of EGFR, was significantly reduced in AP-1-depleted ARPE-19 cells (Supplementary Fig. S7A).”

Depletion of AP-1 suppressed cell growth in ARPE-19 and H1975 cells.

“Accordingly, cell growth was suppressed by the depletion of AP-1 in a non-cancer cell line, ARPE-19, and a NSCLC-derived cancer cell line, H1975, which harbors activating EGFR mutations [17].”

• cites [17] Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2005;2:e73. sourceNot checked yet

Depletion of either AP-1 or GGA2 does not affect expression levels of the other.

“Depletion of either AP-1 or GGA2 did not affect expression levels of the other (Fig. 6A), as reported previously [32, 33], nor did simultaneous depletion of both adapters enhance the reduction of EGFR.”

• cites [32] Uemura T, Sawada N, Sakaba T, Kametaka S, Yamamoto M, Waguri S. Intracellular localization of GGA accessory protein p56 in cell lines and central nervous system neurons. Biomed Res. 2018;39:179–87. sourceNot checked yet
• cites [33] Mardones GA, Burgos PV, Brooks DA, Parkinson-Lawrence E, Mattera R, Bonifacino JS. The trans-Golgi network accessory protein p56 promotes long-range movement of GGA/clathrin-containing transport carriers and lysosomal enzyme sorting. Mol Biol Cell. 2007;18:3486–501. sourceNot checked yet

Depletion of either AP-1 or GGA2 affected the distribution of GGA2 or AP-1, respectively.

“The possible implication of AP-1-GGA2 interaction in the EGFR trafficking is partly supported by in vivo data in the present study showing that the depletion of either AP-1 or GGA2 affected the distribution of GGA2 or AP-1, respectively (Fig. 6).”

AP-1 depletion promotes lysosomal degradation of EGFR.

“AP-1 depletion caused a reduction in the EGFR protein by promoting its lysosomal degradation.”

• Also stated in: § AP-1-depletion accelerates lysosomal degradation of EGFR protein · hedged

  “Together with the increased turnover rate of EGFR by AP-1 knockdown (Fig. 2A), it is suggested that AP-1 depletion does not cause a drastic disturbance in the early endocytic pathway of EGFR, but enhances its transport from the endocytic pathway into lysosomes for degradation.”

GGA2 may also recognize EGFR indirectly through AP-1.

“Although GGA2 associates with the juxtamembrane domain of EGFR [15], it may also recognize EGFR indirectly through AP-1, because the hinge domain of GGA2 can directly bind the ear domain of AP-1 [37].”

• cites [15] Uemura T, Kametaka S, Waguri S. GGA2 interacts with EGFR cytoplasmic domain to stabilize the receptor expression and promote cell growth. Sci Rep. 2018;8:1368. sourceNot checked yet
• cites [37] Doray B, Ghosh P, Griffith J, Geuze HJ, Kornfeld S. Cooperation of GGAs and AP-1 in packaging MPRs at the trans-Golgi network. Science. 2002;297:1700–3. sourceNot checked yet

AP-1/GGA2 targeting could arrest multiple cancer-related signaling pathways that originate from different RTKs.

“Nevertheless, it should be emphasized that potential AP-1/GGA2 targeting as an anti-cancer strategy is not dependent on activation mutations but on the intracellular recycling of EGFR, and that AP-1/GGA2 targeting could arrest multiple cancer-related signaling pathways that originate from different RTKs.”

Both AP-1 and GGA2 have a fundamental influence on cell growth by sustaining the expression of EGFR.

“Together with recent findings that the depletion of GGA2 suppresses cell proliferation in ARPE-19 [15], and two NSCLC-derived cancer cell lines, PC9 and H1975 [16], it is most likely that both AP-1 and GGA2 have a fundamental influence on cell growth by sustaining the expression of EGFR.”

• cites [15] Uemura T, Kametaka S, Waguri S. GGA2 interacts with EGFR cytoplasmic domain to stabilize the receptor expression and promote cell growth. Sci Rep. 2018;8:1368. sourceNot checked yet
• cites [16] O’Farrell H, Harbourne B, Kurlawala Z, Inoue Y, Nagelberg AL, Martinez VD, et al. Integrative genomic analyses identifies GGA2 as a cooperative driver of EGFR-mediated lung tumorigenesis. J Thorac Oncol. 2019;14:656–71. sourceNot checked yet

The recruitment of AP-1 to endosomes may be more relevant than that to the TGN in these cancers.

“These observations suggest that the recruitment of AP-1 to endosomes may be more relevant than that to the TGN in these cancers.”

Growth suppression by AP-1 depletion in H1975 cells both in vitro and in vivo is mediated by EGFR depletion.

“Moreover, since the growth of NSCLC-derived H1975 cells used in this study is known to depend on the EGFR, growth suppression by AP-1 depletion both in vitro and in vivo (Fig. 8A, B) would provide strong evidence that the effect was mediated by the EGFR depletion.”

• AssumptionRests on an assumption: H1975 cell growth depends on EGFR.

These results suggest the involvement of AP-1 in EGFR-dependent cell growth.

“H1975 cells harbor activating EGFR mutations [17], and as such, these results suggest the involvement of AP-1 in EGFR-dependent cell growth.”

• cites [17] Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2005;2:e73. sourceNot checked yet

GGA2 protein is upregulated in HCC and CRC.

“and GGA2 protein has been reported to be upregulated in HCC and CRC [15].”

• cites [15] Uemura T, Kametaka S, Waguri S. GGA2 interacts with EGFR cytoplasmic domain to stabilize the receptor expression and promote cell growth. Sci Rep. 2018;8:1368. sourceNot checked yet

The internalization rate of EGFR is not significantly affected by depletion of AP-1 or GGA2.

“We first confirmed that the internalization rate was not significantly affected by the depletion of AP-1 or GGA2 (Fig. 5B).”

Alterations in intracellular distribution rather than higher expression levels of AP-1 are characteristic for tumor cells.

“suggesting alterations in intracellular distribution rather than higher expression levels of AP-1 are characteristic for tumor cells.”

Accumulating evidence including our data suggests the importance of AP-1/GGA2 in cancer growth.

“Therefore, accumulating evidence including our data suggests the importance of AP-1/GGA2 in cancer growth.”

The reduction of EGFR by γ1-adaptin depletion is not due to off-target effects.

“Importantly, depletion of other subunits, µ1 or β1-adaptin also reduced EGFR (Supplementary Fig. S1D), and reexpression of γ1-adaptin slightly restored EGFR in Western blot analysis (Fig. 1E) and immunofluorescence microscopy (Supplementary Fig. S1E), excluding a possibility of off-target effects.”

The present study provides the first evidence of a molecular link between AP-1/GGA2 and EGFR in Rab11-positive recycling endosomes in both steady-state and EGF-stimulated conditions.

“Therefore, the present study provides the first evidence that shows a molecular link between AP-1/GGA2 and EGFR in Rab11-positive recycling endosomes in both the steady-state and EGF-stimulated conditions.”

Rab11 itself is unlikely to be directly involved in AP-1/GGA2-mediated EGFR recycling.

“It is unlikely that Rab11 itself is directly involved in the AP-1/GGA2-mediated EGFR recycling, because Rab11 knockdown did not cause significant reduction of EGFR (Supplementary Fig. S3B), and Rab11 was not found in the list of EGFR interacting molecules in mass analysis (Supplementary Table S1).”

GGA2 is able to interact with the juxta-membrane domain of EGFR, although this domain does not contain a typical ACLL motif.

“and that GGA2 is able to interact with the juxta-membrane domain of EGFR, although this domain does not contain a typical ACLL motif [15].”

• cites [15] Uemura T, Kametaka S, Waguri S. GGA2 interacts with EGFR cytoplasmic domain to stabilize the receptor expression and promote cell growth. Sci Rep. 2018;8:1368. sourceNot checked yet

H1975 cells harbor activating EGFR mutations.

“H1975 cells harbor activating EGFR mutations [17]”

• cites [17] Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2005;2:e73. sourceNot checked yet

The AP-1-EGFR interaction occurs independently of EGF signaling.

“Interestingly, the PLA signal did not significantly alter after EGF treatment (Supplementary Fig. S4C), suggesting the interaction occurs independently of the EGF signaling.”

The proximity ligation assay specifically detects AP-1-EGFR interaction.

“These results indicate that the PLA specifically detects AP-1–EGFR interaction.”

The specificity of the antibody against γ1-adaptin in recognizing endogenous protein in paraffin-embedded human tissues was confirmed using cell pellets of AP-1-depleted H1975 cells.

“The specificity of the antibody against γ1-adaptin in recognizing the endogenous protein in paraffin-embedded human tissues was confirmed by using cell pellets of AP-1-depleted H1975 cells (Supplementary Fig. S7D).”

AP-1 and GGA2 could act in the recycling pathway of EGFR that evades ubiquitination.

“On the other hand, AP-1 and GGA2 could act in the recycling pathway of EGFR that evades ubiquitination, because they have lower affinities to ubiquitin compared with GGA3 [12].”

• cites [12] Puertollano R, Bonifacino JS. Interactions of GGA3 with the ubiquitin sorting machinery. Nat Cell Biol. 2004;6:244–51. sourceNot checked yet

Both AP-1 and GGA2 regulate cellular contents and cell surface expression of a subset of receptor tyrosine kinases in a cell-dependent manner.

“Together, these assays clearly indicate that, although their preferences are different, both AP-1 and GGA2 regulate cellular contents and cell surface expression of a subset of RTKs in a cell-dependent manner.”

The cellular distribution of either AP-1 or GGA2 depends on the other's expression.

“Together, these results indicate that the cellular distribution of either AP-1 or GGA2 depends on the other’s expression.”

AP-1 and GGA2 play an important role in the maintenance of cell surface EGFR by regulating the receptor recycling pathway.

“AP-1 and GGA2 play an important role in the maintenance of cell surface EGFR by regulating receptor recycling pathway.”