A Sensitive Method to Monitor Ectodomain Shedding of Ligands of the Epidermal Growth Factor Receptor

Şahin, Umut; Weskamp, Gisela; Zheng, Yufang; Chesneau, Valérie; Horiuchi, Keisuke; Blobel, Carl P.

doi:10.1385/1-59745-012-x:99

Cited by 45 publications

(81 citation statements)

References 1 publication

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“…Shedding assays were performed the day after transfection (1,8,9,12). For shedding experiments including inhibitors, cells were preincubated with or without inhibitors for 2-12 h, as indicated.…”

Section: Adam10/17mentioning

confidence: 99%

“…Experiments to test the requirement for extracellular calcium ions were performed in minimal medium (0.814 mM MgSO 4 ⅐7H 2 O, 5.33 mM KCl, 44.05 mM NaHCO 3 , 81.9 mM NaCl, 0.9 mM NaH 2 PO 4 ⅐H 2 O, 25.03 mM Hepes) with or without added CaCl 2 (0.45 mM). AP activity in the supernatants and cell lysates was measured by colorimetry (12). The ratio between the supernatant AP activity and the total AP activity in the cell lysate plus supernatant was calculated from three identically prepared wells and averaged.…”

Section: Adam10/17mentioning

confidence: 99%

“…This value can be used for side by side comparisons of the activity of a given sheddase toward a given AP-tagged protein for the indicated stimulus in a specific cell type. Evaluation of AP activity in the supernatants and cell lysates by SDS-PAGE or by colorimetric assays was performed as described previously (12).…”

Section: Adam10/17mentioning

confidence: 99%

“…In-gel Alkaline Phosphatase Assay-The in-gel detection of alkaline phosphatase-labeled membrane proteins was performed as previously described (12,15). Briefly, lysates of cells expressing AP-tagged human BTC were separated by SDS-PAGE, and the AP was renatured by incubating the gel twice for 30 min in 2.5% Triton X-100 and visualized by adding the AP substrates nitro blue tetrazolium and 5-bromo-4-chloro-3Ј-indolyphosphate.…”

Section: Adam10/17mentioning

confidence: 99%

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The Cytoplasmic Domain of A Disintegrin and Metalloproteinase 10 (ADAM10) Regulates Its Constitutive Activity but Is Dispensable for Stimulated ADAM10-dependent Shedding

Maretzky

Evers

Gall

et al. 2015

Journal of Biological Chemistry

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Background: Release of membrane proteins from the cell surface by the metalloproteinase ADAM10 is post-translationally regulated. Results:The cytoplasmic domain of ADAM10 negatively controls its constitutive activity but is dispensable for its stimulation. Conclusion: Post-translational stimulation of ADAM10 requires its transmembrane and/or extracellular domain. Significance: Elucidating the regulation of ADAM10 is crucial for understanding the control of ADAM10-dependent cell surface proteolysis.

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Section: Adam10/17mentioning

confidence: 99%

Section: Adam10/17mentioning

confidence: 99%

Section: Adam10/17mentioning

confidence: 99%

Section: Adam10/17mentioning

confidence: 99%

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The Cytoplasmic Domain of A Disintegrin and Metalloproteinase 10 (ADAM10) Regulates Its Constitutive Activity but Is Dispensable for Stimulated ADAM10-dependent Shedding

Maretzky

Evers

Gall

et al. 2015

Journal of Biological Chemistry

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“…Fresh Opti-MEM (Invitrogen) medium, with or without PMA and/or GM6001, was added 18-24 h after transfection, and the cells were incubated for 1 h. The supernatants were collected and cleared by centrifugation to remove cell debris. The activity of AP in the supernatant, which reflects the amount of AP-tagged RANK released from the cell surface, was measured by colorimetry, as described previously (15,20). The supernatant from nontransfected cells incubated with the substrate for the same amount of time was used as a spectrophotometric blank to offset background AP activity.…”

Section: Generation Of Rank Expression Vectorsmentioning

confidence: 99%

Receptor Activator of NF-κB (RANK) Ligand Induces Ectodomain Shedding of RANK in Murine RAW264.7 Macrophages

Hakozaki¹,

Yoda²,

Tohmonda³

et al. 2010

The Journal of Immunology

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Osteoclastogenesis is a highly sophisticated process that involves a variety of membrane-bound proteins expressed in osteoblasts and osteoclast precursors. Over the past several years, proteolytic cleavage and release of the ectodomain of membrane-bound proteins, also referred to as ectodomain shedding, has emerged as an important posttranslational regulatory mechanism for modifying the function of cell surface proteins. In line with this notion, several membrane-bound molecules involved in osteoclastogenesis, including CSF-1R and receptor activator of NF-κB ligand (RANKL), are proteolytically cleaved and released from the cell surface. In this study, we investigated whether receptor activator of NF-κB (RANK), one of the most essential molecules in osteoclastogenesis, undergoes ectodomain shedding. The results showed that RANK is released in the form of a soluble monomeric protein and that TNF-α–converting enzyme is involved in this activity. We also identified potential cleavage sites in the juxtamembrane domain of RANK and found that rRANKL induces RANK shedding in a macrophage-like cell line RAW264.7 via TNFR-associated factor 6 and MAPK pathways. Furthermore, we found that RANKL-induced osteoclastogenesis is accelerated in TNF-α–converting enzyme-deficient osteoclast precursors. These observations suggest the potential involvement of ectodomain shedding in the regulation of RANK functions and may provide novel insights into the mechanisms of osteoclastogenesis.

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FGFR2 mutations promote endometrial cancer progression through dual engagement of EGFR and Notch signalling pathways

Dixit

Gonzalez-Bosquet

Skurski

et al. 2023

Clinical & Translational Med

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Background Mutations in the receptor tyrosine kinase gene fibroblast growth factor receptor 2 ( FGFR2 ) occur at a high frequency in endometrial cancer (EC) and have been linked to advanced and recurrent disease. However, little is known about how these mutations drive carcinogenesis. Methods Differential transcriptomic analysis and two‐step quantitative real‐time PCR (qRT‐PCR) assays were applied to identify genes differentially expressed in two cohorts of EC patients carrying mutations in the FGFR2 gene as well as in EC cells harbouring mutations in the FGFR2 . Candidate genes and target signalling pathways were investigated by qRT‐PCR assays, immunohistochemistry and bioinformatics analysis. The functional roles of differently regulated genes were analysed using in vitro and in vivo experiments, including 3D‐orthotypic co‐culture systems, cell proliferation and migration protocols, as well as colony and focus formation assays together with murine xenograft tumour models. The molecular mechanisms were examined using CRISPR / Cas9‐based loss‐of‐function and pharmacological approaches as well as luciferase reporter techniques, cell‐based ectodomain shedding assays and bioinformatics analysis. Results We show that common FGFR2 mutations significantly enhance the sensitivity to FGF7‐mediated activation of a disintegrin and metalloprotease (ADAM)17 and subsequent transactivation of the epidermal growth factor receptor (EGFR). We further show that FGFR2 mutants trigger the activation of ADAM10‐mediated Notch signalling in an ADAM17‐dependent manner, highlighting for the first time an intimate cooperation between EGFR and Notch pathways in EC. Differential transcriptomic analysis in EC cells in a cohort of patients carrying mutations in the FGFR2 gene identified a strong association between FGFR2 mutations and increased expression of members of the Notch pathway and ErbB receptor family. Notably, FGFR2 mutants are not constitutively active but require FGF7 stimulation to reprogram Notch and EGFR pathway components, resulting in ADAM17‐dependent oncogenic growth. Conclusions These findings highlight a pivotal role of ADAM17 in the pathogenesis of EC and provide a compelling rationale for targeting ADAM17 protease activity in FGFR2‐driven cancers.

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A Sensitive Method to Monitor Ectodomain Shedding of Ligands of the Epidermal Growth Factor Receptor

Cited by 45 publications

References 1 publication

The Cytoplasmic Domain of A Disintegrin and Metalloproteinase 10 (ADAM10) Regulates Its Constitutive Activity but Is Dispensable for Stimulated ADAM10-dependent Shedding

The Cytoplasmic Domain of A Disintegrin and Metalloproteinase 10 (ADAM10) Regulates Its Constitutive Activity but Is Dispensable for Stimulated ADAM10-dependent Shedding

Receptor Activator of NF-κB (RANK) Ligand Induces Ectodomain Shedding of RANK in Murine RAW264.7 Macrophages

FGFR2 mutations promote endometrial cancer progression through dual engagement of EGFR and Notch signalling pathways

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