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Analysis of Clathrin-mediated Endocytosis of Epidermal Growth Factor Receptor by RNA Interference

Huang, Fangtian, Khvorova, Anastasia, Marshall, William, Sorkin, Alexander·2004·Journal of Biological Chemistry
DOI 10.1074/jbc.c400046200
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FIG. 1 Depletion of coated pit and endosome proteins by siRNAs. HeLa cells were transfected with siRNA duplexes (four duplexes for each gene indicated as Dup1 , Dup2 , Dup3 , and Dup4 ) or pools of siRNA duplexes ( Pool ), and the cells were lysed 3 days after transfection with the exception of CALM (2 days) and 𝛃 2 (4 days). In experiments with Rab5 siRNAs HeLa cells were transfected with YFPRab5a, YFP-Rab5b, or YFP-Rab5c or not transfected. At the same time, HeLa cells were transfected with siRNA duplexes. The cells were lysed 3 days after transfections. Equal amounts of lysates were resolved by electrophoresis and blotted with the antibody specific to a targeted protein (GFP antibody for YFP fusion proteins) and the antibody to unrelated proteins, PP1 phosphatase or 𝛂 -actinin, and TSG101 (not shown). Note a 𝛃 1-adaptin band (part of AP-1 Golgi adaptor complex) that runs slightly slower than 𝛃 2-adaptin. CALM has two splice variants, 66 and 72 kDa. The full-length Eps15R (a duplet of ∼ 130 kDa) and several truncated spliced forms of ∼ 100 kDa and ∼ 70 kDa were detected in Eps15R immunoprecipitates (shown by arrows ). The siRNA duplexes chosen for internalization experiments are marked by asterisks .
FIG. 1 Depletion of coated pit and endosome proteins by siRNAs. HeLa cells were transfected with siRNA duplexes (four duplexes for each gene indicated as Dup1 , Dup2 , Dup3 , and Dup4 ) or pools of siRNA duplexes ( Pool ), and the cells were lysed 3 days after transfection with the exception of CALM (2 days) and 𝛃 2 (4 days). In experiments with Rab5 siRNAs HeLa cells were transfected with YFPRab5a, YFP-Rab5b, or YFP-Rab5c or not transfected. At the same time, HeLa cells were transfected with siRNA duplexes. The cells were lysed 3 days after transfections. Equal amounts of lysates were resolved by electrophoresis and blotted with the antibody specific to a targeted protein (GFP antibody for YFP fusion proteins) and the antibody to unrelated proteins, PP1 phosphatase or 𝛂 -actinin, and TSG101 (not shown). Note a 𝛃 1-adaptin band (part of AP-1 Golgi adaptor complex) that runs slightly slower than 𝛃 2-adaptin. CALM has two splice variants, 66 and 72 kDa. The full-length Eps15R (a duplet of ∼ 130 kDa) and several truncated spliced forms of ∼ 100 kDa and ∼ 70 kDa were detected in Eps15R immunoprecipitates (shown by arrows ). The siRNA duplexes chosen for internalization experiments are marked by asterisks .CC-BY · From the PDF
FIG. 2 Effects of protein depletion on EGFR and TfnR endocytosis. A , siRNAs selected from the experiments presented in Figs. 1 and 2 were transfected as described in the legend to Fig. 1. After 3 or 4 days (for 𝛃 2, Eps15, and Eps15R siRNAs), the cells plated in 12-well dishes were used to measure the rates of internalization of 125 I-EGF ( white bars ) and 125 I-Tfn ( gray bars ) using 1-5-min incubation of cells with labeled ligands at 37 °C. The internalization rates are expressed as percent to these rates in mock-transfected cells. The k e values of 125 I-EGF and 125 I-Tfn internalization in mock-transfected cells were 0.35-0.42/min and 0.40-0.48/min, respectively. B , cells transfected with siRNAs as in A were grown on coverslips and incubated with 2 ng/ml EGF-Rh for 5 min at 37 °C. The sum projection images made from a Z-stack of 10 two-dimensional images are shown. All fluorescence intensity settings are identical for all images. C , siRNAs for Grb2 and 𝛍 2 subunit of AP-2 were transfected, and the rates of internalization were measured 3 days after initial transfection as in A . The k e values of 125 I-EGF and 125 I-Tfn internalization in mock-transfected cells were 0.38/min and 0.45/min, respectively. D , siRNA for 𝛂 -adaptin was transfected twice using Lipofectamin 2000 ( Lipo2000 ) (as in A and B ) or Oligofectamin, and the rates of 125 I-EGF internalization were measured 4 days after first transfection by incubating cells directly at 37 °C (as in A and B ) ( k e in mock-transfected cells: 0.36/min) or by incubating cells with 1 ng/ml 125 I-EGF for 1 h at 4 °C followed by a chase incubation for 2, 4, and 6 min at 37 °C. In the latter case the apparent rates of uptake were estimated by the ratio of internalized and surface bound 125 I-EGF.
FIG. 2 Effects of protein depletion on EGFR and TfnR endocytosis. A , siRNAs selected from the experiments presented in Figs. 1 and 2 were transfected as described in the legend to Fig. 1. After 3 or 4 days (for 𝛃 2, Eps15, and Eps15R siRNAs), the cells plated in 12-well dishes were used to measure the rates of internalization of 125 I-EGF ( white bars ) and 125 I-Tfn ( gray bars ) using 1-5-min incubation of cells with labeled ligands at 37 °C. The internalization rates are expressed as percent to these rates in mock-transfected cells. The k e values of 125 I-EGF and 125 I-Tfn internalization in mock-transfected cells were 0.35-0.42/min and 0.40-0.48/min, respectively. B , cells transfected with siRNAs as in A were grown on coverslips and incubated with 2 ng/ml EGF-Rh for 5 min at 37 °C. The sum projection images made from a Z-stack of 10 two-dimensional images are shown. All fluorescence intensity settings are identical for all images. C , siRNAs for Grb2 and 𝛍 2 subunit of AP-2 were transfected, and the rates of internalization were measured 3 days after initial transfection as in A . The k e values of 125 I-EGF and 125 I-Tfn internalization in mock-transfected cells were 0.38/min and 0.45/min, respectively. D , siRNA for 𝛂 -adaptin was transfected twice using Lipofectamin 2000 ( Lipo2000 ) (as in A and B ) or Oligofectamin, and the rates of 125 I-EGF internalization were measured 4 days after first transfection by incubating cells directly at 37 °C (as in A and B ) ( k e in mock-transfected cells: 0.36/min) or by incubating cells with 1 ng/ml 125 I-EGF for 1 h at 4 °C followed by a chase incubation for 2, 4, and 6 min at 37 °C. In the latter case the apparent rates of uptake were estimated by the ratio of internalized and surface bound 125 I-EGF.CC-BY · From the PDF
FIG. 3 Effects of simultaneous depletion of several proteins on EGF and Tfn receptor endocytosis. siRNAs targeted to homologous proteins selected from the experiments presented in Figs. 1 and 2 were co-transfected. After 3 (clathrin light chains and Rab5s) or 4 days (Eps15 and Eps15R), the cells plated in 12-well dishes were used to measure the rates of internalization of 125 I-EGF and 125 I-Tfn ( A ) and visualize the extent of protein depletion by Western blotting ( B ). The internalization rates are expressed as percent to these rates in mocktransfected cells. In case of Rab5s depletion, only the amount of Rab5a was controlled due to antibody unavailability.
FIG. 3 Effects of simultaneous depletion of several proteins on EGF and Tfn receptor endocytosis. siRNAs targeted to homologous proteins selected from the experiments presented in Figs. 1 and 2 were co-transfected. After 3 (clathrin light chains and Rab5s) or 4 days (Eps15 and Eps15R), the cells plated in 12-well dishes were used to measure the rates of internalization of 125 I-EGF and 125 I-Tfn ( A ) and visualize the extent of protein depletion by Western blotting ( B ). The internalization rates are expressed as percent to these rates in mocktransfected cells. In case of Rab5s depletion, only the amount of Rab5a was controlled due to antibody unavailability.CC-BY · From the PDF

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Metadata

Authors
Huang, F., Khvorova, A., Marshall, W., & Sorkin, A.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2004-04
Volume
279
Issue
16
Pages
16657-16661
ISSN
0021-9258
DOI
10.1074/jbc.c400046200
Cited by
402
References
2
Language
en
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CoChat. (2026). Citation check: Analysis of Clathrin-mediated Endocytosis of Epidermal Growth Factor Receptor by RNA Interference. https://app.cochat.ai/papers/10.1074/jbc.c400046200
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Huang, F., Khvorova, A., Marshall, W., & Sorkin, A. (2004). Analysis of Clathrin-mediated Endocytosis of Epidermal Growth Factor Receptor by RNA Interference. Journal of Biological Chemistry. https://doi.org/10.1074/jbc.c400046200

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