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First published online 16 August 2005
doi: 10.1242/jcs.02527

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A novel dileucine lysosomal-sorting-signal mediates intracellular EGF-receptor retention independently of protein ubiquitylation

Amy Tsacoumango^1,*, Song Jae Kil^1,*, Liping Ma¹, Frank D. Sönnichsen^1,2,3 and Cathleen Carlin^1,3,4,

¹ Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4970, USA
² Cleveland Center for Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4970, USA
³ Case Western Reserve University Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4970, USA
⁴ The Rainbow Center for Childhood PKD at Rainbow Babies and Children's Hospital of Cleveland, 11100 Euclid Avenue, Cleveland, OH 44106, USA

View larger version (23K): [in a new window]   Fig. 1. (A) Parental NR6 cells that lack endogenous EGFRs and NR6 cell lines stably expressing wild-type human EGFR or a mutant receptor with an inactivating 679-AA substitution, were incubated with 125I-EGF for 2 hours at 4°C, followed by incubation with a chemical cross-linker at room temperature for 15 minutes. (B) NR6 cell lines expressing wild-type or 679-AA-mutant receptors were pulse-labeled with a mixture of [35S]cysteine and [35S]methionine for 30 minutes followed by a 3-hour incubation in chase medium. The pulse-labeled cells were then stimulated with EGF for 0, 1 or 2 hours and cell lysates were immunoprecipitated with a human EGFR-specific antibody. Equal aliquots of total cellular protein (A) or EGFR immunoprecipitates (B) were resolved by SDS-PAGE for autoradiographic detection. Molecular mass standards: 200,000 Da (myosin); 116,300 Da (ß-galactosidase).

View larger version (32K): [in a new window]   Fig. 2. (A-E) Wild-type and 679-AA-mutant cells were lysed and lysates from unstimulated (-) cells and from cells stimulated with EGF for 10 minutes (+) were immunoprecipitated with antibodies against (A-C) human EGFR or (D,E) CBL. Immunoprecipitates were transferred to nitrocellulose filters for immunoblotting after SDS-PAGE. EGFR immunoprecipitates were divided in half and resolved on two gels. One filter was incubated with a phosphorylation-specific EGFR (pTyr1045) antibody (A), and the second with a CBL antibody (B). The filter in (B) was re-probed with an EGFR antibody as a loading control (C). (D,E) The filter with CBL immunoprecipitates was incubated with a phosphotyrosine-specific antibody (pTyr) (D), and then re-probed with a CBL antibody as a loading control (E). IP, immunoprecipitation; IB, immunoblot. Molecular size in Da on the left.

View larger version (110K): [in a new window]   Fig. 3. Confocal images of permeabilized cells that were unstimulated (-EGF) or stimulated with EGF (+ EGF) and co-stained with antibodies against EGFR (green channel) and to CBL (red channel). Green and red channels were merged after both fluorescent signals were adjusted to similar levels. The yellow color indicates the overlap of red and green fluorescence. Areas corresponding to the enlarged insets shown on the extreme right are boxed in all of the panels. Some intracellular vesicles co-stained for EGFR and CBL are highlighted with arrows. Bar, 10 µm.

View larger version (45K): [in a new window]   Fig. 4. (A) Cell lines expressing wild-type or 679-AA-mutant receptors were stimulated with EGF for the times indicated. EGFR immunoprecipitates resolved on a 5% acrylamide gel were transferred to nitrocellulose filters and immunoblotted with a ubiquitin-specific antibody. Solid-line brackets indicate location of ubiquitylated EGFR (high molecular mass), arrow indicates 170-kDa EGFR (non-ubiquitylated). (B) The filter in (A) was re-probed with an EGFR-specific antibody. Arrow indicates 170-kDa EGFR (non-ubiquitylated). High molecular mass ubiquitylated EGFR (dashed-line bracket) was not seen unless the film was overexposed (data not shown).

View larger version (58K): [in a new window]   Fig. 5. (A,B) Cells infected with E3-13.7-positive (+) or E3-13.7-negative (-) adenoviruses were labeled with a mixture of [35S]cysteine and [35S]methionine for 3 hours, and cell lysates were immunoprecipitated with antibodies against E1A (A) or E3-13.7 (B) early adenovirus proteins. (C,D) Adenovirus-infected cells were pulse-labeled for 15 minutes at 16 hours post-infection and were then switched to chase-medium for up to 3 hours (C) or 6 hours (D). Cell lysates were immunoprecipitated with an anti-EGFR antibody. Immunoprecipitates in panels A-D were resolved by SDS-PAGE for autoradiographic detection of radiolabeled proteins. Molecular mass standards: 66,200 (BSA); 45,000 (ovalbumin); 14,400 (lysosyme). (E) Confocal images of permeabilized cells that had been infected with E3-13.7-positive (+ E3-13.7) or E3-13.7-negative (-E3-13.7) adenoviruses and stained with an EGFR-specific antibody. Pos, positive; neg, negative. Bar, 10 µm.

View larger version (60K): [in a new window]   Fig. 6. (A,B) Permanent cell lines stably expressing EGFR-related proteins (A) or IL2R-EGFR chimeras (B), whose genotypes are depicted schematically at the top of each panel, were infected with E3-13.7-positive (+) or E3-13.7-negative (-) adenoviruses. The infected cells were pulse-labeled with a mixture of [35S]cysteine and [35S]methionine for 30 minutes followed by a 5-hour incubation period in chase-medium. Cell lysates were immunoprecipitated with antibodies against external epitopes of EGFR (A) or IL2R (B). Immunoprecipitates were resolved by SDS-PAGE for autoradiographic detection of radiolabeled proteins. (C) Confocal images of permeabilized cells that had been infected with the E3-13.7-positive adenovirus and stained with an IL2R-specific antibody. Areas corresponding to the enlarged insets shown beneath each image are boxed in all of the panels. Bar, 10 µm. TM, transmembrane; 679-LL and 679-AA, wild-type and mutationally inactivated lysosomal sorting-signals, respectively.

View larger version (17K): [in a new window]   Fig. 7. (A,C) The H CSDs for synthetic peptides with (A) 679-LL or (C) 679-AA sequences where a value of 0 corresponds to random coil. CSDs are represented as ppm on the x-axis [ppm=(shift observed/oscillator frequency)x106]. Positive and negative values represent upfield and downfield shifts, respectively. Amino acid residues are labeled on the y-axis, based on the published human EGFR sequence (NCBI accession number P00533). Data were obtained for peptides dissolved in water (open bars) or in the presence of DPC micelles (black bars) at 5°C. (B,D) Summary of intramolecular NOEs observed for (B) wild-type and (D) mutant peptides in the presence of DPC micelles. NOE-intensity-strength is indicated by the thickness of the lines. Dotted lines indicate proline CH-proton resonance effects.

View larger version (44K): [in a new window]   Fig. 8. (A,B) 2D-TOCSY proton spectra for residues Ser671 to Tyr688 in DPC-micelle-bound state (A) without and (B) with a 5-doxyl stearic acid spin-label. Italicized residues in (B) exhibit line-broadening due to the spin label. F1, amide protons; F2, H protons. (C,D) Representation of the predicted orientation of DPC-micelle-stabilized helical structure as a (C) side or (D) front view. EGFR residues also occurring in the adenovirus E3-13.7 protein are underlined. Red and blue residues represent oxygen and nitrogen, respectively, in amino acid side-chains.