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First published online 9 September 2008
doi: 10.1242/jcs.033399

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All EGF(ErbB) receptors have preformed homo- and heterodimeric structures in living cells

Information Processing Biology Unit, Okinawa Institute of Science and Technology, 12-2 Suzaki, Uruma, Okinawa 904-2234, Japan

View larger version (48K): [in this window] [in a new window]   Fig. 1. Subcellular localization of the ErbB family members. (A) CHO cells expressing EGFR-CFP, ErbB2-YFP, ErbB3-CFP or ErbB4-JMa-GFP were serum-starved for 24 hours at 37°C, and then observed by confocal microscopy. Note that the FP-fused EGFR, ErbB2 and ErbB4-JMa were localized in the plasma membrane, whereas ErbB3-CFP was mainly in the nucleus. (B) CHO cells co-expressing EGFR-YFP and ErbB3-CFP, ErbB2-YFP and ErbB3-CFP, or ErbB4-JMa-YFP and ErbB3-CFP were serum-starved, and then observed by confocal microscopy. Note that a large fraction of ErbB3-CFP was localized in the plasma membrane when co-expressed with EGFR-YFP or ErbB2-YFP. By contrast, the majority of the ErbB4-JMa-YFP protein was localized in the nucleus when co-expressed with ErbB3-CFP. Bars, 10 µm.

View larger version (64K): [in this window] [in a new window]   Fig. 2. BiFC analysis of ErbB receptor homodimerization in living cells. (A) Schematic representation of the principle of BiFC. VN and VC are the N-terminal and C-terminal fragments of the Venus FP, respectively. A and B are proteins to be examined for dimerization. (B) EGF-induced phosphorylation of EGFR fused with VN or VC. CHO cells expressing EGFR-VN, EGFR-VC, or both of the constructs were serum-starved and then treated with (+) or without (–) 100 ng/ml EGF for 30 minutes at 4°C. Whole cell lysates were subjected to immunofluorescent staining with anti-phospho-EGFR (p-EGFR) or anti-phospho-Akt (p-Akt) antibody, or with anti-EGFR (EGFR) or anti-Akt (Akt) antibody. (C) Specific homodimerization of EGFR detected by BiFC. CHO cells co-expressing the BiFC constructs indicated were serum-starved, and then observed by confocal microscopy. Cells co-expressing EGFR-VN and –VC were also treated with 100 ng/ml EGF for 30 minutes at 37°C (+ EGF). Bars, 10 µm. (D) Examination of expression levels of BiFC constructs in the positive and negative controls by immunofluorescent staining. CHO cells co-expressing the BiFC constructs indicated were fixed with methanol-acetone, and immunostained with murine anti-FLAG primary antibody and goat anti-HA primary antibody, followed by incubation with Cy3-conjugated donkey anti-mouse secondary antibody and Alexa Fluor 633-conjugated donkey anti-goat secondary antibody. Fluorescence signals of FLAG-tagged proteins (cyan), HA-tagged proteins (red) and Venus (green) were observed by confocal microscopy. Bars, 10 µm. (E) EGF binding does not affect fluorescence intensity of cells co-expressing EGFR BiFC constructs. CHO cells co-expressing EGFR-VN and EGFR-VC were serum-starved, and then treated with ATP synthesis inhibitors for 1.0 hour as described in Materials and Methods. Fluorescence intensities of ROI (region of interest) on the cell membrane were observed by confocal microscopy. 100 ng/ml EGF was added to the cell culture at the time indicated by arrow. The relative intensities were normalized by the average intensity at 3 minutes, and the data points are the means ± s.d. (n=32). (F) Subcellular localization of BiFC construct homodimers of the ErbB receptors in the presence of bound ligand. CHO cells expressing the BiFC constructs indicated were serum-starved, and then observed by confocal microscopy. Cells expressing ErbB3-VN and -VC, or ErbB4-JMa-VN and -VC were also stained with Hoechst 33342 to visualize nuclei. Cells co-expressing ErbB3-VN and -VC, or ErbB4-VN and -VC were treated without (–NRG) or with (+NRG) 1.0 nM NRG for 30 minutes at 37°C. Bars, 10 µm.

View larger version (37K): [in this window] [in a new window]   Fig. 3. Preformed heterodimers of the ErbB receptors in the absence of bound ligand. (A) BiFC analysis of the ErbB receptor heterodimerization in living cells. CHO cells expressing the BiFC constructs indicated were serum-starved, stained with or without Hoechst 33342, and then observed by confocal microscopy. Bars, 10 µm. (B) CHO cells co-expressing the BiFC constructs indicated were serum-starved, treated with 100 ng/ml EGF (+EGF) or 1.0 nM NRG (+NRG) for 30 minutes at 37°C, and then observed by confocal microscopy. Bars, 10 µm. (C) Ligand-induced phosphorylation of preformed ErbB heterodimers. CHO cells co-expressing ErbB2-VN and EGFR-VC, or ErbB3-VN and ErbB2-VC were serum-starved, and then treated with (+) or without (–) 100 ng/ml EGF or 1.0 nM NRG for 30 minutes at 4°C. Whole cell lysates were subjected to immunoblotting using specific antibodies for phosphorylated forms (p-EGFR, p-ErbB2, p-ErbB3 and p-Akt) or for unphosphorylated forms of EGFR, ErbB2, ErbB3 and Akt.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Intracellular domains of the ErbB receptors have dimerization activity, as shown by the mammalian two-hybrid assay. GAL4 and VP16 fusion proteins were encoded by the pBIND and pACT constructs, respectively, as described in Materials and Methods. NIH3T3 cells were co-transfected with a pair of expression plasmids encoding the receptor cytoplasmic domains indicated, and their luciferase activities were measured as described in Materials and Methods. Bars show the fold changes in luciferase activity relative to the value induced by the empty vectors.

View larger version (81K): [in this window] [in a new window]   Fig. 5. The ErbB receptor homo- and heterodimers formed in ER. NIH3T3 cells co-expressing the BiFC constructs indicated were serum-starved, and then treated with (+) or without (–) 10 µg/ml BFA for 8 hours at 37°C. The cells were fixed with methanol-acetone and immunostained with anti-calnexin antibody, followed by incubation with Cy3-conjugated secondary antibody. Nuclei were also stained with Hoechst 33342. The cells were observed by confocal microscopy for calnexin (red), Venus (green) or Hoechst 33342 (cyan) fluorescence. Bars, 10 µm.

View larger version (85K): [in this window] [in a new window]   Fig. 6. Subcellular localization of endogenous ErbB receptors in human carcinoma cells. A431, MDA-MB-453, MDA-MB-468 and MDA-MB-453 cells were serum-starved, fixed with methanol-acetone and immunostained with anti-EGFR, anti-ErbB2, anti-ErbB3 or anti-ErbB4 primary antibody, respectively, followed by incubation with Alexa Fluor 635- or Cy3-conjugated secondary antibody. Nuclei were also stained with Hoechst 33342. Stained cells were observed by confocal microscopy. Note that ErbB3 fluorescence intensity was highest in the nuclei. Bars, 10 µm.

View larger version (21K): [in this window] [in a new window]   Fig. 7. Nuclear ErbB3 is a full-length molecule. (A) MDA-MB-468 cells were serum-starved, and then nuclear and cytoplasmic-membrane fractions were prepared as described in Materials and Methods. The fractions were subsequently analyzed by western blotting using anti-ErbB3 antibody. The efficiency of cell fractionation was confirmed by examination of a cytoplasmic protein, -tubulin and a nuclear protein, histone H3, using anti--tubulin and anti-histone H3 antibodies, respectively. (B) CHO cells expressing ErbB3-CFP were serum-starved, and fluorescence intensities in the nucleus and cytoplasm were measured by confocal microscopy. Relative nuclear and cytoplasmic fluorescence intensities are expressed as the mean ± s.d. (n=24).

View larger version (31K): [in this window] [in a new window]   Fig. 8. Model for molecular mechanism underlying the activation of preformed ErbB receptor dimers by ligand binding. The extracellular domain of the ErbB receptor consists of four sub-domains denoted by 1-4. N and C indicate the N and C lobes of the intracellular kinase domain, which is followed by the C-terminal tail containing five major tyrosine residues for autophosphorylation. The top two molecules are inactive dimers with a closed/tethered form (left) and open/extended/untethered form (right). The bottom two molecules are ligand-bound active dimers, in which unphosphorylated tyrosine (Y) and phosphorylated (pY) are shown.

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