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Comparative Study
. 2002 Jun 10;157(6):929-39.
doi: 10.1083/jcb.200109033. Epub 2002 Jun 3.

c-erbB-3: a nuclear protein in mammary epithelial cells

Martin Offterdinger  1 , Christian Schöfer, Klara Weipoltshammer, Thomas W Grunt
Affiliations
Comparative Study

c-erbB-3: a nuclear protein in mammary epithelial cells

Martin Offterdinger et al. J Cell Biol. .

Abstract

c-erbB receptors are usually located in cell membranes and are activated by extracellular binding of EGF-like growth factors. Unexpectedly, using immunofluorescence we found high levels of c-erbB-3 within the nuclei of MTSV1-7 immortalized nonmalignant human mammary epithelial cells. Nuclear localization was mediated by the COOH terminus of c-erbB-3, and a nuclear localization signal was identified by site-directed mutagenesis and by transfer of the signal to chicken pyruvate kinase. A nuclear export inhibitor caused accumulation of c-erbB-3 in the nuclei of other mammary epithelial cell lines as demonstrated by immunofluorescence and biochemical cell fractionation, suggesting that c-erbB-3 shuttles between nuclear and nonnuclear compartments in these cells. Growth of MTSV1-7 on permeable filters induced epithelial polarity and concentration of c-erbB-3 within the nucleoli. However, the c-erbB-3 ligand heregulin beta1 shifted c-erbB-3 from the nucleolus into the nucleoplasm and then into the cytoplasm. The subcellular localization of c-erbB-3 obviously depends on exogenous stimuli and on the stage of epithelial polarity and challenges the specific function of c-erbB-3 as a transmembrane receptor protein arguing for additional, as yet unidentified, roles of c-erbB-3 within the nucle(ol)us of mammary epithelial cells.

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Figures

Figure 1.
Figure 1.
Nuclear localization of c-erbB-3 in MTSV1-7. (A) Confocal immunofluorescent microscopy demonstrating nuclear c-erbB-3 using the monoclonal RTJ2 antibody directed against a cytoplasmic epitope of c-erbB-3 (a) or polyclonal antibodies against an NH2- (b, Ab-9) or COOH-terminal domain (c, C17). Note in panel b some cytoplasmic and membrane staining is also seen. Bar, 25 μm. (B) Immuno EM using RTJ2 (a–c) and Ab-9 (d) revealing nuclear (a and d), cytoplasmic (b and d), and cell membrane-located (c and d) c-erbB-3 (arrows). np, nucleoplasm; cp, cytoplasm. Bars, 0.1 μm.
Figure 2.
Figure 2.
Effects of LMB on the subcellular distribution of c-erbB-3 using confocal immunofluorescence microscopy. (A) Various cell lines were treated for 24 h with solvent (−) or 20 ng/ml LMB (+) and immunostained using RTJ2. Bar, 25 μm. (B) MCF-7 cells were transiently transfected with FLAG-tagged c-erbB-3, treated for 24 h with solvent (Control) or 5 ng/ml LMB, and immunostained with M2 anti-FLAG antibody. Bar, 5 μm.
Figure 3.
Figure 3.
Immunochemical demonstration of c-erbB-3 in MCF-7 cells. (A) 400 μg protein from untreated (−) or LMB-treated (+) C or N MCF-7 fractions were immunoprecipitated (IP) with SGP1, which detects an extracellular epitope of c-erbB-3, or with mIgG1 and blotted (IB) with C17, which binds to the cytoplasmic part of c-erbB-3. (B) 20 μg total protein from each fraction (C or N) were blotted with anti–c-erbB-3 (C17), antipyruvate kinase, anti–histone H1, anticalnexin, or antitransferrin receptor. Note calnexin has an apparent molecular mass of 90 kD (arrow). The bottom band seen in N represents a nonspecifically stained bulk nuclear protein. (C) Cytoplasmic-enriched fractions were prepared with or without the addition of 0.2% Triton X-100 and blotted for c-erbB-3 (C17). After centrifugation at 100,000 g, an arbitrary amount of the membrane pellet (M) from the fraction prepared without Triton X-100 was loaded as a control.
Figure 4.
Figure 4.
Immunochemical analysis of HRG secretion from MTSV1-7 and MCF10A cells. Concentrated serum-free supernatants from cell-free (0), subconfluent (sub), and confluent (con) MTSV1-7 or MCF10A cultures were blotted with anti-pan HRG.
Figure 5.
Figure 5.
HRGβ1 affects subcellular distribution of c-erbB-3. MTSV1-7 were examined by confocal immunofluorescent microscopy (A,B, and D) and immuno EM (C) for c-erbB-3 (A–C) or HRG (D). (A) Cells on filters pretreated with neutralizing anti-HRG (10 μg/ml, 48 h) were exposed for various times to 1 nM HRGβ1. Note mitotic cells were strongly positive. (B) Endogenous HRG was not blocked by anti-HRG (0 min) before exogenous HRGβ1 (1 nM, 48 h). (C) 0 min, after blocking of endogenous HRG, c-erbB-3 (arrows) was found in the granular component (gc) of the nucleolus (nc). np, nucleoplasm. At 24 h, added HRGβ1 (1 nM) caused relocation of c-erbB-3 into the cytoplasm (cp) and to the cell membrane. (D) Cells were treated as in B but immunostained with anti-pan HRG. Bars: (A, B, and D) 40 μm; (C) 0.1 μm.
Figure 6.
Figure 6.
NLS-2 is functional in c-erbB-3. (A) MTSV1-7 transiently transfected with EGFP–mutant-erbB-3 fusion constructs. Removal of NLS-2 by COOH-terminal truncation completely precluded nuclear localization of the protein in A, panel a (158 kD; larger than the size exclusion limit of the nuclear pore complex, which is ∼60 kD), whereas the smaller construct in A, panel b (36 kD) can be found in both compartments. The COOH-terminal fragment containing NLS-2 (55 kD) is concentrated in the nuclei (A, c). Inactivating mutation of NLS-2 (mutNLS-2) partially reverts nuclear concentration (A, d). Bar, 25 μm. (B) At least four independent points, randomly chosen in each compartment (C, cytoplasm; N, nucleus), were measured in arbitrary units (pixel intensity), and relative antigen staining was expressed for each compartment in percent of total (C + N, means ± SD).
Figure 7.
Figure 7.
NLS-2 is sufficient for nuclear targeting. (A) MTSV1-7 transiently transfected with CPK fusion constructs. CPK alone localizes to the cytoplasm (CPK), whereas the control NLS of human lamin C (CPK-CoNLS) mediates efficient nuclear targeting of CPK. NLS-2 of human c-erbB-3 (CPK-NLS-2), consisting of the sequence RRRRHSP*, efficiently localizes CPK to the nucleus. The same is true for the complete COOH terminus of c-erbB-3 (CPK-erbB3CT). Bar, 25 μm. (B) Quantitation (performed as in 6 B) demonstrated that NLS-2 and the COOH terminus of c-erbB-3 are as effective as the control NLS of human lamin C in directing CPK from the cytoplasm (C) into the nucleus (N).
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