The ErbB-2/HER2 oncoprotein of human carcinomas may function solely as a shared coreceptor for multiple stroma-derived growth factors - PubMed (original) (raw)

The ErbB-2/HER2 oncoprotein of human carcinomas may function solely as a shared coreceptor for multiple stroma-derived growth factors

L N Klapper et al. Proc Natl Acad Sci U S A. 1999.

Abstract

The erbB-2/HER2 oncogene is overexpressed in a significant fraction of human carcinomas of the breast, ovary, and lung in a manner that correlates with poor prognosis. Although the encoded protein resembles several receptors for growth factors, no high affinity ligand of ErbB-2 has so far been fully characterized. However, several lines of evidence have raised the possibility that ErbB-2 can augment signal transduction initiated by binding of certain growth factors to their direct receptors. Here, we contrasted these two models of ErbB-2 function: First, examination of a large series of epidermal growth factor (EGF)-like ligands and neuregulins, including virus-encoded ligands as well as related motifs derived from the precursor of EGF, failed to detect interactions with ErbB-2 when this protein was singly expressed. Second, by using antibodies that block inter-ErbB interactions and cells devoid of surface ErbB-2, we learned that signaling by all ligands examined, except those derived from the precursor of EGF, was enhanced by the oncoprotein. These results imply that ErbB-2 evolved as a shared receptor subunit of all ErbB-specific growth factors. Thus, oncogenicity of ErbB-2 in human epithelia may not rely on the existence of a specific ligand but rather on its ability to act as a coreceptor for multiple stroma-derived growth factors.

PubMed Disclaimer

Figures

Figure 1

Figure 1

ErbB-2 activation depends on coexpression of other ErbB proteins. ErbB-2 phosphorylation was determined in cells expressing the receptor singly (A, D2) or in combination with ErbB-1 and ErbB-3 (B, SKOV3). The indicated ligands (100 ng/ml) or antibodies (20 μg/ml) were used to treat the cells for 5 min at 37°C. Receptor activation in whole cell lysates (A) or immunoprecipitates of ErbB-2 (B) was determined by an antibody directed against phosphorylated tyrosine.

Figure 2

Figure 2

ErbB-2-dependency of growth stimulation by EGF-like ligands. 32D cells expressing ErbB-2 with either ErbB-1 (D12), ErbB-3 (D23), or ErbB-4 (D24) were tested for cell proliferation. Cells deprived of IL-3 were treated with the indicated ligands. Anti-ErbB-2 mAbs belonging to class I (L431), class II (L26, L96), class III (L140), and class IV (L87) or their respective Fab fragments (F26, F431) were added simultaneously. Alternatively, control antibodies were used, including an unrelated mAb (NR), mAbs capable of ligand displacement from ErbB-3 (C105) or ErbB-4 (C72, C36), or an antibody against ErbB-3 that is incapable of displacing NRGs (C379). The extent of cell proliferation was determined 24 h after the addition of stimulating factors by using the colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The results are presented as fold induction over control untreated cells and are the mean ± SD of eight determinations. Note that most mAbs (e.g., L26) have a weak agonist activity of their own.

Figure 3

Figure 3

The effect of surface-expressed ErbB-2 on the kinetics of ligand-induced tyrosine phosphorylation and MAPK activation. ErbB ligands were used to stimulate T47D breast cancer cells and their derivative, T47D-5R, which lacks surface expression of ErbB-2. A comparable number of cells was stimulated at 37°C by the indicated ligands (at 100 ng/ml) for various time intervals. Receptor activation, in whole cell lysates, was detected by immunoblotting (IB) with an antibody directed against phosphorylated tyrosine (P-TYR). MAPK activation in the same preparations was determined by using an antibody against the active doubly phosphorylated form of Erk proteins (Activated MAPK). For control of equal gel loading, the upper part of membranes used to detect MAPK was used to determine the amount of ErbB-2. Note that the 5R cells exhibited up-regulation of the cell-retained ErbB-2.

Figure 4

Figure 4

Activation of ErbB receptors by EGF-like motifs of human proEGF. (A) GST fusion proteins containing EGF-like motifs 1–4, 5–8, or 5–9 of the EGF precursor were immobilized on glutathione-agarose beads. For control, GST fusion proteins containing EGF or NDF were used. The beads were incubated for 1 h at 4°C with conditioned media containing 1 μg of the indicated IgB protein. Protein complexes were immunoblotted with an anti-human Fc antiserum for detection of bound IgBs. (B) Monolayers of the indicated human breast cancer cell lines were incubated, for 10 min at 37°C, in the presence of 100 ng/ml GST fusion proteins or 5 ng/ml ligands (EGF or NDF). Receptor activation was detected by an antiphosphotyrosine antibody.

Figure 5

Figure 5

Viral peptides recruit ErbB-2. (A) Phosphorylation of ErbB-2 by viral peptides [vaccinnia virus growth factor (VGF), Myxoma virus growth factor (MGF), and SFGF] and antibodies (L87, L431) was examined as described in the legend to Fig. 1. (B) IL-3-deprived D23 cells were stimulated by viral peptides in the presence (+L26) or absence _(−_L26) of a class II mAb to the human ErbB-2 (Left). Cells singly expressing ErbB-2 (D2) or ErbB-1 (D1) served as negative and positive controls for ligand activity, respectively. Proliferation induction was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay as described in the legend to Fig. 2. For control of endogenous proliferation signals, we incubated cells with IL-3. (C) The effect of ErbB-2 on downstream activation by SFGF was examined in cells that do (T47D) or do not (T47D-5R) express ErbB-2 on their surface. A time response of activation was detected in whole cell lysates by immunoblotting with an antibody against activated MAPK. The amount of ErbB-2 was verified by immunoblotting the upper part of the membrane with an antibody against the receptor.

Similar articles

Cited by

References

    1. van der Geer P, Hunter T, Lindberg R A. Annu Rev Cell Biol. 1994;10:251–337. - PubMed
    1. Burden S, Yarden Y. Neuron. 1997;18:847–855. - PubMed
    1. Salomon D S, Brandt R, Ciardiello F, Normanno N. Crit Rev Oncol Hematol. 1995;19:183–232. - PubMed
    1. Hynes N E, Stern D F. Biochim Biophys Acta. 1994;1198:165–184. - PubMed
    1. Klapper, L. N., Kirschbaum, M. H., Sela, M. & Yarden, Y. (1999) Adv. Cancer Res., in press. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources