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  • Original Paper
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Unique carboxyl-terminal sequences of wild type and alternatively spliced variant forms of transforming growth factor-α precursors mediate specific interactions with ErbB4 and ErbB2

Oncogene volume 19, pages 3172–3181 (2000)Cite this article

Abstract

We have previously reported that the human transforming growth factor-α (TGF-α) gene encodes three forms of TGF-α precursors, designated wild type (WT), variant I (VaI), and variant II (VaII), derived from alternative splicing. The two carboxyl-terminal valine residues of WT are replaced by 5 (GCRLY) or 4 (ATLG) amino acids in VaI or VaII, respectively. When overexpressed in Chinese hamster ovary (CHO) cells, VaI and VaII, but not WT, support autonomous growth. We detected tyrosine phosphorylation of ErbB2 in the absence of serum, in CHO cells expressing WT, VaI, or VaII, but not in mock transfectants. These observations prompted us to investigate possible interactions between the ErbBs and the TGF-α precursors in CHO cells. All TGF-α precursors were found to co-immunoprecipitate with the ErbBs, but with different specificity. WT co-immunoprecipitated with ErbB4, but not with ErbB1, ErbB2, or ErbB3. VaI and VaII co-immunoprecipitated with ErbB2, but not with ErbB1, ErbB3, or ErbB4. Confocal fluorescent microscopy analysis demonstrated that WT, VaI, and VaII all distribute equally to the cell surface while, as expected, a WT mutant lacking the two C-terminal valine residues does not. Point and deletion mutants involving the unique carboxyl-terminal residues of WT, VaI and VaII, indicated that the interactions between the three TGF-α precursors and the ErbBs were mediated by their carboxyl-terminal regions, which constitute distinct protein-binding motifs. A chimera of the intracellular domain of WT TGF-α linked to exogenous transmembrane and extracellular domains retained both the cell surface distribution and the specific interaction with ErbB4 of full-length WT, confirming that this interaction is mediated by the C-terminus of the TGF-α precursor. While interactions of WT and variant TGF-α with the ErbBs all result in ErbB2 activation, they produce different biological consequences, suggesting that the various TGF-α precursors differentially modulate ErbB signaling.

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Abbreviations

BTC:

β-cellulin

CHO:

Chinese hamster ovary

EGF:

epidermal growth factor

EGFR:

epidermal growth factor receptor

EPR:

epiregulin

FBS:

fetal bovine serum

HB-EGF:

heparin-binding epidermal growth factor

IP:

immunoprecipitation

NRG:

neuregulin

PBS:

phosphate-buffered saline

PDZ:

PSD-95/Dlg/ZO-1

RPA:

ribonuclease protection assay

TGF-α:

transforming growth factor α

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Acknowledgements

We thank TJ Velu for the pCO12-EGFR construct, Nancy Hynes (FMI, Switzerland) for the pBabe-ErbB2, ErbB3, ErbB4 constructs, H Land (ICRF, London, UK) for the pBabe vectors. We thank Craig Woodworth (NCI) for the human tumor-derived cell lines and Elena Mourateva for establishing normal human keratinocyte cultures. This work was supported by National Institutes of Health Grant CA62094 (L Pirisi) and the South Carolina Endowment for Children's Cancer Research (KE Creek). This work was performed in partial fulfilment of the requirements for the PhD degree by X Xu.

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  1. Department of Pathology, University of South Carolina School of Medicine, Columbia, 29208, South Carolina, SC, USA

    Xingzhi Xu, Kevin F Kelleher, Ji Liao, Kim E Creek & Lucia Pirisi

  2. Department of Pediatrics, Children's Cancer Research Laboratory, University of South Carolina School of Medicine, Columbia, 29208, South Carolina, SC, USA

    Kim E Creek

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  1. Xingzhi Xu
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Xu, X., Kelleher, K., Liao, J. et al. Unique carboxyl-terminal sequences of wild type and alternatively spliced variant forms of transforming growth factor-α precursors mediate specific interactions with ErbB4 and ErbB2. Oncogene 19, 3172–3181 (2000). https://doi.org/10.1038/sj.onc.1203645

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