The Relationship between Human Epidermal Growth-like Factor Receptor Expression and Cellular Transformation in NIH3T3 Cells

Bruce D Cohen,Peter A Kiener,Janell M Green,Linda Foy,H Perry Fell,Ke Zhang

doi:10.1074/jbc.271.48.30897

Abstract

A collection of cell lines expressing each human epidermal growth factor receptor (HER) family member alone or in all pairwise combinations in a clone of NIH3T3 cells (3T3-7d) devoid of detectable epidermal growth factor receptor family members has been generated. Transformation, as measured by growth in soft agar, occurred only in the presence of appropriate ligand and only in cells expressing two different HER family members. Transfection of oncogenic neu (Tneu), conferred ligand-independent transformation only in cells which co-expressed HER1, HER3, or HER4, but not when expressed alone or with HER2. Cell lines were also tested for their ability to form tumors in animals. None of the cell lines expressing single HER family members was able to form tumors in animals with the exception of HER1, which was weakly tumorigenic. Although unable to form tumors when expressed alone, HER2 was tumorigenic when expressed with HER1 or HER3, but not HER4. Of all complexes analyzed, cells expressing HER1 + HER2 were the most aggressive. The relationship between HER1 activation, intracellular calcium fluxes, and phospholipase Cgamma1 activation is well established. We found that activation of HER1 was required for the induction of a calcium flux and the phosphorylation of phospholipase Cgamma1. These activities were independent of, and unaffected by, the co-expression of any other family member. Further, heregulin stimulation of all cell lines including those containing HER1 did not demonstrate any effect on intracellular calcium levels or phospholipase Cgamma1 phosphorylation. This demonstrates that heregulin induced cellular transformation by activating HER3- and HER4-containing complexes does not require the activation of either phospholipase Cgamma1 or the mobilization of intracellular calcium.

Highlights

The involvement of tyrosine kinase growth factor receptors and their ligands in the regulation of cell growth and differentiation is well documented
The lack of transformation by homodimer expression was not due to a lack of receptor kinase activity, for all homodimer complexes, especially HER1, demonstrated an increase in receptor tyrosine phosphorylation in response to ligand. This is supported by the fact that expression of Transfection of oncogenic neu (Tneu), a potent transforming oncogene, induced colony formation in cells that co-expressed either HER1, HER3, or HER4 but not when expressed alone or co-expressed with HER2
The complementation of Tneu with HER1, HER3, and HER4 suggests that Tneu can heterodimerize with other family members, the biochemical identification of this heterodimer was not investigated

Summary

Introduction

The involvement of tyrosine kinase growth factor receptors and their ligands in the regulation of cell growth and differentiation is well documented. We were able to demonstrate that transformation, as defined as growth in soft agar, was dependent upon the expression of multiple HER family members in the same cell, and was independent of intracellular calcium mobilization or PLC␥1 phosphorylation. With respect to ligand stimulation of cell lines expressing only one family member, HER1 was partially tyrosine phosphorylated in the absence of ligand and was further stimulated by addition of EGF.

Results

Conclusion