Functional Sites of the erbB-2 Receptor and Its Activator Heregulin

摘要

Over expression of the erbB-2 (HER-2/neu) receptor occurs in up to 30% of human breast cancers and correlates with aggressive disease and poor prognosis for therapy and survival. The growth factor heregulin (HRG) binds to erbB-3 or erbB-4 receptors, promotes dimmer formation with erbB-2 and induces autophosphorylation and activation of erbB-2 signaling. It is generally accepted that HRG and erbB-2 do not interact directly. Depending on its concentration HRG can either inhibit or stimulate cell proliferation in cell lines that overexpress erbB-2. This suggests some type of direct interaction between HRG and erbB-2. Solution structure of HRG and other data support the existence of a low-affinity binding site within the EGF-like domain of HRG. The goal of the proposed experiments is to define the predicted sites of interaction between HRG and the erbB-2 receptor, through generation of HRG and erbB-2 deletion mutants. During the third year of funding, I continued the experiments according to the data obtained in the previous years: a) performed proliferation assays using Ba/ F3 cells infected with deletion mutants of erbB-2 as well as autophosphorylation studies. I also infected with erbB-2 deletion mutants 184B5 immortalized human epithelial cells in order to establish a better in vitro system to study erbB-2 induced tumorigenesis; b) continued to work on purification of recombinant proteins containing alanine substitution in heregulin 1 gene and generated cell lines expressing heregulin mutants in cells coexpressing different levels of erbB receptors. The main result of my work was generation of 8 cell lines expressing the erbB-2 receptor containing different deletions in the extracellular domain, which allowed me to map functional site of this receptor, and 15 cell lines with different levels of erbE receptors expressing heregulin with alanine substitutions within the EGF-like domain.