A crucial role for Epidermal Growth Factor Receptor (erbB) family members in development of breast cancer has been demonstrated over the past 15 years. We have recently cloned a novel protein, EBP1, which interacts with the cytoplasmic domain of erbB3. Treatment of cells with the erbB-3 ligand, heregulin (HRG) induces the translocation of EBP1 from the cytoplasm to the nucleus. Overexpression of EBP1 inhibits proliferation of human breast cancer cells. We propose to study the physiological function of erbB-3EBP1 interactions in human breast cancer cells. Understanding the regulation and consequences of these interactions could lead to a better understanding of erbB-3 signal transduction and to the definition of new therapeutic targets in breast cancer. First, to define the function of erbB-3-EBP1 interactions, we will a) determine HRG-mediated interactions of EBP1 with other erbB receptors and the dependence of such interactions on the expression of erbB-3 heterodimeric binding partners using the yeast-two hybrid system and engineered murine cells expressing defined combinations of erbB receptors 2) establish the colocalization of EBP1 and erbB-3 by confocal microscopy before and after HRG treatment, c) determine the region of EBP1 required for erbB-3 binding and the effects of expression of different regions of EBP1 on HRG-mediated signalling using a series of deletion and single site mutants of EBP1, d) determine the effects of EBP1 overexpression on basal and HRG-induced cell growth and differentiation using stable transfectants with inducible expression. Second, we will determine the role of Protein Kinase C (PKC) in modulating EBP1 activity. The sites of phosphorylation after HRG treatment will be determined using tryptic phosphopeptide mapping and phosphamino acid analysis. Effects of PKC inhibitors on EBP1 function will be assessed in intact cells. Potential PKC sites will be mutated and effects of such mutations on EBP1 function defined. Third, we will define the significance of EBP1 nuclear localization. We will a) determine if EBP1 is part of a multiprotein nuclear localization complex by isolating EBP1 interacting proteins using the yeast two-hybrid system b) define the role of EBP1 and its accessory transcriptional activators using GAL4 DNA binding domain fusions c) identify preferred DNA binding sites for EBP1 using a degenerate oligonucleotide strategy.
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