FUNCTIONAL ANALYSIS OF THE ERBB-3 BINDING PROTEIN EBP1 IN HEREGULIN-MEDIATED SIGNAL TRANSDUCTION.
^1,2 Terri T. Hines, ^2,3 Yuexing Zhang, ² Nicholas Woodford and ^2,3 Anne Hamburger
¹ Department of Biological Sciences, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250
² Greenebaum Cancer Center, ³ Department of Pathology, University of Maryland, Bressler Building, 655 W. Baltimore Street, Baltimore, MD, 21201

Ebp1, was cloned in our laboratory using yeast two hybrid analysis as an ErbB3 binding protein. Ebp1 has also been shown to interact with Rb, a tumor suppressor protein to repress the transcription of proliferation-associated genes in breast cancer cells.  In vitro studies have shown that ectopic expression of Ebp1 inhibits the growth and induces the maturation of breast cancer cells.  Heregulin , a ligand that binds the ErbB3 receptor, also causes maturation of breast cancer cells. We therefore hypothesized that Ebp1 can participate in a Heregulin mediated signal transduction pathways that ultimately result in maturation of breast cancer cells.   To determine whether Heregulin can still function in the absence of Ebp1, we used anti-sense oligonucleotides to inhibit the expression of Ebp1. We also used dominant negative mutants of Ebp1 that inhibit Ebp1 function. Western Blot analysis was used to determine inhibition of Ebp1 expression after treatments with antisense oligos. We used a Dual Luciferase Assay to determine if Heregulin, in the absence of Ebp1 ,can still induce expression of the Heregulin regulated gene, ICAM-1. Luciferase is the enzyme that causes fireflies to glow.  A Dual Luciferase Assay includes luciferase from a firefly and a sea pansy.  The intensity of the glow indicates the amount of protein expressed.  From these studies it has been shown that Heregulin can still induce the expression of ICAM-1 in the absence of functional Ebp1.  Future studies will help us gain further knowledge of the relationship of Ebp1 with Heregulin induced signal transduction pathways that ultimately leads to gene transcription.