In order to delineate the mechanism of signal transduction for growth facto receptors at the initial stage of binding, we focused on defining the tertiary structure of the extracellular domain of epidermal growth factor (EGF) receptor and assessing the existence of potential conformational changes that insulin receptor will undergo when its ligand binds to the receptor. For the former study, the A431 cell line was chosen as the source of the soluble form of EGF receptor. To facilitate the collection of conditioned medium, a new method of large scale adherent cell culture was employed in which cells were grown in a hollow fiber bioreactor. The purified extracellular binding domain will be pooled and analyzed for folde domain analysis. Also, the disulfide bond pairing will be determined. Previously synthesized peptides from the insulin receptor intracellular domains were used to raise rabbit polyclonal antibodies. With Dr. van Obberghen's group, we identified a conformational change in the receptor C-terminus induced by insulin binding to the receptor. Monoclonal antibody preparation is underway to prepare microinjectable antibody to examine the effects of antibody on the receptor signal transduction and identification of second messenger/substrate molecules.
