The long-term objective of this project is to develop a quantitative computer-assisted model of the interaction of polypeptide hormones with responsive human cells. This proposal uses a model system the interaction of the polypeptide mitogen epidermal growth fact (EGF) with cultured human fibroblasts. The rate limiting steps in the cellular pathway involved in the binding, internalization and degradation of the hormone will be defined, quantitated and then incorporated into a computer simulation so that their role in hormone-mediated cellular responses can be more easily investigated. This project will specifically examine the intracellular processing of EGF subsequent to receptor-mediated endocytosis and define those intracellular enzymes that are responsible for the modification of the internalized hormone. Once identified, the processing enzymes will be used as hormone-independent markers for the subcellular compartments through which EGF passes prior to complete degradation which will facilitate a kinetic analysis of the process. An assay for the EGF receptor will be developed to allow an investigation of the effect of EGF on cellular receptor localization. The kinetics of intracellular translocation of both the internalized EGF and its receptor will be determined so that those points in the intracellular pathway that are under regulatory control can be defined. The effect of pH alterations and EGF processing on the ability of the ligand to bind to its receptor will be quantitated in an effort to define how these intracellular processes can affect receptor occupancy. Additional studies will be directed towards quantitating the biosynthetic rate of the EGF receptor and the relationship between this rate and the rate that the EGF receptor appears at the cell surface. This study should reveal if the EGF receptor is recycled to any appreciable extent. The relationship between receptor internalization and degradation will also be investigated in both the presence and absence of EGF to define how hormone addition affects the total cellular content and distribution of receptors. The information derived from all of these experiments will be used as a foundation for constructing a comprehensive model of EGF-cell interactions.
