NMR STUDIES OF EPIDERMAL GROWTH FACTORS

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Epidermal growth factor (EGF), a protein hormone effector molecule is known to regulate cellular development and division. Like all known protein hormones, the mechanism of its action is through binding to a transmembrane receptor molecule. This binding event triggers a series of cellular events (egs. endocytosis; kinase activity, etc...) that lead to such physiological effects as: increased uptake of salts and glucose; increased protein, RNA, and DNA synthesis, and ultimately, cell division. EGF has been, furthermore, implicated as a regulatory substance in tumorigenicity & senescence, as well as in embryogenesis and normal cell and organ growth. Many studies of structure-function relationships of a variety of polypeptide hormones have suggested that the biological function of these molecules is stringently dependent on a precise chemical and physical structure. Yet, little is known about the structure of EGF, about how the conformation of this potent mitogen may be related to its biological effects, or about any specific structural domain(s) essential for interaction with its membrane receptor. Specific aims of this proposal include several steps toward understanding the structure-function relationships in EGF. EGF from three species (i.e. mouse, rat, and human) will be structurally characterized primarily using recently developed 1H two-dimensional NMR methods. Since the binding of any of these EGFs to human fibroblasts is nearly identical although none of the sequences are identical, a receptor binding domain common to all three EGFs is, therefore, proposed. By elucidation and comparison of these three EGF structures, the potential exists for identification of the EGF receptor binding domain. Investigation of conformational perturbations in EGF induced by environmental factors such as pH, temperature, ionic strength and the presence of metabolites is also planned. Amide proton exchange will be studied in order to investigate EGF protein "breathing" dynamics. These data should provide a better understanding of structure-function relations in this important regulatory protein hormone and in growth factors in general.