Protein-tyrosine kinases are important in regulation of growth and differentiation. They are oncogenic when mutated or inappropriately expressed. The mechanism of action of two tyrosine kinase growth factor receptors, the neu protein and the EGF receptor, will be investigated. These proteins are implicated in human breast and squamous cell cancer, respectively. Since they contribute directly to carcinogenesis and are cell surface proteins, they are important therapeutic targets. The present studies will elucidate the mechanism of signalling by tyrosine kinase growth factor receptors and aid in determining how they exert their effects on cells. 1.neu can be activated by a point mutation in the transmembrane domain. A sub-domain within the transmembrane domain has been identified in which second-site mutations inhibit the activity of this point mutation. This region will be further analyzed by site-directed mutation to determine whether it functions in normal signalling stimulated by hormone binding. 2.The oligomerization status of this panel of mutant proteins will be measured to determine whether the same sub-domain is required for neu protein oligomerization as a test of the clustering model for neu activation. 3.A single extra Cysteine residue introduced into the neu protein induces intermolecular crosslinks. These crosslinks will be moved along the molecule to determine whether certain intermolecular orientations activate the protein. 4.Information obtained above will be used to construct structural models for intermolecular interactions mediated by the transmembrane domain. 5.The neu protein and the EGF receptor cross-regulate one another. The mechanisms and consequences of these interactions will be investigated.
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